Taking the Measure

19 of Microbial Systems

I Culture-Dependent Analyses of Microbial

Communities 649
II Culture-Independent Microscopic Analyses
of Microbial Communities 656
III Culture-Independent Molecular Analyses
of Microbial Communities 661
IV Measuring Microbial Activities in Nature 673

MICROBIOLOGYNOW

Touring Microbial Biogeography Using Combinatorial Imaging

Virtually all microorganisms exist as parts of complex com- CLASI-FISH hybridizes each cell with a combination of probes
munities that interact through metabolic cooperation. Since specific for that species but labeled with different fluorescent
complex metabolic interactions are not easily resolved, the dyes, giving each cell a unique fluorescent spectral signature.
microscope is an essential tool for first identifying possible Since the resulting fluorescence at each wavelength is a
cooperation based on colocalization of different species. linear combination of emissions from each fluorescent dye,
However, even the simplest of microbial communities is statistical analysis can determine what combination of dyes
composed of tens if not hundreds of different species. How produced the emission spectrum and therefore identify the
it is possible to visualize the distribution of individual species contributing species.
in such a mixture? The photo shown here was taken from a scraping of the
As we will see in this chapter, microscopic identification human tongue (the specimen is about 140 μm in diameter,
of species commonly uses fluorescence in situ hybridization left to right), showing a remarkable organization of microbial
(FISH), a technique in which cells of individual species are species that almost certainly reflects metabolic interactions
identified by hybridization of fluorescent DNA probes to that can now be further explored using the powerful tools
ribosomal RNA sequences unique to each species. However, we discuss in this chapter. Brown in the photo is human
the method can visualize only a few different species simulta- tissue; the bacteria are: red, Actinomyces spp.; green, Strep-
neously, being limited by the number of dyes that fluoresce tococcus spp.; blue, Rothia spp.; yellow, Neisseria spp.; and
in different colors. A variation on standard FISH technology magenta, Veillonella spp.
that circumvents this limitation is called CLASI-FISH (combi-
natorial labeling and spectral imaging–FISH), which can Source: Welch, J.L.M., Dewhirst, F.E., and Borisy, G.G. 2019. Bioge-
ography of the oral microbome: The site-specialist hypothesis. Annu.
image more than 100 different species simultaneously. Rev. Microbiol. 73. doi: 10.1146/annurev-micro-090817-062503.

648
 CHAPTER 19 • Taking the Measure of Microbial Systems 649

We now begin a new unit devoted to microorganisms in their diversity surveys (Sections 19.4–19.8) indicate that many millions—
natural habitats. We learned in Chapter 1 that microbial communities possibly even trillions—of species exist in nature and have yet to
consist of cell populations living in association with other populations be cultured and formally described. This recognition has stimulated
in nature. The science of microbial ecology is focused on how micro- the development of new methods for isolating microbes from
bial populations assemble to form communities and how these com- nature in order to establish pure cultures. Even though a host of
munities interact with each other and their environments. sophisticated methods are available for studying microbes in their
The major components of microbial ecology are biodiversity and native environments, culturing a microorganism remains the only
microbial activity. To study biodiversity, microbial ecologists must way to fully characterize its properties and predict its impact on its
identify and quantify microorganisms in their habitats. Knowing how environment.
to do this is often helpful for isolating organisms of interest as well, In the first part of this chapter we cover the enrichment
which is another goal of microbial ecology. To study microbial activ- approach, a time-honored and useful method for isolating micro-
ity, microbial ecologists must measure the metabolic processes that organisms from nature, but a method with significant limitations.
microorganisms carry out in their habitats. In this chapter we consider Enrichment is based on culturing in a selective growth medium,
modern methods for assessing microbial diversity and activity. and thus the tools and methods used in this approach are con-
Chapter 20 will outline the basic principles of microbial ecology and sidered culture-dependent analyses. As we will see, considerable
examine the types of environments that microorganisms inhabit. progress has been made in culturing the more elusive microor-
Chapters 21–24 will complete our coverage of microbial ecology by ganisms in natural populations by using robotics and associated
exploring nutrient cycles, applied microbiology, and the role microbes microfabrication technology to establish large numbers of enrich-
play in symbiotic associations with other life forms, including humans. ment cultures that can be monitored simultaneously. In the sec-
We begin with the microbial ecologist’s toolbox, which includes ond and third parts of this chapter we consider culture-independent
a collection of powerful tools for dissecting the structure and func- analyses, techniques that can tell us much about the structure and
tion of microbial communities in relation to their natural habitats. function of microbial communities in the absence of actual labo-
ratory cultures. In the final part of this chapter, we consider meth-
ods for measuring microbial activities in nature and linking them
I • Culture-Dependent Analyses to specific organisms. Collectively, these methods allow the
microbial ecologist to ask both “Who is there?” and “What are
of Microbial Communities they doing?”

M ajor advances in molecular, microscopic, and analyt-

ical methods have revealed important properties of
microorganisms as they exist in nature. However, to fully
19.1 Enrichment Culture Microbiology
For an enrichment culture, a medium and a set of incubation con-
characterize a microbe, there is no substitute for isolating it ditions are established that are selective for the desired organism and
in pure culture. In addition to traditional isolation methods, counterselective for undesired organisms. Effective enrichment cul-
5

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single-cell and high-throughput cultivation methods have tures duplicate as closely as possible the resources and conditions of
greatly advanced success in culturing the “uncultured a particular ecological niche. Hundreds of different enrichment
majority” of microorganisms. strategies have been devised, and Tables 19.1 and 19.2 summarize
some simple and direct ones.

T he vast majority of microorganisms, more than 99% of all spe-

cies by most estimates, have never been grown in laboratory
cultures. To date, about 17,000 species of Archaea and Bacteria have
Inocula
Successful enrichment requires an appropriate inoculum containing
been formally described. By contrast, culture-independent molecular the organism of interest. Thus, the making of an enrichment culture

TABLE 19.1 Some enrichment culture methods for phototrophic bacteria (main C source, CO2)
Incubation condition Organisms enriched Inoculum
Incubation in air
N2 as nitrogen source Cyanobacteria Pond or lake water; sulfide-rich muds; stagnant water;
raw sewage; moist, decomposing leaf litter; moist
soil exposed to light
NO3- as nitrogen source, 55°C Thermophilic cyanobacteria Hot spring microbial mat
Anoxic incubation
H2 or organic acids; N2 as sole nitrogen source Purple nonsulfur bacteria, heliobacteria Same as above plus hypolimnetic lake water
( ▶ Section 20.9); pasteurized soil (heliobacteria);
microbial mats for thermophilic species
H2S as electron donor Purple and green sulfur bacteria
Fe2+, NO2- as electron donor Purple bacteria
 650 UNIT 5 • MICROBIAL ECOLOGY AND ENVIRONMENTAL MICROBIOLOGY

TABLE 19.2 Some enrichment culture methods for nonphototrophic bacteriaa

Electron acceptor Electron donor and nitrogen source Typical organisms enriched Inoculum source
Chemoorganotrophs
Aerobes
O2 Lactate, benzoate + NH4+ Mycobacterium, Nocardia, Pseudomonas Soil, mud, lake sediments, decaying vegetation
Hydrocarbons, toluene + NH4+ Mycobacterium, Nocardia, Pseudomonas
Mannitol or benzoate, N2 as nitrogen Azotobacter
Ethanol + yeast extract, pH 6.0 Acetobacter, Gluconobacter
Cellulose + NH4+ Cytophaga, Sporocytophaga
CH4 + NO3- Methylobacter, Methylomicrobium Lake sediments, thermocline of stratified lake
Chemoorganotrophs
Anaerobic respiration and fermentation
NO3- Organic acids + NO3- Pseudomonas (denitrifying species) Soil, mud, lake sediments, groundwater seeps,
sediments, pasteurized inoculum (80°C for
15 min) for Bacillus enrichments
Yeast extract + NO3- Bacillus (denitrifying species)
ClO3- Acetate Various (per)chlorate-reducing Contaminated groundwater, rivers, soils, sedi-
Bacteria and Archaea ments, wastewater treatment systems
PCE Acetate + H2 + NH4+ Dehalococcoides spp. Tetrachloroethene (PCE)-polluted groundwater
SO42- Lactate, ethanol, organic acids Desulfovibrio, Desulfotomaculum Soil, mud, sediments, sewage sludge
Acetate, propionate, butyrate Fatty acid–oxidizing sulfate reducers
S0 Acetate, ethanol Desulfuromonas Soil, mud, sediments, sewage sludge
None Acetate Methanosarcina, Methanosaeta Mud, lake sediments, rotting plant or animal
material, dairy products (lactic and propionic
acid bacteria); rumen or intestinal contents
(enteric bacteria); sewage sludge; soil, pasteur-
ized inoculum for Clostridium enrichments
Glutamate or histidine Proteolytic Clostridium species
Starch + NH4+ Clostridium spp.
Starch + N2 as nitrogen source Clostridium pasteurianum
Lactate + yeast extract Veillonella spp.
5
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Glucose + yeast extract (pH 5) Lactic acid bacteria (Lactobacillus)

Succinate + NaCl Propionigenium
Oxalate Oxalobacter
Chemolithotrophs
Aerobes
O2 NH4+ Ammonia-oxidizing Bacteria Lake sediments, thermocline of stratified lake
(Nitrosomonas) and Archaea
(Nitrosopumilus), comammoxb bacteria
NO2 - Nitrite-oxidizing Bacteria (Nitrobacter,
Nitrospira)
H2 Hydrogen bacteria
H2S, S0, S2O32- Thiobacillus spp.
Fe2+, low pH Acidithiobacillus ferrooxidans Acid mine drainage
Fe2+, neutral pH Acidovorax, Zetaproteobacteria Groundwater seeps and geothermal seeps
Chemolithotrophs
Anaerobes
Fe+3 Acetate Geobacter, Geospirillum Groundwater
NO3- Fe + 2 + NO3- Iron-oxidizing chemolithotrophs Groundwater, groundwater seeps, sediments
S0, S2O32- Thiobacillus denitrificans Mud, lake sediments, soil
H2 Paracoccus denitrificans
CO2 H2 + NH4+ Methanogens (chemolithotrophic spe- Mud, lake sediments, rotting plant or animal material,
cies only), homoacetogens rumen or intestinal contents, sewage sludge
a
All media must contain an assortment of mineral salts including N, P, S, Mg2+, Mn2+, Fe2+, Ca2+, and other trace elements (◀ Sections 4.1, 4.2). Certain organisms may
have requirements for vitamins or other growth factors. This table is a general overview of enrichment methods focused on substrates and atmospheric conditions and
has not considered the effects that temperature, pH and salinity can have on enrichment culture outcomes.
b
Comammox is the complete oxidation of ammonia all the way to nitrate ( ▶ Section 20.3).
 CHAPTER 19 • Taking the Measure of Microbial Systems 651

begins with collecting a sample from the appropriate habitat to Some enrichment cultures yield nothing. This may be because an
serve as the inoculum (Tables 19.1 and 19.2). Enrichment cultures organism capable of growing under the enrichment conditions
are established by placing the inoculum into selective media and established is absent from the habitat. Alternatively, even though
incubating under specific conditions. In this way, many common the organism of interest exists in the habitat sampled, the resources
microbes can be isolated. For example, the great Dutch microbiolo- and conditions employed in the enrichment may simply be incom-
gist Martinus Beijerinck, who conceptualized the enrichment cul- patible with its growth. Thus, enrichment cultures can yield a firm
ture technique (◀ Section 1.13), used enrichment cultures to isolate positive conclusion (that is, that an organism with certain capacities
the nitrogen-fixing bacterium Azotobacter (Figure 19.1). Because Azo- exists in a particular environment because it was enriched) but Mastering
Microbiology
tobacter is a rapidly growing bacterium capable of N2 fixation in air never a firm negative conclusion (that such an organism is not pres- Art Activity:
(◀ Sections 3.12 and 15.9), enrichment using media devoid of fixed ent because the enrichment failed). Moreover, the isolation of the Figure 19.1 The
isolation of
nitrogen (for example, ammonia or nitrate) and incubation in air desired organism from an enrichment culture says nothing about Azotobacter

selects strongly for this bacterium and its close relatives. Non- the abundance or ecological significance of the organism in its habi-
nitrogen-fixing bacteria and anaerobic nitrogen-fixing bacteria are tat. A positive enrichment proves only that the organism was present
counterselected in this technique. in the sample, and in practice, this can result from even a single
viable cell.
Enrichment Culture Outcomes
For success with enrichment cultures, attention to both the culture The Winogradsky Column
medium and the incubation conditions is important. That is, the The Winogradsky column is an artificial microbial ecosystem and
resources (nutrients) and conditions (temperature, pH, osmotic con- a long-term source of various bacteria for enrichment cultures. Win-
siderations, aerobic or anaerobic, and the like) must closely mimic ogradsky columns have been used to isolate phototrophic purple
those of the habitat to offer the best chance of obtaining the organ- and green bacteria, sulfate-reducing bacteria, and many other anaer-
ism of interest ( ▶ Table 20.1). obes. Named for the famous Russian microbiologist Sergei Wino- Mastering
Microbiology
gradsky (◀ Section 1.13), the column was first used by Winogradsky Art Activity:
in the late nineteenth century in his classic studies of soil Figure 19.2a The
Winogradsky
The absence of NH4+ microorganisms. column
forces cells to fix N2.
Such cells can also
A Winogradsky column is prepared by filling a glass cylinder
use NH4+ if available. about half full with organic-rich, preferably sulfidic mud into which
carbon substrates have been mixed. The substrates determine which
organisms are enriched. Fermentative substrates, such as glucose,
that can lead to acidic conditions and excessive gas formation
Mineral salts medium +NH4+ plate (which can create gas pockets that disrupt the enrichment culture
containing mannitol but
lacking NH4+, NO3–, or and let in air) are avoided. The mud is supplemented with small
amounts of calcium carbonate (CaCO3) as a buffer and gypsum
organic nitrogen
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(CaSO4) as a source of sulfate. The mud is packed tightly in the
cylinder, taking care to avoid trapping air, and then covered with
–NH4+ plate
lake, pond, or ditch water (or seawater if it is a marine column). The
Incubate top of the cylinder is covered to prevent evaporation, and the con-
Soil aerobically.
tainer is placed near a window that receives diffuse sunlight for a
period of months.
NH4+ In a typical Winogradsky column, a diverse community of
microbes develops (Figure 19.2a). Algae and cyanobacteria develop
+NH4+ plate
quickly in the upper portions of the water column; by producing O2
these organisms help to keep this zone of the column oxic much as
they do in the upper zones of a lake. Fermentative processes in the
mud lead to the production of organic acids, alcohols, and H2, suit-
–NH4+ plate able substrates for sulfate-reducing bacteria (◀ Section 15.11).
Adding NH4+ to Hydrogen sulfide (H2S) from the sulfate reducers triggers the devel-
enrichment media opment of purple and green sulfur bacteria (anoxygenic phototrophs,
selects against ◀ Sections 14.3 and 15.4–15.8) that use sulfide as a photosynthetic
N2-fixing bacteria.
electron donor. These organisms typically grow in patches in the mud
Figure 19.1 The isolation of Azotobacter. Selection for aerobic nitrogen-fixing on the sides of the column but may bloom in the water itself if oxy-
bacteria usually results in the isolation of Azotobacter or its relatives. The selective genic phototrophs are scarce (Figure 19.2b). The pigmented cells of
basis of the enrichment is the absence of fixed nitrogen (NH4 + in this case) in the the anoxygenic phototrophs can be sampled with a pipette for
culture medium in the upper flask. Thus the medium selects from the microbial com- microscopy, isolation, and characterization (Table 19.1).
munity those species that can fix N2 aerobically, of which Azotobacter is one of the Winogradsky columns have been used to enrich both aerobic and
most rapidly growing. See Section 1.13 and Figure 1.37 for more on the historical
anaerobic Bacteria and Archaea. Besides supplying a ready source of
importance of Azotobacter.
inocula for enrichment cultures, columns can also be supplemented
 652 UNIT 5 • MICROBIAL ECOLOGY AND ENVIRONMENTAL MICROBIOLOGY

Gradients Column
O2 Foil cap

Lake or pond water Algae and cyanobacteria

Purple nonsulfur bacteria

Sulfur chemolithotrophs

Patches of purple sulfur or

Mud supplemented green sulfur bacteria
with organic nutrients
and CaSO4
Anoxic decomposition
and sulfate reduction
H2S
(a)

Norbert Pfennig
(b)

Figure 19.2 The Winogradsky column. (a) Schematic view of a typical column used to enrich phototrophic bacteria. The column is
incubated in a location that receives subdued sunlight. Anoxic decomposition leading to SO42- reduction creates the gradient of H2S.
(b) Photo of Winogradsky columns that have remained anoxic up to the top; each column had a bloom of a different phototrophic
bacterium. Left to right: Thiospirillum jenense, Chromatium okenii, both of which are purple sulfur bacteria, and Chlorobium limicola
(green sulfur bacterium).

with a specific compound to enrich an organism in the inoculum liquid enrichments established with the same but undiluted inocula.
that can degrade it. Once a crude enrichment has been established It is thought that dilution of the inoculum eliminates quantitatively
in the column, culture media can be inoculated for the isolation of insignificant but rapidly growing “weed” species, allowing develop-
5
UNIT

pure cultures, as discussed in Section 19.2. ment of organisms that are more abundant in the community but
slower growing. Dilution of the inoculum is thus a common prac-
Enrichment Bias tice in enrichment culture microbiology today. As discussed below,
Although the enrichment culture technique is quite useful and still the problem of overgrowth by “weed” species can also be circum-
widely practiced, there exists a bias, and sometimes a very severe bias, vented by physical isolation of the desired organism before intro-
in the outcome of enrichments. This bias is typically most profound ducing it into a growth medium. This can be accomplished by
in liquid enrichment cultures where the most rapidly growing dilution and a variety of classical isolation procedures that we turn
organism(s) for the chosen set of conditions dominate. However, to in the next section. However, more recently, sophisticated meth-
using molecular techniques to be described later, we now know that ods have been developed to physically isolate single cells of interest
the most rapidly growing organisms in laboratory cultures are often (or a single type of cells) and place them in a growth medium that
only minor components of the microbial community rather than the is free of undesired cells. We consider these techniques in Section
most abundant and ecologically relevant organisms carrying out 19.3. Finally, as we will discuss later in this chapter, culture-inde-
the process of interest. This could be for several reasons including the pendent molecular methods have most clearly revealed the limita-
fact that the levels of resources available in laboratory cultures are tions of cultivation in capturing the full microbial diversity of most
typically much higher than those in nature, and the conditions in the environments.
natural habitat, including both the types and proportions of different
organisms present as well as the physical and chemical conditions, Check Your Understanding
are nearly impossible to reproduce and maintain for long periods in • Describe the enrichment strategy behind Beijerinck’s iso-
laboratory cultures. lation of Azotobacter.
This problem of enrichment bias can be demonstrated by com- • Why is sulfate (SO42-) added to a Winogradsky column?
paring the results obtained in dilution cultures (Section 19.2) with • What is enrichment bias? How does dilution reduce enrich-
classical liquid enrichment. Dilution of an inoculum followed by ment bias?
liquid enrichment or plating often yields different organisms than
 CHAPTER 19 • Taking the Measure of Microbial Systems 653

19.2 Classical Procedures

for Isolating Microbes
Once a positive enrichment culture has been obtained, the next step
is typically to attempt to get the enriched organism in pure culture—
one containing a single kind of microorganism. Pure cultures are
valuable because genomes can be quickly isolated and analyzed and
experiments can be done under controlled laboratory conditions to
clearly define the physiology of the isolate. Pure cultures have been
studied since the days of Robert Koch (◀ Section 1.12), and we
considered some of these methods earlier (◀ Section 4.2).

Agar Dilution Tubes and the

Most-Probable-Number Technique
Common isolation procedures include the streak plate, agar dilu-

James Shapiro
tion, and liquid dilution. For organisms that form colonies on agar
plates, the streak plate is quick, easy, and the method of choice
(Figure 19.3a); if a well-isolated colony is selected and restreaked sev- (a) Colonies Paraffin–mineral oil seal
eral successive times, a pure culture is usually obtained. With proper
incubation facilities (for example, anoxic jars or anoxic chambers
for anaerobes, ◀ Section 4.16), it is possible to purify both aerobes
and anaerobes on agar plates by the streak plate method.
In the agar dilution tube method, a mixed culture is diluted in

Marie Asao, Deborhah O. Jung, and Michael T. Madigan

tubes of molten agar medium, resulting in colonies embedded in
the agar. This method is useful for purifying anaerobic organisms
such as phototrophic sulfur bacteria and sulfate-reducing bacteria
from samples taken from Winogradsky columns or other sources. A
culture is purified by successive dilutions of cell suspensions in tubes
of molten agar medium (Figure 19.3b, ◀ Figure 15.28g). Repeating
this procedure using a colony from the highest-dilution tube as
inoculum for a new set of dilutions usually yields pure cultures. A
related procedure called the roll tube method uses tubes containing a
5

UNIT
thin layer of agar on their inner surface. The agar can then be
streaked for isolated colonies. Because the tubes can be flushed with (b)
an oxygen-free gas during streaking, the roll tube method is primar-
Figure 19.3 Pure culture methods. (a) Organisms that form distinct colonies on
ily used for the isolation of anaerobic microbes. plates are usually easy to purify. (b) Colonies of phototrophic purple bacteria in agar
Another purification procedure is the serial dilution of an inocu- dilution tubes; the molten agar was cooled to approximately 45°C before inoculation.
lum in a liquid medium until the final tube in the series shows no A dilution series was established from left to right, eventually yielding well-isolated
growth. When a 10-fold serial dilution is used, for example, the last colonies. The tubes were sealed with a 1:1 mixture of sterile paraffin and mineral oil
tube showing growth should have originated from ten or fewer cells. to maintain anaerobiosis.
Besides being a method for obtaining pure cultures, serial dilution
techniques are widely used to estimate viable cell numbers in the
of colony characteristics on plates or in dilution tubes, and (3) tests of
most-probable-number (MPN) technique (Figure 19.4). MPN
the culture for growth in other media. In the latter, it is important to
methods have been used for estimating the numbers of microorgan-
test the culture for growth in media and under growth conditions in
isms in foods, wastewater, and other samples in which cell numbers
which the desired organism is predicted to grow poorly or not at all
need to be assessed routinely. An MPN count of a natural sample
but in which contaminants will grow vigorously. In the final analysis,
can be done using highly selective media and incubation conditions
the microscopic observation of a single morphological type of cell that
to target one or a small group of organisms or a particular pathogen.
displays uniform staining characteristics (for example, in a Gram stain)
Alternatively, a count can be done using complex media to get a
coupled with uniform colony characteristics and the absence of con-
general estimate of viable cell numbers (but see Section 4.4 for a
tamination in growth tests with various culture media is strong evi-
caveat that applies to such estimates). Use of several replicate tubes
dence that a culture is a pure (axenic) culture.
at each dilution improves accuracy of the final MPN obtained.
Certain molecular methods described in this chapter for charac-
terizing natural microbial communities can also be applied to the
Criteria for Culture Purity verification of culture purity. However, these techniques are comple-
Regardless of the methods used to purify a culture, once a putative pure mentary and do not substitute for the more fundamental observa-
culture has been obtained, it is essential to verify its purity. This is typi- tions of culture characteristics and cellular morphology.
cally done through a combination of (1) microscopy, (2) observation
 654 UNIT 5 • MICROBIAL ECOLOGY AND ENVIRONMENTAL MICROBIOLOGY

Laser Tweezers and Flow Cytometry

Laser tweezers consist of an inverted light microscope equipped
with a strongly focused infrared laser and a micromanipulation
device. Trapping a single cell is possible because the laser beam cre-
1 ml
Enrichment culture ates a force that pushes down on a microbial cell (or other small
(liquid)
or natural sample
or 1 g object) and holds it in place (Figure 19.5a). Then when the laser beam
(solid) is moved, the trapped cell moves along with it. If a mixed sample is
Dilution
in a capillary tube, a single cell can be optically trapped and moved
1 ml 1 ml 1 ml 1 ml 1 ml
away from contaminating organisms (Figure 19.5b). The cell can
then be isolated by breaking the tube at a point between the cell and
No the contaminants and flushing the cell into a small tube of sterile
Growth Growth growth medium. Laser tweezers, when coupled with staining techniques
that identify particular organisms (Sections 19.4 and 19.5), can be
9 ml used to select organisms of interest from a mixture for purification
of
broth and further laboratory study.
1/10 Flow cytometry is a technique for counting and examining a
(10–1) 10–2 10–3 10–4 10–5 10–6 mixture of cells by suspending them in a stream of fluid and passing
them through an electronic detector that sorts them according to
Figure 19.4 Procedure for a most-probable-number (MPN) analysis. Growth in
the 10-4 but not the 10-5 dilution means that cell numbers were at least 104 cells/ml defined criteria; for example, by cell size, shape, or fluorescent prop-
in the sample used for inoculation. Because particle-attached microorganisms can erties. This ability makes cell sorting useful not only for isolating
skew numbers significantly, gentle methods to disassociate microorganisms from single cells but also for enriching a particular cell type from a mix-
particles are often used prior to dilution. In addition, each dilution tube is mixed thor- ture. Cell sorters can deposit individual cells into wells of a microti-
oughly before removing a sample for the next dilution. ter plate where each well contains the same growth medium or a
slightly different growth medium. Because the growth requirements

Check Your Understanding

• What is a pure culture and why is obtaining one useful in b a
microbial ecology?
• How does the agar dilution method differ from streaking
to obtain isolated colonies?
• Why would microscopic examination alone not be suffi- Downward forces Fa
cient to establish that a culture is pure? and Fb allow the laser Fb Fa Cell
beam to drag the cell.
5
UNIT

Beam focus
19.3 Selective Single-Cell Isolation: Laser Objective lens
Tweezers, Flow Cytometry, Microfluidics, a b of microscope

and High-Throughput Methods

The problem of enrichment bias has fueled the development of new
methods for culturing microbes from nature. These advancements
have emerged from the understanding that every microbe has a fun- Laser beam
damental niche and a realized niche. The fundamental niche refers (a)
to the range of environments in which a species will be sustained
when it is not resource-limited, such as may result from competition Once trapped, the separated
cell can be flushed from the
with other species. By contrast, the realized niche refers to the range Optically
capillary into a tube of
trapped cell Severing point
of natural environments supporting a species when it is confronted sterile medium.
with factors such as resource limitation, predation, and competition
from other species.
Establishing laboratory conditions that fall within the fundamental
niche may be sufficient to support an organism once it is in pure Capillary tube Mixture of cells
culture but may fail to selectively enrich the same organism from a Laser
natural sample. Because the realized niche of most microorganisms (b)
is unknown, there has been an increasing emphasis on developing Figure 19.5 The laser tweezers for the isolation of single cells. (a) Mechanism by
methods that physically isolate single cells into separate compart- which individual cells can be isolated. (b) Once a cell has been isolated in a capillary
ments free from competition with other microbes. These include both tube, it can be tested for subsequent growth in pure culture.
manual and robotic methods that function to sort individual cells
from an environmental sample, and we consider these methods now.
 CHAPTER 19 • Taking the Measure of Microbial Systems 655

of some organisms include organic compounds and metabolites clues for the design of laboratory culture media to obtain its growth
produced by other organisms that share their environment, addition in pure culture.
of filter-sterilized source water (for aquatic organisms) or soil water High-throughput cultivation has shown increasing success in iso-
extract (for soil organisms) can be used to supplement the media lating unique bacteria. For example, high-throughput methods were
tested. Each well in the microtiter plate can then be monitored for used for the isolation of one of the most abundant bacteria on
growth or some other property either manually or using robotic Earth, the small marine planktonic bacterium Pelagibacter ubique
methods (high-throughput culture, see next subsection). We explore (Figure 19.6). This bacterium thrives on the very dilute pool of dis-
the mechanism and uses of flow cytometry in more detail in solved organic matter present in the open oceans and eluded classi-
Section 19.12 (see Figure 19.42). cal enrichment methods for years. But with high-throughput
technology, this ecologically important bacterium was brought into
High-Throughput Culture and Microfluidic Devices laboratory culture where its biology could be studied in more detail.
Continuing innovations in single-cell isolation methodology have Microfluidic devices carry the high-throughput concept even fur-
spawned high-throughput culturing methods and related meth- ther by using microfabrication technology to combine channels and
ods for use on an even smaller scale. High-throughput methods wells for fluid transfer and collection on a miniaturized platform.
require dilution (or cell sorting) of a sample to yield a single cell in One such device is less than 10 centimeters long yet holds 3200
each well of a microtiter plate (Figure 19.6). From there, each well is nanoliter-sized wells, with each well serving as a small culture vessel Mastering
Microbiology
robotically monitored over time for cell growth or a specific target (Figure 19.7). An environmental sample is introduced into the micro- Art Activity:

gene. High-throughput methods allow the experimenter to test fluidic device such that each well receives a single cell. Different Figure 19.6
Methodological
many alternative sets of resources and growth conditions systemati- medium formulations can be tested, and the media supplemented pipeline for
high-throughput
cally in an attempt to replicate the realized niche or, alternatively, to with a small amount of filter-sterilized water or soil extract collected cultivation of
previously
allow the organism to occupy its fundamental niche by relieving it from the sampled environment (these additions may stimulate uncultured
from competition. Microtiter wells that are positive for cell growth growth by providing trace nutrients missing in the culture medium). microorganisms

or a target gene of interest identify the acceptable resources and Both cell growth and target genes can be assessed in each well of
conditions for growth of a particular microbe and supply valuable the microfluidic device; growth is assessed by direct microscopic

Sterile seawater Microtiter plates Deposit single cells from seawater

plus minerals into individual wells of microtiter
supplemented with plates. Obtain individual cells by
different nutrients flow cytometric sorting (see Figure
in each well 19.42) or dilution.

UNIT
Inoculum source:
Seawater with 106 cells/ml

Incubate Individual wells

Growth from single-cell inocula

Isolation of Pelagibacter ubique

Adam Deutschbauer and Hans Carlson

Monitor for microbial

growth or specific
target genes. Wells
showing growth
Steve Giovannoni

contain stimulatory
nutrients.

(a) (b)

Figure 19.6 Methodological pipeline for high-throughput cultivation of previously uncultured microorganisms. The method shown
here was used to isolate the marine bacterium Pelagibacter ubique. (a) Robotic system for high-throughput multiplexed pipetting of growth
medium into microtiter plates (arrows point to pipette tips and robotic pipette holder). (b) Following the addition of filter-sterilized seawater
and low nutrient concentrations to the individual wells, and deposition of single cells into individual wells of the microtiter plate, pure cul-
tures of Pelagibacter and other novel marine Bacteria were obtained. Pelagibacter is the most abundant bacterium in the open oceans
( ▶ Section 20.12).
 656 UNIT 5 • MICROBIAL ECOLOGY AND ENVIRONMENTAL MICROBIOLOGY

Inlet for loading sample Outlet for collecting culture

II • Culture-Independent
Microscopic Analyses of
Microbial Communities

Rustem Imagilov and Liang Ma

T he microscope has been the microbiologist’s foremost tool
for studying microbial structure. Today the microscope can
also assist in probing microbial diversity and activity, thanks to
a suite of fluorescent techniques. These advances have greatly
improved our understanding of microbial community structure
and provided unprecedented insight into microbial symbiotic
3200 microwells for
single-cell cultivation relationships with plants, animals, and other microbes.

Figure 19.7 Microfluidic platform for cultivation. An environmental inoculum is

suspended in a cultivation medium and loaded onto this microfluidic device, enabling
confinement of as many as 3200 single cells in nanoliter wells to promote the growth
M icrobial ecologists quantify cells in a microbial habitat to esti-
mate abundance of the entire community or, more specifi-
cally, relative abundances of the different species in the community.
of microcolonies. Following different periods of incubation, cultured populations are Cell stains are necessary to obtain these types of data, and we detail
collected at the outlet and further grown under conditions demonstrated to support
these methods here. Organisms in natural environments can also be
growth on the microfluidic device. The device is about 7 cm wide.
detected by assaying their genes. Genes encoding either ribosomal
RNA (rRNA, ◀ Section 13.11) or enzymes that support a specific
examination of cell numbers in a well under the microscope. If insuf- physiology are the usual targets in these studies. A rapidly develop-
ficient cellular biomass is available for molecular characterization of ing approach to the study of microbial ecology, called multi-omics,
specific target genes by methods such as PCR (see Section 19.6), combines multiple molecular, analytical, and omics meth-
genome amplification as described for single-cell genomics ods (Chapter 10), and is introduced in Section 19.8.
(◀ Section 10.11) can be used prior to further analysis.
A variation on the microfluidics technique employs a microcham- 19.4 General Staining Methods
ber device modified such that each of the tiny chambers is separated
Several general staining methods are suitable for quantifying micro-
from the external environment by a membrane that traps the
organisms in natural samples. Although these methods do not reveal
microbes but allows soluble nutrients to diffuse in and out. Follow-
the physiology or phylogeny of the cells, they are nonetheless reli-
ing the introduction of a single cell into each chamber, the device is
able and widely used by microbial ecologists for measuring total cell
placed back into the environment from which the inoculum was
numbers. One method also allows cell viability to be assessed.
obtained. Then, after incubation for a month or more, microbes that
5
UNIT

initiate growth only when incubated under the conditions and

Fluorescent Staining with Dyes That Bind Nucleic Acids
resources present in their habitats can often be isolated and subse-
quently propagated in the laboratory. or Reveal Viability
Although technology is rapidly advancing the art of isolating new Fluorescent dyes can be used to stain microorganisms from virtually
microbes, patience is still needed in any cultivation effort, as the any microbial habitat. DAPI (4′,6-diamidino-2-phenylindole) is a
discovery of slow-growing or dormant organisms may require popular stain for this purpose, as is the dye acridine orange. There
months of incubation. Also, many microbes in nature are likely is also increasing use of SYBR Green I, a dye that confers very bright
adapted to extremely low nutrient concentrations and may be inhib- fluorescence to all microorganisms, including viruses. These stains
ited by levels of nutrients used to grow organisms commonly stud- bind to DNA and are strongly fluorescent when exposed to ultravio-
ied in the laboratory. Both high-throughput and microfluidic let (UV) radiation (DAPI absorption maximum, 400 nm; acridine
methods overcome these problems by their ability to separate indi- orange absorption maximum, 500 nm; SYBR Green I absorption
vidual cells from other cells that may release inhibitory materials maximum, 497 nm), making the microbial cells in the sample read-
and by surveying a nearly limitless variety of nutrient conditions. ily visible and easy to enumerate. Cells stained with DAPI fluoresce
Currently, these methods offer the best opportunity for culturing the blue, cells stained with acridine orange fluoresce orange or greenish-
most interesting (and likely ecologically relevant) microorganisms orange, and cells stained with SYBR Green I fluoresce green
from nature. (Figure 19.8).
Dyes that stain DNA are widely used for the enumeration of
Check Your Understanding microorganisms in environmental, food, and clinical samples.
• How might you isolate a morphologically unique bacterium Depending on the sample, background staining is occasionally a
present in an enrichment culture in relatively low numbers? problem with fluorescent stains, but because these dyes specifically
stain nucleic acids, they are for the most part nonreactive with inert
• What is meant by “high-throughput” in culturing microorgan-
isms? How has it benefited microbiology? matter. Thus, for many samples, from soil as well as aquatic
sources, they can give a reasonable estimate of the cell numbers
present. Staining with the brightly fluorescent SYBR Green I also
 CHAPTER 19 • Taking the Measure of Microbial Systems 657

Marc Mussman and Michael Wagner

Marc Mussman and Michael Wagner

Willm Martins-Habenna
(a) (b) (c)

Figure 19.8 Nonspecific fluorescent stains. (a) DAPI and (b) acridine orange staining showing microbial communities inhabiting
activated sludge in a municipal wastewater treatment plant. With acridine orange, cells containing low RNA levels stain green.
(c) SYBR Green–stained sample of Puget Sound (Washington, USA) surface water showing green-fluorescing bacterial cells. The
large cells near the center of the field are 0.8–1.0 mm in diameter.

provides excellent enumeration of aquatic virus populations Although useful for research that uses laboratory cultures, the live/
( ▶ Section 20.13). For dilute aquatic samples, cells can be stained dead staining method is not suitable for use in the direct micro-
following collection on a membrane surface by filtration. scopic examination of samples from many natural habitats because
DNA staining is a nonspecific process; all microorganisms in a of problems with nonspecific staining of background materials.
sample are stained. Although this may at first seem desirable, it is However, procedures have been developed to overcome this prob-
not necessarily so. For example, DAPI and acridine orange fail to lem in analyses of aquatic environments; the water sample is fil-
differentiate between living and dead cells or between different spe- tered, and the filters are stained with the live/dead stain and
cies of microorganisms, so they cannot be used to assess cell viability examined microscopically. Thus in aquatic microbiology, live/dead
or to track specific microorganisms in an environment. Viability staining is often used to measure the viability of cell populations in
staining solves one of these problems because it differentiates live the water column of lakes or oceans, or in the flowing waters of
cells from dead ones. Hence, viability stains yield both abundance streams, rivers, and other aquatic environments.
and viability data at the same time. The basis of differentiating
between live and dead cells lies not with a cell’s DNA but whether Fluorescent Proteins as Cell Tags and Reporter Genes
its cytoplasmic membrane is intact or not. Two dyes that fluoresce Bacterial cells can be altered by genetic engineering to make them
green and red are added to a sample; the green-fluorescing dye pen- autofluorescent. As discussed earlier, a gene encoding the green
etrates all cells, viable or not, whereas the red dye, which contains
5

UNIT
fluorescent protein (GFP) can be inserted into the genome of virtu-
the chemical propidium iodide, penetrates only those cells whose ally any cultured bacterium (◀ Sections 8.1 and 12.5). When the
cytoplasmic membrane is no longer intact and that are therefore gene encoding the GFP (gfp) is expressed, cells fluoresce green when
dead. Thus, when viewed microscopically, green cells are scored as observed with ultraviolet microscopy (Figure 19.10; ◀ Figure 12.17).
alive and red cells as dead, yielding an instant assessment of both Although GFP is not useful for the study of natural populations of
abundance and viability (Figure 19.9). microorganisms (because these cells lack the GFP gene), GFP-tagged
cells can be introduced into an environment, such as plant roots, and
then tracked over time by microscopy. Using this method, microbial
ecologists can study competition between the native microbiota and
a GFP-tagged introduced strain and can assess the effect of perturba-
tions of an environment on the survivability of the introduced strain.
The gene gfp and those encoding other fluorescent proteins have
also been used extensively in laboratory cultures of various bacteria
and in controlled environments as reporter genes. When the gene is
Molecular Probes, Inc., Eugene, OR

fused with an operon under the control of a specific regulatory pro-

tein, transcription can be studied by using fluorescence as the indi-
cator (a “reporter”) of activity. That is, when genes containing the
fused fluorescent protein gene are transcribed and translated, both
Live
the protein of interest and the fluorescent protein are made, and
Dead
cells fluoresce the characteristic color. For example, expression of gfp
was used to demonstrate that colonization of alfalfa roots by the
nitrogen-fixing symbiotic bacterium Sinorhizobium meliloti (legume–
Figure 19.9 Viability staining. Live (green) and dead (red) cells of Micrococcus
root nodule symbiosis, ▶ Section 23.4) is promoted by sugars and
luteus (cocci) and Bacillus cereus (rods) stained by the LIVE/DEAD BacLight Bacte-
rial Viability Stain.
dicarboxylic acids released by the plant (Figure 19.10b, c). The
a-galactosidase gene is gene

of interest requires oxygen, not for anoxic environments

 not suitable for small cells, cells can aggregate together

(limitations)

658 UNIT 5 • MICROBIAL ECOLOGY AND ENVIRONMENTAL MICROBIOLOGY

Excite (nm) Emit (nm) FP

399 456 FP1

433 475 FP2

466 507 FP3

467 509 FP4
485 510 FP5 (GFP)
515 528 FP6
516 529 FP7

554 581 FP8

Preston Garcia and Dan Gage

Preston Garcia and Dan Gage

568 592 FP9
587 610 FP10
588 633
FP11
600 650
FP12

(a) (b) (c)

Figure 19.10 Fluorescent protein reporters. (a) Twelve different fluorescent proteins (FP1–FP12) are known that have distinct excita-
tion (Excite) and emission (Emit) properties. (b) Cells of Sinorhizobium meliloti (arrows) carrying a plasmid with an a-galactoside-inducible
promoter fused to the GFP (FP5); the cells are on clover seedling roots. Green fluorescence indicates that a-galactosides are released and
available to support the growth of this bacterium. (c) S. meliloti cells (arrows) carrying a plasmid with a succinate-inducible promoter fused
to GFP; green fluorescence indicates that succinate or other C4 dicarboxylic acids have been secreted by the plant root hairs.

emission properties of GFP and other fluorescent proteins isolated the biggest limitation with the methods we have discussed thus far
from different marine invertebrates (jellyfish, corals, anemones) is that none of them reveal the phylogeny of the microorganisms
have since been altered through mutation to yield a broad palette observed under the microscope. That is, how many species, or phyla,
of fluorescent proteins of varying spectral properties (Figure 19.10a), or even domains, are present in the sample? More specific fluores-
offering the experimenter the capability to monitor several species cent staining methods are required to answer these questions.
simultaneously. When observing unstained or nonspecifically stained natural
One drawback to the use of GFP is that to become fluorescent it populations of microorganisms under the microscope
requires O2, and thus it is not suitable for tracking cells introduced (Section 19.4), one should remember that the sample likely con-
into strictly anoxic habitats. However, flavin-based fluorescent pro- tains a phylogenetically diverse community, even if many cells
teins that do not require O2 are available to overcome this limita- “look” the same. This is because the simple shapes of bacteria can
5
UNIT

tion. These proteins are derived from bacterial and plant conceal their remarkable diversity, and Figure 19.11 shows an
photosensory flavoproteins and are more thermally stable than the
GFP, making them useful for tracking mildly thermophilic species.
In addition to these fluorescent tags for tracking microbes, phyloge-
netic stains (Section 19.5) are widely used for identifying microbes,
and a wide variety of new fluorescent “super-resolution” microscopy
techniques are available for tracking individual molecules within a
microbial cell (◀ Section 8.1). Thus, fluorescence technology has
come a long way since the days when only DAPI and acridine orange
were available for visualizing microbial cells in nature.

Check Your Understanding

Alex T. Nielsen

Alex T. Nielsen

• How does viability staining differ from stains like DAPI?

• What types of environments limit the application of GFP?
• Why is it incorrect to say that the GFP is a “staining” method?
(a) (b)

Figure 19.11 Morphology and genetic diversity. The photomicrographs shown

19.5 Microscopic Specificity: Fluorescence here, produced by (a) phase contrast and (b) a technique called phylogenetic FISH
(Section 19.5), are of the same field of cells. Although the large oval cells are of a
In Situ Hybridization (FISH) rather unusual size for prokaryotic cells and all look similar by phase-contrast
As we have just seen, the microscope is an essential tool for enumer- microscopy, the phylogenetic stains reveal that there are two genetically distinct
types (one stains yellow and one stains blue). Both cell types are about 2.3 mm in
ating and assaying microorganisms in a general way—their mor-
diameter. The smaller, green cells in pairs or clusters are about 1 mm in diameter.
phology, their Gram reaction, their viability, and the like. However,
 CHAPTER 19 • Taking the Measure of Microbial Systems 659

fluorescent probes bound to a cell reflects the number of its ribo-

somes. As single microbial cells can contain tens of thousands of
ribosomes, strong signals can be achieved. And, because ribosomes
are scattered throughout the cell in prokaryotic cells, the entire cell
becomes fluorescent (Figures 19.11b and 19.12). One limitation of
the FISH technique is that slow-growing organisms often have very
low numbers of ribosomes and mRNAs, and therefore fluoresce very
poorly. However, new staining methods have been developed that
Bacillus measure protein synthesis directly, and provide essential informa-
tion about the viability and activity of cells in their natural environ-
Yeast
ments, even when growing slowly (see Figure 19.15).
By targeting sites in the rRNA sequence that are variable between
Norman Pace
Norman Pace

Norman Pace
different organisms, phylogenetic stains can be designed to be very
specific and react with only one species or a handful of related
microbial species. Alternatively, by targeting conserved sequences in
(a) (b) (c)
the rRNA they can be made more general and react with, for exam-
Figure 19.12 Fluorescently labeled rRNA probes: Phylogenetic stains. (a) Phase- ple, all cells of a given phylogenetic domain (Bacteria, Archaea,
contrast photomicrograph of cells of Bacillus megaterium (rod, Bacteria) and the Eukarya) or a given phylum within a domain. Using FISH, an inves-
yeast Saccharomyces cerevisiae (oval cells, Eukarya). (b) Same field; cells stained tigator can identify or track a specific organism of interest or an
with a yellow-green universal rRNA probe (this probe hybridizes with rRNA from entire domain of interest in a natural sample. For example, if one
organisms of any phylogenetic domain). (c) Same field; cells stained with a eukaryal
wished to determine the percentage of a given microbial population
probe (only cells of S. cerevisiae react). Cells of B. megaterium are about 1.5 mm in
diameter and cells of S. cerevisiae are about 6 mm in diameter.
that are Archaea, an archaeal-specific phylogenetic stain could be
used in combination with DAPI (Section 19.4) to assess Archaea and
total numbers, respectively, and a percentage derived by
example of this. The two photos are of the same microscope field, calculation.
one taken in black and white of cells in a microbial community and FISH technology can also employ multiple phylogenetic probes
the other taken in color of the same cells stained in a way that at once. With a suite of probes, each designed to react with a par-
reveals their phylogeny. If only unstained cells (Figure 19.11a) were ticular organism or group and each containing its own fluorescent
viewed, it would be easy to conclude that the large ovoid-shaped dye, FISH can image multiple taxa in a habitat in a single experi-
cells are all of a single species. But staining to reveal phylogeny ment (Figure 19.13 and see MicrobiologyNow on page 648). Simi-
(Figure 19.11b) shows that they are not; the blue and yellow fluo- larly, the method easily distinguishes between genetically distinct
rescent stains target different species of large ovoid cells while the cells of similar morphology (Figure 19.11b). If FISH is combined
green stain reveals yet a third species in the community. Micro- with confocal microscopy (◀ Section 1.9), it is possible to explore
5

UNIT
scopic assessments of phylogenetic diversity such as this can only
be made using phylogenetic stains, and we consider these here along
with some related staining techniques.

Phylogenetic Identification Using FISH

Because of their great specificity, nucleic acid probes can be har-
nessed as powerful tools for identifying and quantifying microor-
ganisms. Recall that a nucleic acid probe is a DNA or RNA
oligonucleotide complementary to a sequence in a target gene or
RNA; when the probe and the target come together, they hybrid-
Michael Wagner and David A. Stahl

Michael Wagner and Jiri Snaidr

ize (◀ Section 12.1). Nucleic acid probes can be made fluorescent

by attaching fluorescent dyes to them. The fluorescent probes can
then be used to identify organisms that contain a nucleic acid
sequence complementary to the probe. This technique is called
fluorescence in situ hybridization (FISH), and different appli-
cations are described here, including methods that target phylog-
eny (Figure 19.12 and see Figure 19.13) or gene expression (see (a) (b)
Figure 19.14).
Figure 19.13 FISH analysis of activated sludge from a wastewater treatment
Phylogenetic FISH stains are fluorescing oligonucleotides comple- plant. (a) Nitrifying bacteria. Red, ammonia-oxidizing bacteria; green, nitrite-oxidiz-
mentary in base sequence to sequences in ribosomal RNA (16S or ing bacteria. (b) Confocal laser scanning micrograph of a sewage sludge sample
23S rRNA in Bacteria and Archaea or 18S or 28S rRNA in eukaryotes, treated with three phylogenetic FISH probes, each containing a fluorescent dye
Chapter 13). Phylogenetic stains penetrate cells without lysing them (green, red, or blue) that identifies a particular group of Proteobacteria (Chapter 16).
and hybridize with rRNA directly in the ribosomes. The number of Green-, red-, or blue-stained cells reacted with only a single probe; other cells re-
acted with two (turquoise, yellow, purple) or three (white) probes.
 660 UNIT 5 • MICROBIAL ECOLOGY AND ENVIRONMENTAL MICROBIOLOGY

microbial populations with depth, as, for example, in a bio- is also useful in phylogenetic studies of microbes that may be grow-
film ( ▶ Section 20.4). In addition to microbial ecology, FISH is ing very slowly, such as organisms inhabiting the open oceans where
also an important tool in the food industry and in clinical diag- cold temperatures and low nutrient concentrations limit growth
nostics for the microscopic detection of specific pathogens in food rates (Figure 19.14). Because such cells have few ribosomes com-
products or clinical specimens. pared with more actively growing cells, standard FISH methodology
often yields only a weak signal.
Single-Cell Metabolic Activity Staining Using CARD-FISH Although cells that fluoresce brightly by FISH are likely actively
and BONCAT-FISH synthesizing proteins (as indicated by their high number of ribo-
FISH methods have been developed for measuring the metabolic somes), a direct measure of translational activity can be made using
activity of single cells by using probes for specific messenger RNAs bioorthogonal noncanonical amino acid tagging (BONCAT) combined
(transcriptional activity) or for protein synthesis (translational activ- with FISH (BONCAT-FISH). Bioorthogonal compounds are syn-
ity). Besides characterizing different taxa in a habitat as phylogenetic thetic molecules that mimic natural metabolites and when assimi-
stains do, these forms of FISH measure gene expression. These micro- lated and metabolized by a living cell do not interfere with normal
scopic methods thus complement phylogenetic staining—tech- physiological processes. Similarly, bioorthogonal analysis refers to
niques that answer the microbial ecologist’s important question of a chemical reaction that can occur inside a living cell without inter-
“Who is there?”—by answering the other important question in fering with normal function.
microbial ecology: “What are they doing?” In BONCAT (Figure 19.15), cells are first incubated with a modified
Measuring transcription requires targeting messenger RNA version of a normal cellular metabolite, such as the amino acid
(mRNA), which is much less abundant in cells than rRNA; because methionine containing a reactive alkyne group (Figure 19.15a). This
of this, standard FISH techniques cannot be applied. Instead, the methionine analog is called a noncanonical (meaning nonstan-
signal (fluorescence) must be amplified. A FISH method that dard) amino acid. Following incorporation of this analog into pro-
enhances the signal is called catalyzed reporter deposition FISH (CARD- teins of actively growing cells, the cells are treated with a fluorescent
FISH). In CARD-FISH the specific nucleic acid probe contains a reporter molecule containing a chemically reactive azide group that
molecule of the enzyme peroxidase conjugated to it instead of a bonds specifically to the methionine analog by way of its reactive
fluorescent dye. After there has been time for hybridization between alkyne group (Figure 19.15b). The phylogenetic identity of transla-
the probe and a specific mRNA, the preparation is treated with a tionally active cells is then determined using standard FISH technol-
fluorescently labeled compound called tyramide, which is a substrate ogy in which the rRNA probe is labeled with a different fluorescent
for peroxidase. Within cells containing the nucleic acid probe, the dye from that attached to the azide group. An example of BONCAT-
tyramide is converted by the activity of peroxidase into a very reac- FISH to identify translationally active cells in sediments of a marsh
tive intermediate that covalently binds to adjacent proteins; this estuary is shown in Figure 19.15c. Note that only some of the cell
amplifies the signal sufficiently to be detected by fluorescence clusters stained green by a bacteria-specific FISH probe are also
microscopy (Figure 19.14). Each molecule of peroxidase activates translationally active, as indicated by BONCAT magenta staining.
Thus, both a phylogenetic snapshot and an activity snapshot are
5
UNIT

many molecules of tyramide so that even mRNAs present at very low

abundance can be visualized. Besides detecting mRNA, CARD-FISH obtained in a single experiment.
After BONCAT-FISH, further genetic analysis is possible by using
cell sorting to recover labeled cells for genomic analysis of sorted
populations or of single cells (see Section 19.12). Once cells have
been concentrated by cell sorting techniques, sequencing can be
done on single genes, specific gene families, or entire genomes to
yield an increasingly more detailed picture of the genetic makeup
of the targeted cells.
Although the techniques we have just discussed do not require
laboratory culture, in Part III we consider what microbial ecologists
typically mean when they say their microbial community analyses
are “culture-independent.”
Michael Wagner and Marc Mussman

Check Your Understanding

• What structure in the cell is the target for fluorescent probes
in phylogenetic FISH?
• FISH and CARD-FISH can be used to reveal different things
about cells in nature. Explain.
• Compare the utility of CARD-FISH versus BONCAT-FISH for
evaluating cellular activity.
Figure 19.14 Catalyzed reporter deposition FISH (CARD-FISH) labeling of
Archaea. Archaeal cells in this preparation fluoresce intensely (green) relative
to DAPI-stained cells (blue).
 CHAPTER
C HA
APTER 19 • Taking the Measure of Microbial Systems 661

OH
+ Incubation and
NH2
dye coupling
Methionine analog (HPG)
Environmental Only translationally
sample active cells retain the
fluorescent dye
(a) Overview of the method

Incorporated
alkyne groups

Ribosome N
+

Roland Hatzenpichler
tRNA N N
N3 dye

U
G G A G
A
G
U U C C U G A
G G A G Azide group
G G A C U G A C C
U C C
3¿ (c)

Incorporation of HPG into Fluorescent dye Fluorescently labeled

growing peptide chains coupling reaction intracellular proteins
during sample incubation

(b) Details of the chemistry

Figure 19.15 BONCAT labeling of translationally (b) Chemistry of the process. HPG is incorporated by Blue, cells stained with the nonspecific nucleic acid dye
active microbial cells. (a) Overview. Cells in the en- the cell as methionine in growing polypeptides. When DAPI (Section 19.4). Green, cells stained by FISH for the
vironmental sample take up the methionine analog treated with the fluorescent dye-labeled azide, the phylogenetic domain Bacteria. Magenta, translational-
L-homopropargylglycine (HPG). Following incubation to azide group on the dye binds to the alkyne group on ly active cells stained using BONCAT. The large green
allow active cells to take up the HPG and incorporate it HPG to yield a fluorescent protein. (c) BONCAT-FISH ovoid cells are about 4 mm in diameter, the large ma-
into growing peptide chains, cells are treated with a analysis of activity and diversity of microbial cells in genta cells are about 6 mm in diameter, and the small
dye that makes translationally active cells fluoresce. Little Sippewissett Salt Marsh (Massachusetts, USA). magenta rod-shaped cells are about 1.5 mm long.

III • Culture-Independent of gene expression (transcription and translation) with measure-

5

UNIT
ments of active processes using the tools of metabolomics
Molecular Analyses of (◀ Section 10.10) and isotopic tracers to unravel the food webs sus-
taining complex microbial communities.
Microbial Communities Many surveys of microbial diversity and potential activity are per-
formed at the gene level using the polymerase chain reaction (PCR),

R evolutionary changes in DNA sequencing technology have

made it possible to rapidly recover vast amounts of gene
and genome sequence information from the environment.
DNA fragment analysis by gel electrophoresis (DGGE, T-RFLP,
ARISA, each discussed below) or molecular cloning, and DNA
sequencing and analysis (Chapters 10 and 12). Increasingly, as a
However, fundamental ecological understanding is possible result of changing technologies in DNA sequencing, entire genomes
only when that sequence information is paired with appro- extracted from cells present in an environmental sample are ana-
priate measures of cellular and community activities. lyzed as a more comprehensive measure of the biodiversity of
microbial communities. We begin with an overview of single gene
M icrobial biodiversity studies often forgo isolating organisms
or even quantifying or identifying them microscopically.
Instead, complementary molecular methods are used to measure the
analysis and then introduce multi-omics, the integration of data
from multiple omics platforms to more fully characterize microbial
community structure and function (see Figure 19.21). The term
biodiversity, potential activities, and realized activities of complex
omics (Chapter 10) is increasingly used to describe individual or
communities. As we now review in this section, gene and genome
various combinations of genomics, transcriptomics, proteomics,
sequencing serve as an important measure of diversity and potential
and metabolomics for analysis of organisms and communities of
activity, with the caveat that annotation of all novel gene functions
organisms. When these methods are used for the study of complex
remains a daunting challenge (◀ Section 10.2). However, detection
microbial communities, the terminology is often modified with the
of a gene is not the same as gene expression. As we will discuss in the
prefix “meta” (e.g., metagenomics, metatranscriptomics, metaproteomics)
following two sections, metabolic activity can be measured at the
as discussed in Section 19.8.
level of both the community and single cells by combining analyses
 662 UNIT 5 • MICROBIAL ECOLOGY AND ENVIRONMENTAL MICROBIOLOGY

19.6 PCR Methods of Microbial are called a phylotype. In microbial ecology, the phylotype concept
is primarily used to provide a natural (phylogenetic) framework for
Community Analysis describing the microbial diversity of a given habitat, regardless of
We discussed the principle of the polymerase chain reaction (PCR) in whether the identified phylotypes are cultured organisms or not.
Section 12.1. Recall the major steps involved: (1) Two nucleic acid prim- Thus, the word phylotype is widely used to describe the microbial
ers are hybridized to a complementary sequence in a target gene; diversity of a habitat based solely on nucleic acid sequences. It is
(2) DNA polymerase copies the target gene; and (3) multiple copies of only when additional physiological and genetic information
the target gene are made by repeated melting of complementary strands, becomes available, typically after the organism is brought into labo-
hybridization of primers, and new synthesis (◀ Figure 12.2). From a ratory culture (Sections 19.2 and 19.3), that proposing a genus and
Mastering single copy of a gene, several million copies can be made for subsequent species name for a phylotype becomes possible.
Microbiology
Art Activity:
analyses. PCR finds wide applications in microbial ecology. In a typical molecular community analysis experiment, total DNA
Figure 19.15 is isolated from a microbial habitat (Figure 19.16). Commercially
Steps in
single-gene PCR and Microbial Community Analysis
biodiversity
analysis of a Which genes are best suited to be target genes for microbial com-
microbial
community munity analyses? Because genes encoding the small subunit ribo- mRNA should also be targeted
Microbial
somal (SSU) rRNAs are phylogenetically informative and techniques community
for their analysis are well developed (◀ Section 13.11), they are
widely used in community analyses. Moreover, because rRNA genes
are universal and contain several regions of high sequence conserva- Extract total
tion, it is possible to amplify them from all organisms using only a community DNA.
few different PCR primers, even though the organisms may be phy- DNA
logenetically distantly related. In addition to rRNA genes, genes that
encode enzymes for metabolic functions unique to a specific Using fluorescently tagged
primers, amplify a region Amplify a region of the
organism or group of related organisms can be the target genes of the 16S or 23S rRNA 16S or 23S rRNA gene
(Table 19.3). Many of the organisms that harbor these genes are gene and cut with a using general or specific
restriction enzyme primers (for example,
described in Chapter 15. PCR Bacteria-specific) or more
(T-RFLP), OR amplify a
Genes such as those encoding rRNAs that have retained ancestral region connecting the 16S restrictive primers (to
function while changing in sequence over time as species have diverged and 23S rRNA genes target endospore-forming
(ARISA). bacteria).
are called orthologs (◀ Sections 13.8 and 13.11 and Figure 13.16).
Assuming absence of horizontal gene transfer (◀ Section 13.9), organ- Run pooled 16S
isms that share the same or very closely related orthologous genes rRNA gene
fragments from
variable to allow discrimination each sample on Next-
5
UNIT

Resolve DNA DGGE. generation

TABLE 19.3 Genes commonly used for evaluating specific fragments by sequencing
length by
microbial processes in the environment electrophoresis, Sample
using PCR generally using 1 2 3 4
an automated DGGE gel
Metabolic processa Target gene Encoded enzyme system with Different
fluorescence 16S rRNA Millions of amplified
Denitrification narG Nitrate reductase detection. gene fragment sequences
nirK, nirS Nitrite reductase fragments
Phylogenetic
norB Nitric oxide reductase Excise analysis
nosZ Nitrous oxide reductase bands
Fluorescence intensity

Bacillus subtilis
Env 1
Nitrogen fixation nifH Nitrogenase
Sequences of Bacillus cereus

Nitrification amoA Ammonia monooxygenase individual bands Bacillus megaterium

Env 2
Methane oxidation pmoA Methane monooxygenase Phylogenetic Clostridium histolyticum

Sulfate reduction apsA Adenosine phosphosulfate Fragment length

analysis Env 3

reductase
dsrAB Sulfite reductase Figure 19.16 Steps in single-gene biodiversity analysis of a microbial commu-
nity. From total community DNA, 16S rRNA genes are amplified using primers
Methane production mcrA Methyl coenzyme M
reductase
that target only Firmicutes, a group of gram-positive Bacteria that includes the
endospore-forming genera Bacillus and Clostridium. The 16S rRNA gene products
Degradation of petro- nahA Naphthalene dioxygenase obtained from PCR are then either separated by DGGE or sequenced directly by
leum compounds
next-generation sequencing; from the sequence data, a phylogenetic tree is ge-
alkB Alkane hydroxylase nerated. “Env” indicates an environmental sequence (phylotype). Alternatively, in
Anoxygenic pufM M subunit of photosynthetic T-RFLP analyses, the PCR-amplified products are labeled with a fluorescent dye and
photosynthesis reaction center fragmented by cutting with a restriction enzyme before electrophoresis. The number
a
of peaks identified by fluorescence corresponds to the number of phylotypes.
All of these metabolic processes are discussed in Chapter 14.
 CHAPTER 19 • Taking the Measure of Microbial Systems 663

available kits that yield high-purity DNA from soil or other complex phylotypes that can differ in base sequence significantly or by as
habitats are available for this purpose. The DNA obtained is a mix- little as a single base change.
ture of genomic DNA from all of the microorganisms that were in Once DGGE has been performed, the individual bands are excised
the sample from the habitat (see Figure 19.17). From this mixture, and sequenced (Figure 19.17). With 16S rRNA as the target gene, for
PCR is used to amplify the target gene and make multiple copies of example, the DGGE pattern immediately reveals the number of
each variant (phylotype) of the target gene. If RNA is isolated phylotypes (distinct 16S rRNA genes) present in a habitat (Fig-
instead of DNA (to detect those genes being transcribed), the RNA ure 19.17c). Although not suited for the analysis of highly complex
can be converted into complementary DNA (cDNA) by the enzyme communities, since individual bands may not be fully resolved, the

GC%
reverse transcriptase (◀ Section 11.11) and the cDNA subjected to method provides an excellent mechanism to quickly evaluate tem-
PCR as for isolated DNA. However, regardless of whether DNA or poral and spatial shifts in microbial community structure
RNA is originally isolated, the different phylotypes need to be (Figure 19.17c). If PCR primers specific for genes other than 16S
sorted out following the PCR step before they can be sequenced. rRNA are used, such as a metabolic gene (Table 19.3), the variants of
Sorting can be accomplished using one of three different methods: this specific gene that exist in the sample can also be assessed. Thus,
(1) physical separation by gel electrophoresis (◀ Section 12.2), although the number of bands on a DGGE gel is an overview of the
(2) clone library construction (◀ Sections 12.2 and 12.9), and biodiversity in a habitat (Figure 19.17c), sequence analysis is still
(3) next-generation sequencing technology (◀ Section 10.2). We required for identification and to infer phylogenetic relationship.
consider these methods now.
T-RFLP and ARISA
Denaturing Gradient Gel Electrophoresis: A rapid method of microbial community analysis is terminal restric-
Separating Very Similar Genes tion fragment length polymorphism (T-RFLP). In this method a target
One method to resolve phylotypes is denaturing gradient gel gene (usually an rRNA gene) is amplified by PCR from community
electrophoresis (DGGE), which separates genes of the same size DNA using a primer set in which one of the primers is end-labeled
that differ in their melting (denaturing) profile because of differ- with a fluorescent dye. The PCR products are then treated with a
ences in their base sequence (Figure 19.17a, b). DGGE employs a gradi- restriction enzyme (◀ Section 12.2) that cuts the DNA at specific
ent of a DNA denaturant, typically a mixture of urea and formamide. sequences. This generates a series of DNA fragments of varying
When a double-stranded DNA fragment moving through the gel length, the number of which depends on how many restriction cut
reaches a region containing sufficient denaturant, the strands begin sites exist in the DNA. The fluorescently labeled terminal fragments
to “melt”; at this point, their migration stops (Figure 19.17b, c). are then separated by size on an automated DNA sequencing instru-
Differences in base sequence cause differences in the melting prop- ment that detects fluorescent fragments (thus, only the terminal
erties of DNA. Thus, the different bands observed in a DGGE gel are dye-labeled fragments are detected). The pattern obtained shows the

1 2 3 4 5 6 7 8 1 2 3 4 5
5

UNIT
Jennifer A. Fagg and
Michael J. Ferris

This phylotype appears

(a) PCR amplification to be universal.

1 2 3 4 5 6 7 8 This phylotype is unique

to treatment facility 2.

This phylotype is present

Jennifer A. Fagg and Michael J. Ferris

in both treatment
Gerard Muyzer and Abdelaziz Belila

facilities 1 and 2.

(b) DGGE (c) DGGE of wastewater treatment plants

Figure 19.17 PCR and DGGE gels. Bulk DNA was isolated from a microbial community and amplified by PCR using primers for 16S rRNA
genes of Bacteria (a, lanes 1 and 8). Six bands later resolved by DGGE (b, lanes 2–7) were excised and reamplified and each gave a single
band at the same location on the PCR gel (a, lanes 2–7). However, by DGGE analysis, each band migrated to a different location on the gel
(b, lanes 2–7). Note that all bands migrate to the same location in the nondenaturing PCR gel because they are all of the same size, but they
migrate to different locations on the DGGE gel because they have different sequences. (c) DGGE profiles of microbial communities from
different wastewater treatment facilities amplified using primers for the 16S rRNA genes of Bacteria.
 664 UNIT 5 • MICROBIAL ECOLOGY AND ENVIRONMENTAL MICROBIOLOGY one band indicates that organism doesn't contain

restriction enzyme
rRNA sequence variation and general abundance of different A technique related to T-RFLP that provides more detailed analysis
sequence types (fragment fluorescence intensity) in the microbial of microbial communities is automated ribosomal intergenic spacer analy-
community sampled (Figure 19.16). sis (ARISA), which exploits the typical proximity of 16S and 23S rRNA
DGGE and T-RFLP both measure single-gene diversity, but in dif- genes in the genomes of Bacteria and Archaea. The DNA separating
ferent ways. The pattern of bands on a DGGE gel reflects the number these two genes, called the internal transcribed spacer (ITS) region, differs
of same-length sequence variants of a single gene (Figure 19.17), in length among species and often also differs in length among the
whereas the pattern of bands on a T-RFLP gel reflects variants differ- multiple rRNA operons of a single species (Figure 19.18a). The PCR
ing in DNA sequence of a single gene as measured by differences in primers for ARISA are complementary to conserved sequences in the
restriction enzyme cut sites. The information obtained from a 16S and 23S rRNA genes that flank the spacer region. Amplification
T-RFLP analysis, in addition to providing insight into the diversity (Figure 19.18b) and analysis (Figure 19.18c) are conducted as
and population abundances of a microbial community, can also be described for T-RFLP, resulting in a complex pattern of bands that can
used to infer phylogeny. Diagnostic information for each fragment be used for community analysis. However, ARISA differs from T-RFLP
includes knowledge of sequences near both ends (primer sequence in that ARISA does not require a restriction enzyme digestion following
and restriction enzyme cut site), knowledge that a second restriction PCR amplification. The word “automated” in the ARISA acronym refers
site does not exist within the fragment, and fragment length. Using to the use of a DNA sequencing instrument that automatically identi-
specialized software, this information can be used to search for fies and assigns sizes to each dye-labeled fragment (Figure 19.18c), as
matching 16S rRNA sequences in public databases. Although this is can also be done in T-RFLP analyses. ARISA has received greatest
of some predictive value, closely related sequences are often not application in the study of microbial community dynamics by moni-
differentiated by these criteria. Thus, T-RFLP generally underesti- toring, for example, changes in the presence and relative abundance
mates the diversity within a microbial community. of a specific community member through time and space.

Position 1 1540 1 2900

16S rRNA gene ITS region 23S rRNA gene

Forward PCR primer 50–1500bp Reverse PCR primer

containing fluorescent
tag ( ) Position 1513 Position 23 Each fragment of a unique
(a) length corresponds to a
unique community member.

5
UNIT

PCR total community

DNA using ITS primers
Community DNA

Separate fragments by size

(b) by gel electrophoresis.

1000
Fluorescence

750

500

250

0
400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250

Fragment size (base pairs)

(c)

Figure 19.18 Automated ribosomal intergenic spacer analysis (ARISA). (a) Structure of rRNA operon spanning the 16S rRNA gene (posi-
tions 1–1540), an internal transcribed spacer (ITS) region of variable length, and the 23S rRNA gene (positions 1–2900). The PCR primers,
one labeled with a fluorescent dye, are complementary to conserved sequences near the ITS region. (b) Amplified DNA fragments of differ-
ent lengths, each corresponding to a community member. (c) Fragment analysis determined by an automated DNA sequencer. The peaks,
which correspond to different ITS regions, can be identified by cloning and sequencing the amplified products.
 Genomic DNA library method

CHAPTER 19 • Taking the Measure of Microbial Systems 665

Diversity Studies Using Clone Libraries Technology change, 2005 to present

or Next-Generation Sequencing Pyrosequencing (100–800 nt reads)
Illumina (50–300 nt reads)
Early molecular microbial diversity research relied on the construc- Pacific Biosciences (60,000 nt reads)
tion of clone libraries to separate individual amplified DNA mole- MinION Nanopore Technology
(200,000 nt reads)
cules (amplicons); each clone in the library contained a unique
sequence that was then used as a template for sequence determina-
tion (◀ Sections 10.2 and 12.2). Figure 19.17a shows that a 16S 107
rRNA gene amplicon mixture appears as a single band when run on
a nondenaturing gel. However, because the amplified target gene 106
came from a mixture of different cells, the different phylotypes in the

Throughput (million nt/run)

105 30X Human Genome
single band have different sequences and need to be sorted out
before they are sequenced. Today this is almost exclusively done by
104
next-generation sequencing systems (◀ Section 10.2) rather than by
DGGE or by cloning. There are many such sequencing platforms Read length
103 100 nt
available (Figure 19.19), and each has advantages and disadvantages.
Long sequence reads are particularly useful for environmental >200 nt
102
metagenomic studies where the goal is to reconstruct genomes from >400 nt
members of the microbial community (Section 19.8). The MinION 10 >60,000 nt
(nanopore technology, ◀ Explore the Microbial World, “DNA
Sequencing in the Palm of Your Hand,” Chapter 10) sequencing
system is widely useful for this purpose because it can generate the Time
long sequence reads necessary for genome assembly purposes. Once (a)
a genome is assembled, sequence accuracy can be improved using
other platforms better capable of sequencing short DNA fragments
Percent of each phylotype in the

5
at high fidelity (Figure 19.19a).
environmental DNA sample

Low-sensitivity Sensitivity of phylotype detection

MinION and these other next-generation sequencing systems do detection using Sanger or fluorescent dye
4 technology to sequence
not require a cloning step, as individual DNA fragments are
Medium- approximately 100 (low sensitivity)
separated and amplified on the sequencing device itself; thus, PCR 3 or 1000 (medium sensitivity) clones
sensitivity
products can be used directly for sequencing. Since millions of detection from an environmental library
amplification reactions are then conducted simultaneously, the total 2
number of sequencing reads vastly exceeds what is possible by
High-sensitivity detection
sequencing individual clones obtained in a clone library one at a 1
(next-generation sequencing) allows
5

UNIT
time (Figure 19.19). This tremendous volume of sequence data for detection of the rare biosphere
allows for what has been called deep sequence analyses, meaning that 0
100 200 300 400 500
minor phylotypes that were possibly missed by the more limited
Unique phylotypes, ordered by decreasing abundance
and expensive clone library method are now revealed (Figure
(b)
19.19b). For example, if a particular phylotype were present at only
0.01% in a library of cloned sequences, sequencing of a thousand Figure 19.19 Community diversity analyses using next-generation sequencing
clones or more would be needed to ever detect it. By contrast, next- technology. (a) Current sequencing platforms (◀ Section 10.2) have the capacity to
generation sequencing would detect this low-abundance phylotype generate 1012 nucleotides (nt) of sequence in a single sequencing run (requiring a
along with its more abundant neighbors. This collection of minor week or less), with individual read lengths varying from 100 to 800 nucleotides. The
two segments in the rightmost bar show that technologies generating longer reads
phylotypes, which represent a substantial fraction of total diversity
have lower throughput per sequencing run. (b) This enormous sequencing capacity
but only a minor component of total organism abundance in most revealed many unique phylotypes that were not detected using DGGE or clone li-
environments, has been called the rare biosphere (Figure 19.19). Once brary sequencing. Fewer than 100 unique phylotypes would be detected by Sanger
next-generation sequencing reveals these rare but potentially very (first-generation) sequencing of 1000 clones in a library of 16S rRNA gene PCR am-
important components of a microbial community, various tools in plicons. Jed Fuhrman is acknowledged for input to part b.
the microbial ecologist’s toolbox can be deployed to learn more
about their activities and overall role in their ecosystem.
variant of PCR that allows each phylotype to be quantified as well as
amplified, ◀ Section 12.1), it has often been observed that the most
Results of PCR Phylogenetic Analyses
abundant phylotypes in a natural microbial community are ones that
Phylogenetic analyses of microbial communities have yielded surpris- have thus far defied laboratory culture. These sobering results make
ing results. For example, using the gene encoding 16S rRNA as the it clear that our knowledge of microbial diversity from enrichment
target, analyses of natural microbial communities typically show that cultures is far from complete and that enrichment bias (Section 19.1)
many phylogenetically distinct Bacteria and Archaea (phylotypes) are is a serious problem in culture-dependent biodiversity studies. Obvi-
present whose rRNA gene sequences differ from those of all known ously, much work remains to put our abilities to culture microbes on
laboratory cultures. Moreover, using quantitative PCR (qPCR, a a par with our existing abilities to detect and identify them in nature.
 666 UNIT 5 • MICROBIAL ECOLOGY AND ENVIRONMENTAL MICROBIOLOGY

Check Your Understanding The GeoChip—also called a functional gene microarray—targets

• What could you conclude from PCR/DGGE analysis of a sample genes that function in a measurable environmental process. How-
that yielded one size band by PCR and one band by DGGE? ever, because genes encoding enzymes of similar function can vary
One size band by PCR and four bands by DGGE? significantly in sequence, the arrays must contain many thousands
• What surprising finding has come out of many molecular stud- of probes in order to achieve reasonable coverage of natural diver-
ies of natural habitats using 16S rRNA as the target gene? sity. Even then, such arrays may only sample a fraction of the actual
functional diversity in a habitat. Successful use of a GeoChip
• How has next-generation sequencing technology altered our
understanding of microbial community diversity? requires several steps: (1) isolation of total community DNA from
the sample, (2) random sequence amplification (as is also used for
amplification of DNA from a single cell, ◀ Section 10.11), (3) fluo-
rescence labeling of the environmental DNA, and (4) hybridizing
19.7 Microarrays for Analysis of Microbial the environmental DNA to probes on the GeoChip. The GeoChip
Phylogenetic and Functional Diversity functional gene microarray contains over 160,000 probes covering
We previously considered the use of DNA chips—a type of microarray— more than 1400 gene families encoding proteins that participate in
for assessing overall gene expression in a microbial pure culture carbon, nitrogen, and sulfur cycling processes. By measuring the
(◀ Section 10.8). Specific microarrays can also be constructed for hybridization signal conferred by binding of environmentally
rapid gene-based analyses of biodiversity and the functional poten- derived DNA to each probe on the microarray (Figure 19.20), a
tial of natural microbial communities. Microarrays designed to rapid appraisal of the potential metabolisms operating in a particu-
exclusively target different 16S rRNA sequence types (phylotypes), lar habitat can be obtained.
called PhyloChips, have been used in the past, but microarrays GeoChips circumvent many of the time-consuming steps of
employed for environmental studies now focus on metabolic genes molecular microbial ecology—PCR, DGGE, cloning, and sequencing
of biogeochemical significance. This type of microarray, the Geo- (Figure 19.16). In addition, the GeoChip is useful for detecting low-
Chip, detects genes required for selected metabolic processes, includ- abundance genes that would be difficult to identify by sequencing
ing sulfate reduction, ammonia oxidation, denitrification, and and also offers some information on actual gene abundance. How-
nitrogen fixation (Figure 19.20 and Table 19.3). ever, an important caveat to the use of microarrays is the possibility

Functional category Gene Total Database gene

families probes coverage (%)
Carbon cycling 149 26922 49
Nitrogen cycling 32 6493 52
Sulfur cycling 27 4739 64
Phosphorus 7 3260 52
5 Metal homeostasis 121 43432 47
UNIT

Viruses 115 2857 55

Other 81 10380 42
Organic remediation 104 11591 41
Virulence 639 21152 45
Jizhong Zhou, Zhili He, and Joy Van Nostrand

Secondary metabolism 68 4032 56

Electron transfer 15 797 65
Stress response 89 26306 33

TOTAL 1447 161,961 44

(b)

Figure 19.20 GeoChip analysis of functional gene diversity. The GeoChip contains
GeoChip
over 161,000 probes covering more than 365,000 gene sequences in public databases,
encompassing most major biogeochemical processes. The image shows green fluo-
rescence of varying intensity (approximating gene abundance) following hybridi-
zation of fluorescent dye–labeled environmental DNA to the individual probes in one
region of the high-density array of probes. The red spots correspond to repeated ap-
plications of a known amount of a reference DNA standard. A red-dye-labeled probe
complementary to the reference standard was added to the environmental DNA
prior to hybridization. Red fluorescence of equal intensity among the reference stan-
(a) dard spots confirms that hybridization was uniform throughout the array.
 CHAPTER 19 • Taking the Measure of Microbial Systems 667

of nonspecific hybridization. That is, gene variants that are closely 19.8 Environmental Multi-omics: Integration
related in sequence may not be resolved in a microarray because of
overlapping hybridization patterns. Moreover, totally unrelated of Genomics, Transcriptomics,
genes may yield false positive results if they are sufficiently comple- Proteomics, and Metabolomics
mentary in base sequence to hybridize to the probe. And finally,
As was presented in Chapter 10, a more complete understanding of
unlike nucleic acid sequencing, whose costs keep plummeting yearly,
how a microorganism functions requires an integrated accounting
designing and manufacturing gene chips are not yet inexpensive
of all central cellular processes. These include gene expression, func-
endeavors. Combined with culture-independent phylogenetic diver-
tional knowledge of all gene products and product activities, and all
sity studies (Section 19.6), functional gene arrays offer the microbial
metabolites produced during growth. The analytical tools used to
ecologist a detailed picture of both the diversity of organisms and
characterize those processes in pure cultures (◀ Sections 10.11–
the diversity of metabolisms in any given environment. However,
10.13) are now being applied to the study of complex microbial
what the GeoChip and related functional gene arrays do not reveal is
communities to yield an unprecedented view of microbial ecology.
actual metabolic activity; they show only the genetic potential for
Characterization of a natural system is a much more complex prob-
metabolic activity. We got a taste of how metabolic activity can be
lem than the analysis of a single organism. Not only must processes of
measured using BONCAT, a microscopic method of detecting meta-
each organism be measured, but the many changing biotic and abiotic
bolic activity (Figure 19.15). In the next section, we explore other
interactions that modify those processes must also be resolved. Here
methods of assessing microbial activity that use omics, the most
we consider the methods of genomic, transcriptomic, proteomic, and
powerful tools in the microbial ecologist’s toolbox.
metabolomic analysis required to unveil patterns of microbial diversity
that will ultimately lead to a more predictive understanding of micro-
Check Your Understanding bial community function and response to environmental change. Col-
• What are some caveats of interpreting a hybridization signal on lectively called omics or, when used in combination, multi-omics, these
the GeoChip? rapidly evolving methods are having a dramatic impact on our under-
• What are the advantages and disadvantages of microarray tech- standing of the structure and function of natural microbial communi-
nology compared to sequencing PCR products? ties. We begin with an overview of individual omics (Figure 19.21), and
then present exemplar case studies of their application.

Microbial community

UNIT
DNA RNA Proteins Metabolites Stable/radioactive Probe-based
isotopes single-cell
microscopic
13
C 15N 34S or cell-sorting
18O 14C 3H 32P 35S
analyses

Targeted Metagenome Metatranscriptome Metaproteome Metabolome

gene analysis
Mapping
Mapping

16S, amoA,
dsrAB, etc. Gene expression Protein Community food Stable isotope probing FISH
profiles identification webs
Single-cell activity BONCAT
• MAR
• (Nano) SIMS FACS
Phylogeny, diversity, and gene/
pathway function Isotopic tracers for Single-cell
• Single-gene amplification process measurements genomics
• Genome sequencing
• Microarrays

Potential activity Gene expression Activities of communities and single cells

Figure 19.21 Multi-omics methods overview. The combined use of advanced genetic, molecular, microscopic, and isotopic methods is
revolutionizing the study of natural microbial communities. Assignment of protein and transcript sequences to specific community members is
accomplished by mapping their sequences on unique genomes assembled from metagenomic sequences. Abbreviations: FISH, fluorescence in
situ hybridization (Figures 19.11–19.14); BONCAT, bioorthogonal noncanonical amino acid tagging (Figure 19.15); FACS, fluorescence-activated
cell sorting (see Figure 19.42); MAR, microautoradiography (see Figure 19.41); SIMS, secondary ion mass spectrometry (see Figure 19.39).
 668 UNIT 5 • MICROBIAL ECOLOGY AND ENVIRONMENTAL MICROBIOLOGY

Metagenomics and Reconstructing

Environmental Genomes
An encompassing approach to the genetic characterization of micro- Microbial
community
bial communities is environmental genomics, also called
metagenomics. Before the metagenomics era, microbial commu- Extract total
nity analyses typically focused on the diversity of a single gene in an community DNA.
environmental sample. By contrast, in environmental genomics, all
DNA
genes in a given microbial community can be sampled and, if the Community Environmental
experiment is properly designed, the information obtained can sup- sampling approach genomics approach
port a much deeper understanding of the structure and function of Amplify single gene, for example, Sequence directly (without
the community than can single-gene analyses. gene encoding 16S rRNA or a gene cloning) using high-throughput
The goal of a metagenomics study today is to use next-generation encoding a specific function (e.g., next-generation sequencing
ammonia oxidation) methods
DNA sequencing (◀ Section 10.2) to identify as many genes as pos-
sible from an environmental DNA sample and determine the phy-
Sequence and Assemble and
logeny of the organism(s) to which the genes belong. Although generate tree. annotate.
complete and finished genomes are often not the goal of metage-
nomics, there is increasing interest in assembling individual
genomes from large metagenomic datasets to at least the draft stage. Partial to
Outcomes complete
Rather than simply generating a list of all genes present in an envi-
genomes
ronment, nearly complete genomes can better connect functional
and phylogenetic aspects of a microbial habitat. An example of this Single-gene phylogenetic tree Total gene pool of the community
is shown in the “connection graph” of Figure 19.22 that depicts an
assembly of genomes from a coastal marine water sample. A total of 1. Phylogenetic snapshot 1. Identification of all gene categories
of most members of the 2. Discovery of new genes
58.5 billion nucleotides in the metagenome were used to stitch community 3. Linking of genes to phylotypes
together these complete and near-complete genomes. Such massive 2. Phylogenetic diversity of a
metagenomic undertakings can be highly complex enterprises but microbial guild such as the
ammonia-oxidizing Archaea
often reveal links between physiologies and phylogenies not obtain-
able in the absence of reconstructed genomes. Figure 19.23 Single-gene versus environmental genomic approaches to microbial
community analysis. In the environmental genomic approach, all community DNA is
sequenced, but the assembled genomes may not all be complete. Total gene recov-
ery is variable and depends on several factors including the complexity of the habi-
tat and the amount of sequence determined. Recovery is typically better when

5 diversity is low and sequence redundancy is high.

UNIT

A problem with genome assembly from a mixture of environmen-

tal DNA sequence reads, however, is that the genomes obtained are
unlikely to be either complete or clonal, instead being composed of
fragments of DNA from closely related strains of a species
(Figure 19.23). This has proven to be a major problem in the assembly
of soil microbial genomes, for example, from large pools of metage-
nomic data. A single gram of fertile soil contains about 1012 bacterial
and archaeal genes and 109 genomes; complete coverage of these
genes is not yet feasible with available technology, even with the
sequencing of 300 billion nucleotides in one soil study! Single-cell
Vaughn Iverson and Ginger Armbrust

genomics (◀ Section 10.11 and Figure 10.30) may ultimately over-

come this problem (see also Section 19.12), but by definition, this
method only provides information from a single microbial cell.
Of critical importance in evaluating a genome reconstructed from
metagenomic DNA is to assess whether it contains all of the genes
required by a cell (for example, all necessary tRNA and rRNA genes
and genes encoding essential proteins such as DNA and RNA poly-
merases) and is therefore a legitimate genome candidate. In addi-
Figure 19.22 Genome assembly from a coastal marine metagenome consisting of tion, an assessment of the relative abundance of genes encoding
58.5 billion nucleotides of sequence. This “connection graph” is intended as a visual
specific functions is equally valuable, since abundance changes sug-
representation of the complexity and abundance of partial and complete genomes
assembled from the water sample. The long strands, colored by differences in the
gest interactions among species or a common response to a particu-
percentage of guanine plus cytosine content, correspond to prokaryotic genomes lar environmental variable. For example, if a high number of genes
and the small circular strands are most likely from viruses or plasmids. were recovered in the pathway for nitrogen fixation, this would
 CHAPTER 19 • Taking the Measure of Microbial Systems 669

suggest that the environment sampled was limited in NH4+, NO3-, DNA directly without the need to amplify it or resolve different
and other forms of fixed nitrogen, thus selecting for nitrogen-fixing phylotypes before sequencing.
bacteria. Figure 19.23 contrasts the environmental genomic ap- The Sargasso Sea metagenome study revealed several novel find-
proach with single-gene analysis of microbial communities. ings such as the presence of candidate ammonia-oxidizing genes in
Although metagenomics can reveal much about a microbial habi- archaeal genomes, a result that was later confirmed with the isola-
tat, there are many things metagenomics cannot tell us about envi- tion and description of a new group of Archaea, the Thaumarchaeota
ronmental microbial communities. Currently no methods are (◀ Section 17.5). Moreover, genes encoding proteorhodopsin, a light-
available for translating metagenomic sequence data into funda- sensitive proton pump present in certain Proteobacteria and related
mental physiological information about the microbial community, to bacteriorhodopsin of extreme halophiles (◀ Section 17.1), were
such as the maximum specific growth rate of different species, their found in the genomes of several new phylogenetic lineages of Bac-
affinities for nutrients, their optimum, minimum, and maximum teria. However, despite this major sequencing undertaking, much
pH or temperature for growth, or their speed of recovery from star- was missed. This is because 1 milliliter of seawater contains approxi-
vation. Moreover, any metabolisms never before encountered are mately 5 trillion base pairs (bp) of bacterial genomic DNA and
unlikely to be deduced from nucleotide sequence data alone. These would therefore require 5000 times the sequencing effort just to
realities once again underscore why it is important to culture new cover each base pair once on average! Hence, even with current tech-
microbes from nature; at present, there is simply no substitute for nology (see Table 10.2)—which can generate over a trillion bp of
culture-based characterization to define many critical aspects of a sequence in a few days (Figure 19.19)—no one natural environment
microbe’s functional biology. has yet been sequenced completely.
Genomic/metagenomic approaches have also revealed variations
Some Examples of Environmental Genomics in genes associated with a single phylotype; that is, in strains that
Environmental genomics can detect both new genes in known contain identical, or nearly identical, rRNA genes. For example, in
organisms and known genes in new organisms. A large number of studies of Prochlorococcus, the most abundant cyanobacterium (oxy-
interesting microbial communities have been probed using early genic phototroph) in the ocean ( ▶ Section 20.11), comparison of
metagenomic tools. In an early study of bacterial and archaeal diver- the genome sequences of cultured strains with Prochlorococcus genes
sity in the Sargasso Sea (a low-nutrient region of the Atlantic Ocean obtained from metagenomic analyses of ocean water identified
near Bermuda), over one billion nucleotides were sequenced and extensive regions shared between the cultured and environmental
from this over 1800 bacterial and archaeal species were detected, populations (Figure 19.24). This high level of gene conservation con-
including 148 previously unknown phylotypes and many novel firms that the organisms in culture are typical of environmental
genes. Many of these species had previously been missed using rRNA populations. In addition, however, these analyses also identified
community analyses that employed PCR and cloning or PCR and several highly variable regions in which the genomes of cultured
DGGE (Section 19.6). Genes that fail to amplify, of course, remain strains differed significantly from those of environmental popula-
undetected in community analyses, and cloning efficiency is far tions. These variable regions were clustered in the genome as genomic
islands (chromosomal islands, ◀ Section 13.10), and likely encode
5

UNIT
from 100%. Metagenomics sidesteps these problems by sequencing

Genomic islands ISL1 ISL2 ISL3 ISL4 ISL5

100

80
Percent identity

1
Genomic
2 Fold coverage
islands
1.65 Mbp 5 8.0
3
4.0
Fold coverage 1.0
4 0.5
0.25
0
2 4 6 8 10 12 14 16
Position along the Prochlorococcus genome (105 bp)

Figure 19.24 Metagenomic analysis. Sequences (represented as green dashes) from the Sargasso Sea metagenome that align to the
genome sequence of a cultured Prochlorococcus, showing regions where the cultured strain has genes of high similarity (high percent
identity) with sequences in the metagenome, and other regions (shaded) where it lacks genes in common (genomic islands, ISL1–ISL5).
Since the DNA sequence contained within the genomic islands is thought to encode niche-specific functions, the cultured strain would
likely not exhibit the same environmental distribution as strains containing all the island genes. Fold coverage is a measure of how com-
pletely the various regions in the Prochlorococcus genome are accounted for by similar sequences in the metagenome.
 670 UNIT 5 • MICROBIAL ECOLOGY AND ENVIRONMENTAL MICROBIOLOGY

functions that control the growth response of particular Prochlorococ- genes in the community are actually being expressed and the relative
cus populations to environmental variables unique to their habitat, level of that expression, at a specific time and place (Figure 19.21).
such as temperature or light quality and intensity ( ▶ Section 20.11 Because the expression of most genes in Bacteria and Archaea is
and Figure 20.26). controlled at the level of transcription (Chapter 7), mRNA abun-
dance can be taken as a census of individual gene expression levels.
Metatranscriptomics, Metaproteomics, Thus, gene transcript abundance determined for an entire com-
and Metabolomics munity can be used to infer the operation of major metabolic pro-
As we discussed in Chapter 10, the genomics era has spawned sev- cesses catalyzed by that community at the time of sampling
eral additional “omics,” in particular, metatranscriptomics, metapro- (Figure 19.25).
teomics, and metabolomics. Metatranscriptomics is analogous to Metaproteomics, the measure of the diversity and abundance of
metagenomics but analyzes the sequences of community RNA different proteins in a community, is an even more direct measure of
rather than community DNA. The isolated RNA is converted into cell function than is metatranscriptomics. This is because different
cDNA by reverse transcription (◀ Section 11.11) before sequencing mRNAs have different half-lives and efficiencies of translation, and
and analysis as for DNA. Whereas metagenomics describes the thus will not all yield the same number of protein copies. However,
functional capacities of the community (for example, the relative metaproteomics is a greater technical challenge than either metage-
abundance of specific genes), metatranscriptomics reveals which nomics or metatranscriptomics. Protein identification, usually by

phoR
Pi High-affinity transport pstS
pstC/pstA
phoU
Phosphorus Pi Low-affinity transport pitA
assimilation
Polyphosphate ppk

phoX
Alkaline phosphatase phoD
Cell growth phoA

nifK
Nitrogen fixation nifH
nifD
Nitrogen nasA/narB
assimilation Assimilative nitrate nasD
reduction nasF
nirB
5
UNIT

Ammonium transport amt

narK
narG
Dissimilative nitrate narH
reduction narJ
(membrane/cytoplasmic) narI
Anaerobic
respiration napA
Energy napB
napC
conservation Dissimilative nitrate napH
reduction napG
(periplasmic) napD
napF
amoA
Chemolithotrophy Ammonia oxidation amoB
amoC

6 7 8 9 9.4

Log10 transcripts/liter
Limit of detection

Figure 19.25 Metatranscriptomic analysis of coastal marine surface waters. Expression of genes for key steps in the N and P cycle in
a seawater sample determined by sequencing environmental mRNA. These data showed that the microbial community was using both in-
organic (high expression of P transporters) and organic (alkaline phosphatase) forms of phosphate (PO43-). Low levels of transcripts for
genes required for nitrate (NO3 -) assimilation contrasted with the high expression of genes for ammonia (NH3) transport and chemolitho-
trophic NH3 oxidation. Also, as expected for oxic marine surface waters, there was little expression of genes for NO3 - respiration. Data
courtesy of Mary Ann Moran, University of Georgia Marine Sciences.
 CHAPTER 19 • Taking the Measure of Microbial Systems 671

mass spectrometric characterization of peptides released from enzy- ◀ Section 14.15) and oxidizing methane (methanotrophy,
matic digestion of the total protein pool by specific proteases ◀ Section 14.16) as well as oxidizing and reducing carbon monox-
(◀ Section 10.9), relies on naturally available material since it is not ide (CO dehydrogenases), were also detected in the bog microbial
possible to amplify protein sequences as one does using PCR to community.
amplify nucleic acids for sequencing. Protein identification also This combined metagenomic and metaproteomic snapshot of
requires at least partial separation of the individual peptides in order a major microbial community highlights the power of omics for
to reduce complexity of the sample and a reference genome or resolving the “who” and “how” of complex microbial communi-
metagenome to identify potential coding sequences (see Figure ties and for using the results to predict the response(s) of these
19.21). Metabolomics is the comprehensive analysis of cellular and communities to environmental changes. In the case of permafrost
extracellular metabolites (also called exometabolites) of a micro- melting, the potential for the release of large amounts of CO2
bial community and is the most direct measure of the food web (due to increased respiration of organic carbon) and CH4 (due
structure of a community. As discussed in Chapter 10 (◀ Section to increased activities of methanogens) from permafrost as cli-
10.10), advanced methods in mass spectrometry are now providing mate change progresses (see Section 21.9) was clearly apparent
the analytical power required to resolve and quantify the many hun- (Figure 19.26).
dreds of metabolites that form the food webs of natural microbial
communities. Utility of Metabolomics: An Environmental
The utility and power of integrating omics methods for the study Case Study of Biocrusts
of natural microbial communities has been on display in many eco- A study of the microbial ecology of biological soil crusts (biocrusts)
logical studies published in the scientific literature in recent years and offers an example of how metabolomics can provide new under-
was evident in the metatranscriptomic study shown in Figure 19.25. standing of food webs that sustain natural microbial communities.
We continue this theme here with two case studies, one employing Biocrusts are microbe-dominated communities that develop in arid
metaproteomics and the other metabolomics. environments of limited vegetation that in part function to reduce
soil erosion (Figure 19.27; ▶ Section 20.6). They are relatively simple
Utility of Metaproteomics: An Environmental communities in which a dominant filamentous cyanobacterium, in
Case Study of Permafrost this case Microcoleus vaginatus (a member of the Oscillatoriales,
Permafrost covers an estimated 20% of the land on Earth and ◀ Section 15.3), is the primary producer and the ultimate source of
sequesters large amounts of organic carbon. Because climate change dissolved carbon used by associated heterotrophic microbes. These
will likely lead to the release of two major “greenhouse gases”—CO2 communities persist in a desiccated, dormant state for extended
and CH4—from permafrost, identifying the microbes inhabiting periods but rapidly become active after a rainfall. Analysis of the
actively melting permafrost along with their metabolic potential is biocrust metabolome using advanced methods of mass spectrom-
essential for climate predictions. Using a combination of metage- etry identified and quantified hundreds of metabolites (e.g., ad-
nomics (◀ Section 10.7) and metaproteomics (◀ Section 10.9), a enine, aspartate, uracil, methylguanine, 2-isopropylmalate, methio-
5

UNIT
revealing snapshot of the genes and proteins of the microbial com- nine, guanidinobutanoate, glycine betaine, malate, succinate). Many
munity present in a permafrost meltwater bog has been obtained of these metabolites are produced by Microcoleus and released to the
(Figure 19.26). environment for consumption by other members of the soil crust
During the metagenomic analysis of the bog, DNA sequences community. Since this cyanobacterium is unable to fix nitrogen, its
were assembled and annotated into complete and partial genomes nitrogen requirement is largely supplied by nitrogen-containing
(Figure 19.26a). This analysis indicated that Bacteria of the phyla metabolites supplied by heterotrophic members of the
Proteobacteria, Actinobacteria, and Chloroflexi predominated (Chap- community.
ters 15 and 16), while methanogens (Euryarchaeota) were the domi- Characterization of the intracellular metabolites and exometabo-
nant Archaea (Chapter 17). From the large amount of sequence data lites of a pure culture of Microcoleus showed that a significant frac-
obtained, the genomes of three novel (and as yet uncultured) meth- tion of its intracellular metabolites were released during growth and
anogens could be assembled without any cultures of the organisms thus made available to heterotrophic community members as exo-
having been obtained. This indicated that unique methanogens metabolites (Figure 19.28a). In turn, the heterotrophic community
resided in the permafrost—most likely species that function well in produced metabolites used selectively by the Microcoleus or by other
the cold (psychrophiles)—and therefore that the potential for heterotrophs (Figure 19.28b). Bacterial cultures isolated from the
increasing rates of methanogenesis as the permafrost melted was crust were found to have strong substrate preferences, each deplet-
high. Using the annotated DNA sequences and the output from the ing a certain fraction of the metabolites while ignoring others.
mass spectrometry detection of peptides, the identity of proteins There was also little overlap in utilization; only two metabolites,
extracted from the bog sample was determined along with their glutamate and a second, unidentified compound, were used by all
microbial sources (Figure 19.26b). The proteomics detected an members of a group of bacterial isolates. Thus, each isolate from
abundance of functionally diverse proteins including those that par- the biocrust likely occupies a specialized nutritional niche, using
ticipate in cellular housekeeping, transport, and organic carbon res- only a subset of the total pool of metabolites as nutrients. Analysis
piration. But importantly, and in agreement with the metagenomics of the patterns of metabolite uptake and release suggested 80
data, several proteins associated with C1 metabolism, including potential cross-feeding patterns. In addition to providing an exam-
those necessary for both making methane (methanogenesis, ple of the power of metabolomics for untangling complex food
 672 UNIT 5 • MICROBIAL ECOLOGY AND ENVIRONMENTAL MICROBIOLOGY

Environmental sample

Extract Extract total

total DNA protein

Protein digestion and

Sequence DNA
mass spectrometry

G A C T T A
DNA analysis Peptide identification and quantification
Assembly of
genomes

Identify protein source

by screening genomes
16S rRNA gene
for genes encoding
analysis
peptide sequence.
(a)

ed o ids rs
ch in ac tide me
e r ran n am o l y
los ga B ai i n
pe
p o
Xy Su ch ids Am Di op lla
NapC PSP Bi nB ge Figure 19.26 Meta-omics of permafrost.
Opa
SusD ac To trB Fla
M (a) Metagenomic and metaproteomic analy-
p
sis of an environmental sample. Total DNA
Lem
and protein are extracted from a sample and
GlnA GlnB CO dehydrogenase
5 Ptol analyzed to identify microorganisms and
UNIT

Bacterioferritin
GlnB (aerobic-type) Ccp their associated proteins. Following
Translation DsrA sequencing, the DNA is assembled into par-
Glycolysis/ CO dehydrogenase
elongation CO2 tial and complete individual genomes. The
Gluconeogenesis
CO dehydrogenase factors Tu, 16S rRNA gene can also be profiled to deter-
EF1, GTPase Cold
1 2 3 4 5 mine the phylogenetic affiliation of Bacteria
Mtd shock
3 Thr Hup and Archaea present in the sample. After
Ribosomal proteins Mer
6 9 identifying the digested proteins by mass
Citric RNase MtrA MtrH
acid Mo spectrometry, their microbial source can be
7 10
cycle McrA McrB McrG determined by searching for corresponding
GroEL GroES
8 chaperones RNA polymerase DNA sequences in the metagenomic data.
(b) Visualization of a subset of the proteins
CO2 Housekeeping functions CH4 identified from metagenomic and metapro-
teomic analysis of a bog sample. Ccp: cyto-
chrome c peroxidase, DsrA: sulfite reductase
Key enzymes
Porins Bog metagenome subunit A, GlnA/GlnB: nitrogen storage, Hup:
1. 6-Phosphofructokinase
heterosulfide reductase, Lem: peptidoglycan-
Bradyrhizobium 2. Glyceraldehyde-3-phosphate dehydrogenase
ATP synthase 3. Acetyl-CoA acetyltransferase associated lipoprotein, Mtd/MtrA/MtrB/
CO2 Greenhouse 4. NAD-dependent aldehyde dehydrogenases MtrH/McrA/McrB/McrG/Mer/Mo: methane
ABC transporter 5. Phosphoenolpyruvate synthase/pyruvate phosphate metabolism, NapC: NapC/NirT cytochrome c,
CH4 gases dikinase (EMP in Archaea) Opa: opacity protein, PSP: phosphate-selec-
6. Pyruvate phosphate dikinase tive porin, Ptol: periplasmic component of
Other transporter
7. Dihydrodipicolinate synthase
the Tol biopolymer transport system, Thr:
8. Glucose/sorbosone dehydrogenase
9. Acetoacetate decarboxylase thermosome, SusD: sulfate ABC transport
10. Pyruvate/2-oxoglutarate dehydrogenase system ATP-binding protein, TonB: periplas-
mic protein. Data adapted from Hultman, J.,
(b) et al. 2015. Nature 521: 208.
 CHAPTER 19 • Taking the Measure of Microbial Systems 673

(a) (b) webs, the use of this exometabolomic

approach for identifying metabolites used by
uncultured community members yields a sec-
ond scientific benefit: It can guide researchers
in formulating selective culture media for
enrichment of heterotrophic bacteria that
eluded initial cultivation efforts.
In the final part of this chapter, up next, we
look at some other tools in the microbial ecol-
ogist’s toolbox that can measure total micro-
(c) bial community activities.

Check Your Understanding

• What is a metaproteome, and how does
it differ from a metagenome and from a
metatranscriptome?

Trent Northern and Mia Jones

• How do environmental multi-omics
approaches differ from environmental
genomics or single-gene analyses,
such as that based on 16S rRNA gene
analysis for microbial community
characterization?
Figure 19.27 Desert biocrust of the Colorado Plateau. (a) The thin desert biocrust is stabilized by the filamentous • How can the most metabolically active
cyanobacterium Microcoleus, which binds together soil particles and reduces erosion. (b, c) Visible Microcoleus cell populations in a community be
filaments (arrows) show the fine architecture of the crust. Microcoleus nourishes a rich community of primarily identified using environmental omics
heterotrophic bacteria, releasing metabolites consumed by community members, which in turn produce metabo- methods?
lites consumed by the cyanobacterium. Environmental metabolomics (see Figure 19.28) has the power to unravel
the complex food web that sustains these critical microbial communities in arid lands.

106 IV • Measuring Microbial

Estimated abundance

104
102
Total cellular
metabolites Activities in Nature
M icrobial communities manifest their presence through the
5

UNIT
0 25 50 75 100 125 150 175 200 225
concerted activities of individual microorganisms that
2
10 Released metabolites together drive major biogeochemical processes. A powerful
104 (200 exometabolites) suite of isotopic and genetic methods combined with high-
106 resolution microscopy and sensor technology have made it
(a) possible for the microbial ecologist to map these processes
from the level of individual cells to entire environmental
metabolites relea systems.
200 by Microcoleus sed

W e wrap up this chapter by considering methods for measuring

microbial activities in natural samples beyond those already
discussed in Parts II and III. The techniques we consider here include
the use of radioisotopes, microsensors, nanosensors, stable isotopes,
Microcoleus Heterotrophic and genomic methods. Activity measurements in a natural sample
microbial
community are collective estimates of the physiological reactions occurring in the
130 ed
m e t a b o l i te s c o n s u m entire microbial community, although several techniques to be dis-
b y M icro c ole u s cussed later (Sections 19.10–19.12) allow for a more targeted assess-
(b) ment of physiological activity. Activity measurements reveal both
Figure 19.28 Metabolic network supporting the desert biocrust microbial the types and rates of the major metabolic reactions occurring in a
community. (a) Abundance of each metabolite arranged in decreasing order (up- habitat, and the various techniques can be used alone or in combi-
per panel) The primary producer, the cyanobacterium Microcoleus, releases a large nation in microbial community analyses. In conjunction with
fraction of its intracellular metabolites (green) into the environment (blue). (b). These multi-omics analyses, these methods help define the structure and
metabolites nourish a complex heterotrophic community that in turn produces metabo- function of microbial ecosystems—the ultimate goal of microbial
lites nourishing Microcoleus. In addition, hundreds of additional metabolites consumed ecology—and often inform the design of enrichment cultures to
by the crust community were identified indicating additional metabolic interactions and
bring microbial community members into the laboratory.
cross-feeding among heterotrophs (arrows between bacterial cells). Data adapted
from Baran, R., et al. 2015. Nat. Commun. 6 : 8289, doi: 10.1038/ncomms9289.
 674 UNIT 5 • MICROBIAL ECOLOGY AND ENVIRONMENTAL MICROBIOLOGY

19.9 Chemical Assays, Radioisotopic followed, radioisotopes are more useful than strictly chemical assays.
For instance, if measuring photoautotrophy is the goal, the light-
Methods, Microsensors, dependent uptake of radioactive carbon dioxide (14CO2) into
and Nanosensors microbial cells can be measured (Figure 19.29b). If sulfate reduction
is of interest, the rate of conversion of 35SO42- to H235S can be
In many studies, direct chemical measurements of microbial reac-
assessed (Figure 19.29c). Heterotrophic activities can be measured
tions are sufficient for assessing microbial activity in an environ-
by tracking the release of 14CO2 from 14C-labeled organic com-
ment. For example, the fate of lactate oxidation by sulfate-reducing
pounds (Figure 19.29d), and so on.
bacteria in a sediment sample can be tracked easily. If sulfate-reducing
Both isotopic and chemical methods are widely used in microbial
bacteria are present and active in a sediment sample, then lactate
ecology. To be valid, however, these must employ proper controls
added to the sediment will be consumed and SO42- will be reduced
because some isotopic transformations might be due to abiotic pro-
to hydrogen sulfide (H2S). Since lactate, SO42-, and H2S can all be
cesses. The killed cell control is an essential control in such experi-
measured specifically and with high sensitivity by chemical assay,
ments. That is, it is essential to show that the transformation being
the transformations of these substances relative to one another in a
measured stops when chemical agents or heat treatments that kill
sample can easily be followed (Figure 19.29a). However, when very
microorganisms are applied to the sample. Formalin at a final con-
high sensitivity is required, or when turnover rates need to be deter-
centration of 4% is commonly used as a chemical sterilant in micro-
mined, radioisotopes are more useful than strictly chemical assays.
bial ecology studies. This kills all cells, and transformations that are
Another approach to the sensitive measurement of certain micro-
insensitive to the presence of 4% formalin can be ascribed to abiotic
bial process rates takes advantage of selective chemical inhibition of
processes (Figure 19.29a). Additional controls are also necessary. For
an enzyme specific to the process. For example, a widely employed
example, if H2-driven sulfate reduction is to be measured in a bottle,
chemical inhibitor uses acetylene to inhibit some processes, such as
an essential control is to have a separate bottle identical in every way
nitrification, or as an alternative substrate in another, such as nitro-
except that it lacks H2 (Figure 19.29c). If photosynthesis is to be
gen fixation. The final set of tools we will describe in this section are
measured, a dark control is essential (Figure 19.29b), and so on. The
different types of microsensors and nanosensors used to measure an
art of doing good experimental science is in designing experiments
active microbial process by quantifying changes in the concentration
with the correct controls, and this is especially true of bulk assays of
of specific chemicals in time and space.
microbial activity where particular subsets of the entire microbial
community are targeted.
Radioisotopes
When very high sensitivity is required, or turnover rates need to be
Acetylene as an Inhibitor and an Alternative Substrate
determined, or the fate of portions of a molecule needs to be
In some activity measurements, it is useful to inhibit the activities of
certain organisms in order to focus on the activities of others. For exam-
Sulfate reduction Photosynthesis ple, the short-chain hydrocarbon acetylene (HC ‚ CH) is an inhibitor
2 incorporation

of several metabolisms, including nitrification (◀ Section 14.9), deni-

5
UNIT

Formalin-killed control
Lactate or H2S

Light trification (◀ Section 14.11), and methanogenesis (◀ Section 14.15).

H 2S In nitrification, acetylene inhibits the first step, ammonia oxidation
to nitrite, while in denitrification, acetylene inhibits the terminal
step, the reduction of nitrous oxide (N2O) to N2. Thus, acetylene
14CO

Killed can be used to focus on particular steps in these processes with the
Lactate Dark
knowledge that the inhibited steps are not occurring. For example,
Time Time the use of acetylene to block reduction of N2O is a common assay
(a) (b) for denitrification, using the production rate of N2O (an easily
assayed gas) in the presence of acetylene as a measure of the deni-
Sulfate reduction 14
C-Glucose respiration trification rate in an environmental sample. Acetylene and other
metabolic inhibitors are useful in microbial ecology but must be
CO2 evolution

H2 present used with the caveat that the inhibitor might also affect some unrec-
H235S

ognized metabolic process that contributes to the activity being

measured.
H2 absent Acetylene is also commonly used for another purpose. The capac-
14

Killed
ity for nitrogen fixation is widely distributed among Bacteria and
Killed
Archaea (Table 19.4; ◀ Section 3.12). Nitrogenases are not specific for
Time Time N2 and also reduce other triply bonded compounds, such as acety-
(c) (d) lene, as alternative substrates. Unlike the reduction of N2 to 2 NH3
—a six-electron process—the reduction of acetylene by nitrogenase
Figure 19.29 Microbial activity measurements. (a) Chemical measurements of lac-
tate and H2S transformations during SO42- reduction. Radioisotopic measurements:
is only a two-electron process, ethylene (H2C “ CH2) being the final
(b) photosynthesis measured with 14CO2; (c) SO42- reduction measured with 35SO42- product. Hence, the reduction of acetylene to ethylene provides a
conversion to H235S. (d) production of 14CO2 from 14C-glucose. straightforward method for measuring nitrogenase activity
 CHAPTER 19 • Taking the Measure of Microbial Systems 675

activity of nitrogenase—reduction of a triple bond—provides high

TABLE 19.4 Some nitrogen-fixing organismsa confidence that acetylene reduction to ethylene measured in a natu-
Free-living aerobes
ral sample is due to the activity of nitrogen-fixing bacteria.

Chemoorganotrophs Phototrophs Chemolithotrophs

Microsensors
Azotobacter Cyanobacteria (e.g., Alcaligenes
Microsensors in the form of glass needles containing a sensing
Azomonas Anabaena, Nostoc, Acidithiobacillus
Azospirillum Gloeothece, mechanism at the tip have been used to study the activity of micro-
Klebsiellab Aphanizomenon) organisms in nature. Microsensors have been constructed that mea-
Methylomonas sure many chemical species including pH, O2, NO2-, NO3-, nitrous
Free-living anaerobes oxide (N2O), CO2, H2, and H2S. As the name microsensor implies,
Chemoorganotrophs Phototrophs Chemolithotrophsc these devices are very small, their tips ranging in diameter from 2 to
Clostridium Purple bacteria (e.g., Methanosarcina 100 mm (Figure 19.31). The sensors are carefully inserted into the habi-
Desulfotomaculum Chromatium, Methanocaldococcus tat in small increments to follow microbial activities over very short
Methanococcus, distances.
Rhodobacter)
Microsensors have many applications. For example, O2 concen-
Green sulfur bacteria
(e.g., Chlorobium) trations in microbial mats ( ▶ Figure 20.8c), aquatic sediments, or
Heliobacteria soil particles ( ▶ Figure 20.3) can be accurately measured over
Symbiotic extremely fine intervals using microsensors. A micromanipulator is
With leguminous plants With nonleguminous plants used to insert the sensors gradually through the sample such that
measurements can be taken every 50–100 mm (Figure 19.32). Using
Soybeans, peas, clover, Alder, bayberry, autumn olive, many other
etc. with Rhizobium, bushy plants, with the actinomycete Frankia a bank of microsensors, each sensitive to a different chemical,
Bradyrhizobium, simultaneous measurements of several transformations in a habitat
Sinorhizobium can be made.
a
Only some common genera are listed in each category; many other genera are known. Except Microbial processes in the sea are extensively studied because they
where indicated, all organisms are Bacteria.
b
Nitrogen fixation occurs only under anoxic conditions.
have a profound impact on nutrient cycles and the overall health of
c
All are Archaea. the planet. As it is difficult to reproduce in the laboratory the condi-
tions found at great depths, it is useful to use microsensors on
robotic devices to analyze microbial activities on the seafloor.
(Figure 19.30). This technique, known as the acetylene reduction assay,
Figure 19.33 shows deployment of an instrument “lander” equipped
is widely used in microbiology to detect and quantify nitrogen fixa-
with a suite of microsensors to detect various chemicals in the sedi-
tion. Nitrogen fixation can also be assayed directly using the stable
ment. These data can then be analyzed and compared with that in
(nonradioactive) isotope 15N2 and measuring its reduction to
15 overlying ocean water to explore the exchange of nutrients and other
NH3; however, acetylene reduction is a more rapid and sensitive
chemicals between water and sediment.
5

UNIT
method for measuring N2 fixation and can easily be used in labora-
One of the biologically most important chemical species in the
tory studies of pure cultures or ecological studies of nitrogen-fixing
oceans is nitrate (NO3-), but electrochemical sensors cannot mea-
bacteria directly in their habitat. To do this, a sample, which may be
sure NO3- in seawater, as the high concentrations of salts interfere.
soil, water, or a culture, is incubated in a vessel with HC ‚ CH and
To circumvent this problem, a “living” microsensor was designed
the gas phase is later analyzed by gas chromatography for the pro-
duction of H2C “ CH2 (Figure 19.30). The unique enzymatic

Atmosphere, 10% C2H2 in air (aerobes) or

10% C2H2 in N2 or argon (anaerobes) Chart recorder for
gas chromatograph

( )
2H
HC CH H2C CH2
C2H2 C2H2
Acetylene Ethylene C2H2
C2H4
(C2H2) (C2H4)
C2H4
Nitrogenase

Incubation Sample headspace Time 0 1h 2h

periodically and inject
into gas chromatograph.
Stoppered vial containing
cell suspension

Figure 19.30 The acetylene reduction assay of nitrogenase activity in nitrogen-fixing bacteria. The results show no ethylene (C2H4)
at time 0 but increasing production of C2H4 as the assay proceeds. As C2H4 is produced, a corresponding amount of C2H2 is consumed.
Because the reduction of N2 to 2 NH3 requires 6 e- and the reduction of C2H2 to C2H4 only 2 e-, each three C2H4 produced is equivalent
to the reduction of one N2.
 676 UNIT 5 • MICROBIAL ECOLOGY AND ENVIRONMENTAL MICROBIOLOGY

Membranes Glass

Gold
Silicone rubber cathode Glass Platinum
Electrolyte
50–100 om NO3– N2O 2e– + N2O + H2O N2 + 2 OH–

4 om Bacteria Gold cathode Nutrient solution

(a) (b)

Figure 19.31 Microsensors. (a) Schematic drawing of an oxygen (O2) microsensor. Oxygen diffuses through the silicone membrane in the
microsensor tip and reacts with electrons on the gold surface of the cathode, forming hydroxide ions (OH-); the latter generates a current
proportional to the O2 concentration in the sample. Note the scale of the electrode. (b) Biological microsensor for the detection of nitrate
(NO3 -). Bacteria immobilized at the sensor tip denitrify NO3 - or NO2 - to N2O, which is detected by electrochemical reduction to N2 at the
cathode. Based on drawings by Niels Peter Revsbech.

that contains bacteria within its tip that reduce NO3- (or NO2-) to Sensor Nanoparticles for Spatially
N2O. The N2O produced by the bacteria is then detected following Resolved Chemical Imaging
its abiotic reduction to N2 at the cathode of the microsensor (Figure
An entirely different sensor approach to measuring biologically sig-
19.31b); this provides an electrical impulse signaling the presence
nificant processes in the environment uses nanotechnology. Tradi-
of NO3-. In the oxic layer of marine sediments, NO3- is produced
tional microsensors only generate a single point measurement, and
from the oxidation of NH4+ (nitrification, ◀ Section 14.9), so there
because of this, multiple measurements must be made, for example,
is often a peak of NO3- in the sediment surface layer (Figure 19.32).
In the deeper, anoxic layers of the sediment, NO3- is consumed by
denitrification and dissimilative nitrate reduction to ammonia
(DNRA) (◀ Section 14.11), and NO3- therefore disappears a few
millimeters below the oxic–anoxic interface (Figure 19.32).

Oxygen (O2) concentration (oM)

0 100 200 300

5
UNIT

Seawater
Denitrification Nitrification

0
Depth in sediment (mm)

O2 NO3–

Oxic sediment

5
and DNRA

Anoxic sediment

10
Niels Peter Revsbech

0 4 8 12
Nitrate (NO3–) concentration (oM)

Figure 19.32 Depth profiles of O2 and NO3 -. Data obtained using the lander shown
in Figure 19.33 equipped with microelectrode sensors for remote chemical charac-
terization of deep-sea sediments. Note the zones of nitrification and denitrification
Figure 19.33 Deployment of a deep-sea lander. The lander is equipped with a bank
and the fine scale resolution. DNRA, dissimilative nitrate (NO3 -) reduction to
of microsensors (arrow) to measure distribution of chemicals in marine sediments.
ammonia (NH4 +). Based on data and drawings by Niels Peter Revsbech.
 CHAPTER 19 • Taking the Measure of Microbial Systems 677

when measuring depth-related changes in O2 concentration in a distribution of O2 concentration was determined by taking fluores-
marine sediment (Figure 19.32). Hence, this approach is essentially cent images of the coral under different levels of illumination, where
unusable for examining chemical gradients in more than one oxygen levels can be seen to change quickly (Figure 19.34). When
dimension, such as on an uneven biological surface. To explore such combined with nanosensors that respond to temperature differ-
microbial habitats, sensor nanoparticles have been developed that can ences, similar experiments should also be possible to measure coral
be sprayed in a thin layer over the uneven surface. These tiny parti- responses to increasing ocean temperatures, an environmental vari-
cles (from a few to several hundred nanometers in diameter) made able thought to be a major trigger of the massive coral die-offs seen
from silica or synthetic polymers incorporate fluorescent or phos- in recent years. By establishing links between coral structural orga-
phorescent compounds that have a concentration-dependent lumi- nization, light, temperature, and chemical microenvironments,
nescent response to specific chemicals. Biological nanosensors have these analyses should yield important insights into factors that con-
been developed for assaying temperature, pH, O2, and a few ions of trol the coral symbiosis and allow for more accurate predictions of
biological importance, such as Ca2 + and K + . how this important marine symbiosis will respond to climate
A study to measure changing O2 distribution over a coral surface change.
demonstrates the power of sensor nanotechnology (Figure 19.34). As Other applications of nanosensor technology have been deployed
we will learn in Chapter 23, stony corals are marine animals whose or are on the horizon. For example, nanoparticles sensitive to both
cells contain the symbiotic dinoflagellate Symbiodinium, an alga that O2 and pH have been used to examine the delivery of oxygen to the
nourishes the coral cells by carrying out photosynthesis. However, root systems of the seagrass Zostera marina. The observation of
Symbiodinium is extremely sensitive to changes in temperature and reduced pH and elevated oxygen close to the seagrass roots was
irradiation associated with climate change. When growth conditions attributed to oxygen delivered by the plant to support the growth of
become unfavorable, the symbiont can be expelled and the coral sulfur-oxidizing chemolithotrophs (◀ Sections 14.7 and 15.12),
bleached, and this has resulted in the extensive loss of living coral bacteria that oxidize the toxic hydrogen sulfide (H2S); the resulting
reefs worldwide in recent years ( ▶ Figure 23.41). To document these H2SO4 acidifies the roots, lowering the pH.
changes experimentally, ecological studies of oxygen-producing Although ecological applications of nanotechnology are still in
coral tissues were undertaken to measure the response of the sym- their infancy, it is likely that in coming years more chemically
biotic alga to changes in light intensity. To do this, a coral surface diverse sensor particles will be developed and their use will become
was covered with a thin layer of nanoparticles containing a fluores- more widespread because of their unique ability to track gradients
cent indicator for oxygen. Following calibration using an inert fluo- of microbial activity along uneven surfaces.
rescent reference dye also incorporated in the particles, the spatial
Check Your Understanding
(a) (b) (c)
• How can a microbial ecologist be
sure that transformation of a radio-
Dark isotope is actually caused by
0 microbes?
5

UNIT
• If a large pulse of organic matter
entered the sediment, how would
that change the profiles of NO3- and
Philippe Laissue

O2 shown in Figure 19.32?

O2 (%)

• Suggest additional applications of

10
Light sensor nanoparticles in environmental
20
18 Polyp mouth microbiology.
16
O2 (%)

14
12
10
19.10 Stable Isotopes and
30
8
6
Stable Isotope Probing
0.0 0.2 0.4 0.6 0.8 1 1.2 1.4 Many of the chemical elements have more
Distance (mm) than one isotope, which differ in their
number of neutrons. Certain isotopes are
Figure 19.34 Oxygen nanosensor analysis of coral photosynthetic activity. (a) Calibration of nanosensor response to
unstable and break down as a result of
different dissolved oxygen concentrations using a fragment of a coral skeleton painted with the nanosensor octaethyl-
porphine ketone platinum (II). (b) Living coral response to transition from darkness (top panel) to light (bottom panel).
radioactive decay. Others, called stable iso-
Note how the oxygen-depleted region due to coral respiration in the dark panel (arrow) quickly becomes oxygenated topes, are not radioactive, but are metabo-
in the light. (c) Upper panel, photomicrograph of a coral polyp showing the polyp mouth (arrow). The red color is not lized differently by microorganisms and
from nanoparticles but is due to autofluorescence of chlorophyll in Symbiodinium cells present in the polyp and sur- can be used to study microbial transfor-
rounding coral tissue. Lower panel, high-resolution profile from nanosensor data of an oxygen gradient across a polyp mations in nature. There are two methods
similar to that shown in the upper panel following transition to darkness. The scale on the abscissa indicates the small in which stable isotopes can yield infor-
size of a polyp. The figure data and components in parts a and b were generated by Dr. Klaus Koren (Department of
mation on microbial activities, isotopic
Bioscience, University of Aarhus) and Dr. Michael Kühl (Department of Biology, University of Copenhagen) with techni-
cal assistance from Sofie Lindegaard Jakobsen (Department of Biology, University of Copenhagen).
fractionation and stable isotope probing.
 678 UNIT 5 • MICROBIAL ECOLOGY AND ENVIRONMENTAL MICROBIOLOGY

Enzyme substrates
Enzyme that
Fixed carbon Use of Isotopic Fractionation in Microbial Ecology
fixes CO2 The isotopic composition of a material can reveal its biological or
geological past. For example, plant material and petroleum (which
12CO is derived from plant material) have similar isotopic compositions
2
12C (Figure 19.36). Carbon from both plants and petroleum is isotopi-
organic
cally lighter than the CO2 from which it was formed because the
biochemical pathway used to fix CO2 discriminated against 13CO2
13CO 13C (Figures 19.35 and 19.36). Moreover, methane (CH4) produced by
2 organic
methanogenic Archaea (◀ Section 17.2) is isotopically extremely
light, indicating that methanogens discriminate strongly against
Figure 19.35 Mechanism of isotopic fractionation with C as an example. En- 13
zymes that fix CO2 preferentially fix the lighter isotope (12C). This results in fixed CO2 when they reduce CO2 to CH4 (◀ Section 14.15). By contrast,
carbon being enriched in 12C and depleted in 13C relative to substrate CO2. The carbon in isotopically heavier marine carbonates is clearly of geo-
size of the arrows indicates the relative abundance of each isotope of carbon. logical origin (Figure 19.36).
Because of the differences in the proportion of 12C and 13C in
carbon of biological versus geological origin, the 13C> 12C ratio of
Isotopic Fractionation rocks of different ages has been used as evidence for or against past
The two elements most useful for stable isotope studies in microbial biological activity in Earth’s ancient environments. Organic C in
ecology are carbon (C) and sulfur (S), although the heavy isotope rocks as old as 3.5 billion years shows evidence of isotopic fraction-
of nitrogen, 15N, is also widely used. Carbon (C) exists in nature ation (Figure 19.36), supporting the idea that autotrophic life
primarily as 12C, but about 5% exists as 13C. Likewise, S with its four existed at this time. Indeed, we now believe that the first life on
stable isotopes exists primarily as 32S. Some S is found as 34S, and Earth appeared somewhat before this, about 3.8–3.9 billion years
very small amounts as 33S and 36S. The relative abundance of these ago (◀ Sections 1.5 and 13.1).
isotopes changes when certain C or S compounds are metabolized The activity of sulfate-reducing bacteria is easy to recognize from
by microorganisms because the enzymes that act on these com- their fractionation of stable S isotopes in sulfides (Figure 19.37). As
pounds typically favor the lighter isotope of C or S. That is, relative compared with an H2S standard, sedimentary H2S is highly enriched
to the lighter isotope, the heavier isotope is discriminated against in 32S (depleted in 34S, Figure 19.37). Fractionation during sulfate
when both are metabolized by an enzyme (Figure 19.35). reduction allows one to identify biologically produced S and has
For example, when CO2 is fixed into cell material by an autotro- been widely used to trace the activities of sulfur-cycling Bacteria and
phic organism, the cellular C becomes enriched in 12C and depleted Archaea through geological time. Sulfur isotopic analyses have also
in 13C, relative to an inorganic carbon standard of known isotopic been used as evidence for the lack of life on the Moon. For example,
composition. Likewise, the S atom in H2S produced from the bacte- the data in Figure 19.37 show that the isotopic composition of
rial reduction of SO42- is isotopically lighter than H2S that has
5 formed geochemically. These discriminations are called isotopic
UNIT

fractionations (Figure 19.35) and are typically the result of biologi-

cal activities. Thus this technique can be used as a measure of Marine carbonate
whether a particular transformation has been catalyzed by
Atmospheric CO2
microorganisms.
The isotopic fractionation of C in a sample is calculated as the Calvin cycle plants
extent of 13C depletion relative to a standard having an isotopic
composition of geological origin. The standard for C isotope anal- Petroleum
Methane
ysis is rocks from a Cretaceous (65- to 150-million-year-old) lime-
stone formation (the Pee Dee belemnite). Because the magnitude
of fractionation is usually very small, depletion is calculated as Cyanobacteria
“per mil” (‰, or parts per thousand) and reported as the d13C
(pronounced “delta C 13”) of a sample using the following Purple sulfur bacteria
formula:
Green sulfur bacteria
(13C> 12C sample) - (13C> 12C standard)
d C =
13
* 1000‰ Recent marine sediments
(13C> 12C standard)
3.5-billion-year-old rocks
The same formula is used to calculate the fractionation of S isotopes,
in this case using iron sulfide (FeS) mineral from the Canyon Diablo
–80 –70 –60 –50 –40 –30 –20 –10 0 +10
meteorite as the standard:
f 13 0
C ( /00)
(34S> 32S sample) - (34S> 32S standard)
d34S = * 1000‰ Figure 19.36 Isotopic geochemistry of C and 12C. Note that C fixed by autotrophic
13
(34S> 32S standard)
organisms is enriched in 12C and depleted in 13C. Methane formed from the reduction
of CO2 with H2 by methanogenic Archaea shows extreme isotopic fractionation.
 CHAPTER 19 • Taking the Measure of Microbial Systems 679

organisms) that metabolized the benzoate (Figure 19.38). Thus,

Igneous rocks although all DNA from the sample would be isolated, the 13C-DNA
Marine is heavier, albeit only slightly heavier, than 12C-DNA, and this dif-
sulfate ference is sufficient to separate the DNAs by a special type of cen-
Sedimentary sulfide trifugation technique (Figure 19.38). Once the 13C-DNA is isolated, it
can be analyzed for phylogenetic genes or metabolic genes to yield
Meteoritic sulfide genomic information about the benzoate degrader(s) in the sample.
If instead of an organic compound, the experimental study focused on
Lunar sulfides
nitrogen fixation (conversion of N2 to cell nitrogen, ◀ Section 3.12),
15
Sulfide from marine mud N2 could be supplied to a sample. When nitrogen fixers in the
sample incorporate this, they will produce slightly heavier DNA
Elemental
sulfur than organisms that cannot fix nitrogen, and the heavier DNA can
be isolated and analyzed for genes of interest.
–30 –20 –10 0 10 20 30 SIP can also be used in combination with metagenomics to pin-
point organisms carrying out a specific metabolism (from SIP
f 34S (0/00)
results) in the context of all the other species and metabolisms pres-
Figure 19.37 Isotopic geochemistry of 34S and 32S. Note that H2S and S0 of bio- ent in the sample (as revealed by metagenomic results). Besides a
genic origin are enriched in 32S and depleted in 34S. phylogenetic “hit” from the SIP results, additional genomic analyses
of the labeled DNA could reveal functional genes required for the
specific metabolism. Moreover, the phylogenetic and functional
sulfides in lunar rocks closely approximates that of the H2S stan-
results from the SIP experiment could be further confirmed from the
dard, which represents primordial Earth, and differs from that of
metagenomic profile. Clearly many opportunities exist for blending
microbially produced H2S.
SIP and metagenomics to study important ecological problems.
Stable Isotope Probing
Check Your Understanding
Beyond stable isotope fractionation, an alternative stable isotope
• What is the simplest explanation for why lunar sulfides are
method called stable isotope probing (SIP) can be used to iden-
isotopically similar to those of the primordial Earth?
tify an organism or organisms carrying out the transformation of a
• What is the expected isotopic composition of carbon in
nutrient labeled with a specific stable heavy isotope, such as 13C or
15 methanotrophs (bacteria that consume CH4)?
N or even 18O (the lighter, more common isotopes of these ele-
ments are 12C, 14N, and 16O, respectively). The idea behind SIP is • How might exchange of metabolites among members of a
microbial community complicate interpretation of a stable
that the label will be selectively incorporated only into the cellular
isotope probe experiment?
material of organisms actively metabolizing the nutrient. Then, fol-
5

UNIT
lowing the isolation and sequencing of isotopically labeled DNA,
the organisms carrying out the transformation can be identified.
Stable isotope probing can be used to ask general to more specific 19.11 Linking Functions to Specific Organisms
questions. For example, if 13C-labeled benzoate were added to a The isotopic methods described thus far used samples containing
sediment sample and the sample incubated for an appropriate large numbers of cells to infer that specific metabolisms were occur-
period, the 13C label would end up in the DNA of the organism (or ring within a community or in particular species within the

13
C-DNA
This cell
metabolizes
13
C substrate
12
C-DNA

Feed 13C Extract Separate Remove and

substrate. DNA. light (12C) 13
C-DNA analyze (PCR
from heavy 16S rRNA or
Colin Murrell

Environmental These cells do not (13C) DNA. metabolic genes,

sample metabolize 13C or do genomics).
12
substrate C-DNA
Ultracentrifuge
tube with DNA

Figure 19.38 Stable isotope probing. The microbial community in an environmental sample is fed a 13C substrate. Organisms that can
metabolize the substrate produce 13C-DNA as they grow and divide; 13C-DNA can be separated from lighter 12C-DNA by density gradient
centrifugation (photo). The isolated DNA is then subjected to specific gene analysis or entire genomic analysis.
 680 UNIT 5 • MICROBIAL ECOLOGY AND ENVIRONMENTAL MICROBIOLOGY

community. These methods give an overview of community activi- is directed such that a two-dimensional image of the distribution of
ties but do not reveal the contribution of individual cells. To do this, specific ions on the sample surface is obtained. In addition, by
new methods have been developed that can measure the activity and focusing the ion beam on the same spot during repeated cycles of
the elemental and isotopic composition of single cells. These are sputtering, material can be slowly burned away to expose deeper
powerful methods for connecting cells of a specific microbial popu- regions of the sample. This high-resolution SIMS analysis is where
lation with a specific activity or ecological niche, but in most cases, the term NanoSIMS got its name. NanoSIMS instruments have mul-
the phylogeny of the organisms of interest must be known in order tiple detectors that provide for the simultaneous analysis of ions of
to develop the necessary FISH probes (Section 19.5). Single-cell different mass-to-charge ratios originating from the same sample
methods have been of particular importance to the study of metabo- location (Figure 19.39a).
lite exchange among microorganisms, such as exchanges involved When NanoSIMS is combined with FISH (Section 19.5) in a tech-
in syntrophic relationships (◀ Section 14.22), where close physical nology called FISH-SIMS, the incorporation of different elements,
association is essential for efficient exchange. natural isotopes, or isotope-labeled substrates can be tracked into
individual cells of specific cell populations. A variation on the FISH-
Single-Cell Metabolisms Imaged by Secondary SIMS method that simplifies the identification of cells scanned by
Ion Mass Spectrometry (SIMS) NanoSIMS uses probe-conferred deposition of a halide (Br, F, I),
Secondary ion mass spectrometry (SIMS) is based on the detection of either through direct incorporation of the halide into an oligonucle-
ions released from a sample placed under a focused high-energy otide probe or by using a halide-containing tyramide substrate (see
primary ion beam, for example, of cesium (Cs+); from the data gen- CARD-FISH, Section 19.5). Halogens possess a high ionization yield
erated, the elemental and isotopic composition of released materials compared with other elements and are thus easy to detect and are
can be obtained. When the primary ion beam impacts the sample, typically of low natural abundance. Using this technology, one of
most chemical bonds are broken and atoms or polyatomic frag- the NanoSIMS detectors is dedicated to identifying cells to which
ments are ejected from a very thin layer (1–2 nm) of the surface as the probe has hybridized (Figure 19.39d) by halogen ionization
either neutral or charged particles (secondary ions), a process called while the remaining detectors are used for assessing elemental com-
sputtering. These secondary ions are directed to a mass spectrometer, position (Figure 19.39c).
an instrument that can determine their mass-to-charge ratio. Because of its excellent spatial resolution, NanoSIMS technology
NanoSIMS instruments are SIMS devices designed to yield infor- is being increasingly used to examine metabolite transfer among
mation on single cells. The instrument is equipped with Cs+ and O2 single cells of interacting microorganisms. For example, labeling
primary beam sources with a resolution of 50 nm for the Cs+ ion with 15N2 followed by NanoSIMS was used to demonstrate the trans-
beam and 200 nm for the O2 beam. The O2 beam generates positive fer of N2 fixed by filamentous cyanobacteria to attached heterotro-
secondary ions and is used to analyze metals (e.g., Fe, Na, Mg) while phic bacteria (Figure 19.39c). Labeling with 15NH4 and 13C-labeled
the Cs+ beam generates negative secondary ions for the analysis of CO2 or organic substrates is also being used to explore the assimila-
major cellular elements (C, N, P, S, O, H) and halogens. The Nano- tion of key nutrients and the transfer of metabolites among micro-
bial species in both aquatic and soil microbial communities.
5
UNIT

SIMS instrument also records where on the specimen the ion beam

Mass spectrometer Although nitrogen is fixed in the heterocyst, this

Ion Magnet cell is terminally differentiated and nongrowing.
source Thus, only the epibiont and adjacent vegetative
cell (white arrows) are 15N labeled.

High
Primary Secondary
ions ions A B C D E
N or 19F enrichment

Multiple detectors
Jennifer Pett-Ridge and Peter K. Weber

Jennifer Pett-Ridge and Peter K. Weber

Jennifer Pett-Ridge and Peter K. Weber

15

Specimen
stage Heterocyst Low
(a) (b) (c) 15 (d) 19
N F

Figure 19.39 NanoSIMS technology. (a) Sche- cyanobacterium (Anabaena) to a Rhizobium spe- allows for simultaneous fluorescence and halogen
matic of NanoSIMS operation showing the beams of cies attached to the cyanobacterial heterocyst. The labeling) and NanoSIMS. (b) Total 12C abundance
primary (red) and secondary (blue) ions and five dif- coculture was incubated with 15N2, and the transfer shown by gray tones. (c) 15N enrichment. (d) 19F
ferent detectors, each of which identifies ions of a of 15N-labeled compounds from Anabaena to Rhizo- abundance conferred by a probe that hybridizes
different mass-to-charge ratio. (b–d) Demonstration bium was imaged using a combination of enhanced only to the attached rhizobial cells (EL-FISH).
of interspecies nutrient transfer from a filamentous elemental labeling–FISH (EL-FISH, a technique that
 CHAPTER 19 • Taking the Measure of Microbial Systems 681

of interest can be further manipulated, for example, through capture

Eva Heinz and Matthius Horn by laser tweezers (Section 19.3). In the example of Raman shown in
Figure 19.40, the incorporation of phenylalanine (an amino acid)
13C
by an obligate chlamydial symbiont of an amoeba has been tracked
Phenylalanine 967 cm–1
following addition of phenylalanine labeled with 15N and 13C to the
Unsaturated amoeba growth medium. A less specific but still useful application
Raman intensity (arbitrary units)

lipids 1664
Protein of Raman is based on the incorporation of heavy protons from deu-
833 terated water (2H2O). Since water is a reactant in many biochemical
* reactions, metabolically active microorganisms that incorporate the
heavy hydrogen atom can be identified by Raman microspectros-
120 h
copy for further processing, including characterization by single-cell
genomics (◀ Section 10.11).
Nucleic acids 781
Radioisotopes in Combination with FISH:
72 h
Microautoradiography-FISH
Carbohydrates ~400–580 Radioisotopes are used as measures of microbial activity in a micro-
scopic technique called microautoradiography (MAR). In this
0h
method, cells from a microbial community are exposed to a sub-
Carbohydrates ~1030–1090
strate containing a radioisotope, such as an organic compound or
400 600 800 1000 1200 1400 1600 1800 CO2. Heterotrophs take up the radioactive organic compounds and
–1
Raman shift wave number (cm )

Figure 19.40 Raman microspectroscopic analysis of single cells. A cell of

Michael Wagner, Kilian Stöcker, and Holger Daims

Acanthamoeba containing FISH-stained chlamydial symbionts (inset photo, arrows
point to blue chlamydial cells) and Raman spectroscopy of isolated chlamydial cells.
After incubation for 72 and 120 h in medium containing 13C phenylalanine, symbionts
were released by lysis of the amoeba and their Raman spectra recorded. Peaks diag-
nostic for labeled phenylalanine are shown in red. Since the Raman wave number of
13
C phenylalanine at 967 cm-1 is well resolved from other spectral features, the ratio of
1003 cm-1 (unlabeled phenylalanine, at *) to 967 cm-1 (labeled phenylalanine) peak
areas corresponds to the relative amount of labeled phenylalanine incorporated by
single cells (note how this increases with time). Other red peaks correspond to spec-
tral peaks specific for different chemical bonds in 13C phenylalanine. Also shown are
selected peaks and their wave numbers for other cellular components of the symbiont.
5

UNIT
(a)

Raman Microspectroscopy
Raman microspectroscopy can be used to characterize the molecular
and isotopic composition of single cells by nondestructive illumina-
tion with monochromatic light generated by a laser. Raman is a
form of spectroscopy that measures light scattering and can yield
Michael Wagner, Per Nielsen, and Natuscka Lee

Michael Wagner, Per Nielsen, and Natuscka Lee

both qualitative and quantitative results. Compositional analysis is

based on photon scattering after interaction with different cellular
components. Although most of the scattered photons have the same
energy as the incident photons, a small fraction of scattered photons
are shifted in wavelength (relative to the incident wavelength) to
either a longer wavelength (a phenomenon known as Stokes Raman
scattering) or shorter wavelength (anti-Stokes scattering).
Raman spectrometers separate the more abundant Stokes scat-
tered photons for analysis and, when combined with confocal
microscopy (◀ Section 1.9), can generate a compositional spectrum (b) (c)
of a single microbial cell (Figure 19.40). Although the spectrum is
complex, several compounds and molecules have characteristic Figure 19.41 MAR-FISH. Fluorescence in situ hybridization (FISH) combined
with microautoradiography (MAR). (a) An uncultured filamentous cell belonging to
peaks that can identify specific cell types, physiological states, or
the Gammaproteobacteria (as revealed by FISH) is shown to be an autotroph (as
metabolic activities following incorporation of compounds labeled revealed by MAR-measured uptake of 14CO2). (b) Uptake of 14C-glucose by a mixed
with stable isotopes. culture of Escherichia coli (yellow cells) and Herpetosiphon aurantiacus (filamen-
Major advantages of Raman microspectroscopy include the fol- tous green cells). (c) MAR of the same field of cells shown in part b. The radio-
lowing: It is nondestructive and can be used on living cells; water activity of incorporated glucose exposes the film and shows that glucose was
does not cause interference; it can be combined with FISH; and cells assimilated mainly by cells of E. coli.
 682 UNIT 5 • MICROBIAL ECOLOGY AND ENVIRONMENTAL MICROBIOLOGY

autotrophs take up the radioactive CO2. Following incubation in the counting cells microscopically, and this problem is compounded if
substrate, cells are affixed to a slide and the slide is dipped in pho- populations are present in low numbers. However, flow cytometry
tographic emulsion. While the slide is left in darkness for a period, (Section 19.3) offers an alternative to more labor-intensive micro-
radioactive decay from the incorporated substrate induces forma- scopic methods.
tion of silver grains in the emulsion; these appear as black dots Flow cytometers can examine specific cell parameters such as size,
above and around the cells. Figure 19.41a shows a MAR experiment shape, or fluorescent properties as the cells pass through a detector
in which an autotrophic cell has taken up 14CO2. at rates of many thousands of cells per second (Figure 19.42). Fluo-
Microautoradiography can be done simultaneously with FISH (Sec- rescence may be intrinsic (for example, chlorophyll fluorescence of
tion 19.5) in MAR-FISH, a powerful technique that combines identi- phototrophic microorganisms); or it may be conferred by DNA
fication with activity measurements. MAR-FISH allows a microbial staining, by differential staining of live versus dead cells (vital
ecologist to determine (by MAR) which organisms in a natural sample stains), or by fluorescent DNA probes (FISH or BONCAT-FISH), all
are metabolizing a particular radiolabeled substance while at the same methods discussed earlier in this chapter.
time identifying these organisms (by FISH) (Figure 19.41b, c). MAR- A major advantage of flow cytometry is the ability to carry out
FISH thus goes a step beyond phylogenetic identification by revealing multiparametric analyses, that is, the capacity to combine multiple
physiological information about the organisms, as is also true of parameters in the analysis of a microbiological sample or to sort
NanoSIMS. Such data are useful not only for understanding the activ-
ity of the microbial ecosystem but also for guiding enrichment cul-
tures. For example, knowledge of the phylogeny and morphology of
Cells
an organism metabolizing a particular substrate in a natural sample
labeled
can be used to design an enrichment protocol to isolate the organism. by FISH
In addition, MAR-FISH results can be quantified by counting the silver or BONCAT
grains as a measure of the amount of substrate consumed by single
cells, allowing the activity distribution in a community to be described.
The technique is limited only by the availability of suitable radioactive Sample
isotopes. For example, although C-labeled substrates work well, it is stream
not feasible to track N incorporation using MAR-FISH because the
Light scatter and
radioactive isotope 13N has a very short half-life. However, it is feasible fluorescence detector
to track N incorporation using the nonradioactive stable isotope 15N Nozzle
with NanoSIMS, as we saw earlier (Figure 19.39).
Laser
Check Your Understanding
• How could NanoSIMS be used to identify a nitrogen-fixing
bacterium?
5
UNIT

• Why is Raman microspectroscopy suited for the selective Induces charge on

isolation of microorganisms and NanoSIMS is not? selected droplets
+ –
• How does MAR-FISH link microbial diversity and activity? Deflection
plates

19.12 Linking Genes and Cellular Properties

to Individual Cells
We have seen in the previous section how the combination of FISH
with MAR or FISH with NanoSIMS allows for analyses of both Sorted
samples
microbial diversity and activity. Coupled with advanced DNA
sequencing methods that can determine a genome sequence from
the DNA contained in a single cell (◀ Section 10.11), these tech- Assay for Assay for
niques are at the cutting edge of microbial ecology today. Improve- specific genes specific genes
(e.g., 16S (e.g., 16S
ments in single-cell DNA sequencing technology, combined with rRNA genes Waste rRNA genes
high-throughput analysis and isolation of single cells by flow cytom- or metabolic (nonlabeled cells) or metabolic
etry, now allow for gene identification and selected physiological genes). genes).
analyses (e.g., size and intrinsic fluorescence) to be performed on
Figure 19.42 Flow cytometric cell sorting. As the fluid stream exits the nozzle,
selected populations and single cells in the environment. it is broken into droplets containing no more than a single cell. Droplets containing
desired cell types (detected by fluorescence or light scatter) are charged and col-
Mastering
Microbiology
Flow Cytometry and Multiparametric Analyses lected by redirection into collection tubes or microtiter plates by positively or neg-
Art Activity: Because of the large population sizes of natural microbial communi- atively charged deflection plates. DNA recovered from a specific population of
Figure 19.36 cells or amplified from a single cell (◀ Figure 10.30) is characterized by PCR amplifi-
Flow cytometric ties, methods that rely on microscopy can examine only a very small
cell sorting cation and sequencing of specific genes or by full genome sequence analysis.
part of a whole community. It is difficult to assess cell numbers by
 CHAPTER 19 • Taking the Measure of Microbial Systems 683

cells in order to find a specific population. A good example of this Cells labeled by
was the discovery in the late 1980s of a novel and abundant com- FISH or BONCAT.
munity of marine cyanobacteria, all species of the genus Prochloro-
coccus. Prochlorococcus cells are smaller and have different fluorescent Isolate fluorescent cells
properties than another common marine cyanobacterium, Syn- by flow cytometry (see
Figure 19.42).
echococcus. Based on differences in size and fluorescence, flow
cytometry resolved these two populations and Prochlorococcus was
subsequently shown to be the predominant oxygenic phototroph Extract DNA.
in ocean waters between 408S and 408N latitude, reaching concen- DNA
trations greater than 105 cells>ml. Metagenomics has also been used Gene Genome
to identify the unique genomic features of different natural popula- route route
Perform multiple
tions of Prochlorococcus (Figure 19.24). These findings have led to displacement
the conclusion that Prochlorococcus is the most abundant photo- Use PCR to amplification.
amplify specific
trophic organism on Earth. We discuss the biology of Prochlorococcus genes (16S rRNA Phage
in more detail in Section 20.11. genes, metabolic DNA
genes, etc.). Primers polymerase
Single-Cell Genomics A
A major stumbling block in a PCR-based gene recovery method is 3′ 5′
the requirement that a specific gene that will react with the primers
B 5′
used in the amplification be identified prior to analysis. Newer
3′ 5′
methods of DNA amplification now provide an alternative method
for associating specific genes with a specific organism without the C
problems and biases associated with PCR. These methods employ
single-cell genomics (◀ Section 10.11), one of the more recent tools 5′
to enter the microbial ecologist’s toolbox. As discussed in Chapter 3′ 5′
10, when combined with methods for recovery of single cells and
D
high-throughput DNA sequencing methods, single-cell genome
sequencing provides a powerful tool for linking specific metabolic
Sequence genome. 5′ 5′
functions to individual cells that have never been grown in labora-
5′
tory culture.
3′ 5′
Multiple displacement amplification (MDA) (Figure 19.43) is key
to single-cell genomics because it can amplify chromosomal DNA
Figure 19.43 Genetic analyses of sorted cells. DNA is recovered from a specific
from a single cell isolated from a natural environment using a cell
5

UNIT
population of cells following FISH labeling and flow cytometric sorting (Figure 19.42).
sorting technique, such as flow cytometry (Figure 19.42). MDA uses DNA is characterized by PCR amplification and sequencing of specific genes, or by
a specific bacteriophage DNA polymerase to initiate replication of amplification of the entire genome by multiple displacement amplification (MDA)
cell DNA at random points in the chromosome, displacing the com- followed by sequencing. For MDA, an amount of DNA sufficient for full genome
plementary strand as each polymerase molecule synthesizes new sequence determination is produced using short DNAs of random sequence as
DNA. The phage polymerase has strong strand displacement activity, primers (A) to initiate genome replication by a bacteriophage DNA polymerase. The
resulting in the synthesis of numerous high-molecular-weight DNA bacteriophage polymerase copies DNA from multiple points in the genome and also
displaces newly synthesized DNA (B, C), thereby freeing additional DNA for primer
products at very high fidelity. The number of genome copies pro-
annealing and (D) initiation of polymerization.
duced by MDA is usually sufficient to assemble the complete, or
nearly complete, genome of the cell that yielded the DNA using
next-generation sequencing platforms and powerful sequence analy- develop strategies to recover them by either classical enrichment and
sis software. In this way, both phylogenetic and metabolic functions isolation methods (Sections 19.1 and 19.2) or by any of the several
can be inferred from the genome sequence and PCR is not required. single-cell isolation culturing techniques now available to tease out
PCR can be a problem if there exists a bias in the genome against the individual cells and get them growing in the laboratory (Section
primers used. MDA avoids this by annealing random rather than 19.3 and Figure 19.42).
specific primers to the genomic DNA such that amplification of the
entire genome is highly likely. A Preview of What’s to Come
Not surprisingly, MDA requires stringent control over purity to Now that we have some background on how we can track and iden-
eliminate contaminating DNA, but when combined with high- tify microbes in nature and assess their activities, we will use these Mastering
Microbiology
throughput DNA sequencing methods, MDA provides a powerful tools as we move on to the next chapter to consider where microbes Art Activity:
tool for linking specific metabolic functions to individual cells that actually live in nature. In Chapter 20 we will link microbial diversity Figure 19.37
Genetic analysis
have thus far eluded laboratory culture. Information about the met- with major habitats to answer the questions “Who is where?” and of sorted cells
abolic capacities of these uncultured organisms can then be used to
684 UNIT 5 • MICROBIAL ECOLOGY AND ENVIRONMENTAL MICROBIOLOGY

“Why are they there?” In Chapter 21 we will connect diversity with where we focus on microbe–human relationships, both the highly
metabolism to explore the major nutrient cycles in nature that are beneficial and the life-threatening.
driven by microbial activities. In Chapter 22 we will consider
microbes and their habitats once again but in the context of Check Your Understanding
microbes that reside in our buildings, subways, and other major • Compared with microscopy, what are the advantages and
structures constructed by humans. Chapters 23 and 24 also link disadvantages of flow cytometry for characterizing a microbial
microbes to habitats but in the context of the many symbiotic asso- community?
ciations that microbes have developed with plants and animals, • What key method is required to do genomics on a single cell?
including ourselves. Background on where microbes live in nature
and why is the natural introduction to the final two units in this text

Go to Mastering Microbiology for videos,

animations, practice tests, and more.
CHAPTER REVIEW

I • Culture-Dependent Analyses of Microbial into the environment and reporting gene expression.
In natural samples, morphologically identical cells may
Communities actually be genetically distinct.
19.1 The enrichment culture technique is a means of obtain-
ing microorganisms from natural samples. Successful Q What are dyes that stain DNA used for? Is background
staining a problem when using such dyes?
enrichment and isolation prove that an organism of a 19.5 FISH methods have combined the power of nucleic acid
specific metabolic type was present in the sample, but probes with fluorescent dyes and are thus highly specific in
do not indicate its ecological importance or abundance. their staining properties. FISH methods include phyloge-
Enrichments following dilution of the sample often yield netic stains, CARD-FISH, and BONCAT-FISH. Using
different organisms than enrichments with undiluted fluorescence microscopy, CARD-FISH can identify tran-
samples. scriptionally active cells while BONCAT-FISH can identify

Q What exactly constitutes an enrichment culture? What

should an effective enrichment culture duplicate?
translationally active cells.

19.2 Once a successful enrichment culture has been established, Q Compare and contrast CARD-FISH and BONCAT-FISH.
Why are these methods more suitable than FISH for
characterizing very slowly growing microorganisms in the
pure cultures can often be obtained by conventional micro-
biological procedures, including streak plates, agar dilution, environment?
and liquid dilution methods.
III • Culture-Independent Molecular Analyses
Q Describe how an appropriate inoculum can be used to
successfully start an enrichment culture.
of Microbial Communities
19.3 Several methods are available to isolate and culture single
cells. Laser tweezers allow one to isolate a cell from a 19.6 PCR can be used to amplify specific target genes such as
microscope field and move it away from contaminants. rRNA genes or key metabolic genes for subsequent analy-
Flow cytometric sorting combined with high-throughput sis of community structure and potential functions. DGGE
culturing technology allow for isolated cells to be cultured and T-RFLP can identify the different variants of these genes
in a large variety of culture media simultaneously to identify among the species in a community. Application of ARISA is
the resources and conditions best suited to the growth of the limited to amplification of the internal transcribed spacer
isolated cell. region separating the 16S and 23S rRNA genes.

Q It is thought that every microbe has a fundamental

niche and a realized niche. How does a fundamental Q Which method, ARISA or T-RFLP, would provide more
detail about microbial community complexity? Why?
niche differ from a realized niche? 19.7 In current application, microarrays used for environmental
studies primarily consist of thousands of DNA probes that
II • Culture-Independent Microscopic Analyses of hybridize to specific genes encoding key biochemical
processes.
Microbial Communities
19.4 DAPI, acridine orange, and SYBR Green are general stains for Q Why might a microarray be superior to using high-
throughput sequencing to quantify both abundant and
rare gene sequences in a complex microbial community?
quantifying microorganisms in natural samples. Some stains
can differentiate live versus dead cells. The GFP makes cells What are the limitations of functional microarrays, such as
autofluorescent and is a means for tracking cells introduced the GeoChip, for analysis of microbial communities?
 CHAPTER 19 • Taking the Measure of Microbial Systems 685

19.8 Environmental multi-omics uses different combinations to identify microorganisms metabolizing and assimilating
of metagenomics, metatranscriptomics, metaproteomics, the compound added to a community.
and metabolomics to derive deeper understanding of how
microbial diversity relates to the complex metabolic food
webs sustaining natural microbial communities and asso-
Q Will autotrophic organisms contain more or less 12C in
their organic compounds than was present in the CO2
that fed them? Why would SIP using 15NO3- not be useful
ciated biogeochemical processes. for identifying bacteria carrying out nitrate respiration?

Q Give an example of how multi-omics has contributed to

a better understanding of microbial food web structure.
Why is the use of environmental genomic information alone
19.11 A variety of advanced technologies such as NanoSIMS,
MAR-FISH, BONCAT-FISH, and Raman microspectros-
copy make it possible to examine metabolic activity, gene
often insufficient to discover new biochemical properties?
content, and gene expression of single cells in natural micro-
bial communities. NanoSIMS employs secondary ion mass
IV • Measuring Microbial Activities in Nature spectrometry technology. MAR-FISH combines the uptake
of radiolabeled substrates (MAR) along with phylogenetic
19.9 The activity of microorganisms in natural samples can be identification (FISH). Raman microspectroscopy is a non-
assessed very sensitively using radioisotopes or microsensors, destructive method (retaining cell viability).
or both. The measurements obtained give the net activity of
the microbial community.
Q What can MAR-FISH tell you that FISH alone cannot?
How might you combine SIP and NanoSIMS to identify

Q What are the major advantages of radioisotopic

methods in the study of microbial ecology? What type
of controls (discuss at least two) would you include in a
novel methane-consuming cells in a natural community?
19.12 Flow cytometry combined with cell sorting can rapidly
radioisotopic experiment to show 14CO2 incorporation by evaluate many thousands of single cells in a natural en-
cyanobacteria or to show 14C-glucose incorporation by vironment for basic cellular properties (size, shape) or
anaerobic bacteria? gene content (using specific fluorescent probes). Single-cell
genomics incorporates methods for use on individual cells
19.10 Natural isotopic composition, the result of isotopic from a natural microbial community, for example, by flow
fractionation by enzymes that discriminate against the cytometric isolation of cells and multiple displacement
heavier form of an element, can reveal the biological ori- amplification to obtain genome sequences of single cells.
gin and/or biochemical mechanisms involved in the forma-
tion of various substances. Stable isotope probing (SIP) uses
compounds labeled with isotopes not naturally abundant
Q How would you use cytometric cell sorting to evaluate
genome sequence variation among a population of ma-
rine bacteria present in low abundance?

APPLICATION QUESTIONS

1. Design an experiment for measuring the activity of sulfur- anoxic lake sediments and whether or not it is H2-limited.
oxidizing bacteria in soil. If only certain species of the sulfur Also, determine the morphology of the dominant methanogen
oxidizers present were metabolically active, how could you tell (recall that these are Archaea, ◀ Section 17.2). Finally, calculate
this? How would you prove that your activity measurement what percentage the dominant methanogen is of the total ar-
was due to biological activity? chaeal and total prokaryotic populations in the sediments. Re-
2. You wish to know whether Archaea exist in a lake water sam- member to specify necessary controls.
ple but are unsuccessful in culturing any. Using techniques 4. Design a SIP experiment that would allow you to determine
described in this chapter, how could you determine whether which organisms in a lake water sample were capable of ox-
Archaea exist in the sample, and if they do, what proportion idizing the hydrocarbon hexane (C6H14). Assume that four
of the cells in the lake sample are Archaea? different species could do this. How would you combine SIP
3. Design an experiment to solve the following problem: Determine with other molecular analyses to identify these four species?
the rate of methanogenesis (CO2 + 4 H2 S CH4 + 2 H2O) in

CHAPTER GLOSSARY

Acridine orange a nonspecific fluorescent incorporating a noncanonical amino acid DAPI a nonspecific fluorescent dye that stains
dye used to stain DNA in microbial cells in into their proteins followed by coupling it DNA in microbial cells; used to obtain total
a natural sample to a fluorescent reporter molecule (BON- cell numbers in natural samples
BONCAT-FISH a method to identify trans- CAT stands for bioorthogonal noncanoni- Denaturing gradient gel electrophoresis
lationally active cells labeled by FISH by cal amino acid tagging) (DGGE) an electrophoretic technique
686 UNIT 5 • MICROBIAL ECOLOGY AND ENVIRONMENTAL MICROBIOLOGY

capable of separating nucleic acid frag- High-throughput culturing methods compounds such as O2, H2S, or NO3- that
ments of the same size that differ in base the use of microtiter plates whose wells can be immersed into a microbial habitat
sequence contain various culture media that can be at microscale intervals
Enrichment bias a problem with enrich- inoculated with single cells whose growth or Most-probable-number (MPN) technique
ment cultures in which “weed” species tend target gene content is measured robotically the serial dilution of a natural sample to
to dominate in the enrichment, often to the Isotopic fractionation the discrimination determine the highest dilution yielding
exclusion of the most abundant or ecologi- by enzymes against the heavier isotope of growth
cally significant organisms in the inoculum the various isotopes of C or S, leading to Multi-omics the integration of data from
Enrichment culture a culture that employs enrichment of the lighter isotopes multiple omics platforms to more fully
highly selective laboratory methods for Laser tweezers a device for obtaining pure characterize microbial community struc-
obtaining microorganisms from natural cultures by optically trapping a single cell ture and function
samples with a laser beam and moving it away Multiple displacement amplification
Environmental genomics (metagenomics) from surrounding cells into sterile growth (MDA) a method to generate multiple
the use of genomic methods (sequencing medium copies of chromosomal DNA from a sin-
and analyzing genomes) to characterize MAR-FISH a technique that combines iden- gle organism
natural microbial communities tification of microorganisms by FISH with Nucleic acid probe a strand of nucleic acid
Exometabolite a product of metabolism measurement of metabolic activities by that can be labeled and used to hybridize
released from a cell that may be metabo- microautoradiography (MAR) to a complementary molecule from a mix-
lized by other organisms Metabolomics the comprehensive analysis ture of other nucleic acids
Flow cytometry a technique for counting of cellular and extracellular metabolites of Phylotype one or more organisms with
and examining microscopic particles by sus- a cell, organism, or microbial community the same or related sequences of a phylo-
pending them in a stream of fluid and pass- Metaproteomics the measurement of genetic marker gene
ing them by an electronic detection device whole-community protein expression Realized niche the range of natural envi-
Fluorescence in situ hybridization (FISH) using mass spectrometry to assign peptides ronments supporting a species when that
a method employing a fluorescent dye to the amino acid sequences encoded by organism is confronted with factors such
covalently bonded to a specific nucleic unique genes as resource limitation, predation, and
acid probe for identifying or tracking Metatranscriptomics the measurement of competition from other species
organisms in the environment whole-community gene expression using Stable isotope probing (SIP) a method
Fluorescent protein any of a large group RNA sequencing for characterizing an organism that incor-
of proteins that fluoresce different colors, Microautoradiography (MAR) the mea- porates a particular substrate by supplying
including the green fluorescent protein, for surement of the uptake of radioactive sub- the substrate in 13C or 15N form and then
tracking genetically modified organisms strates by visually observing the cells in an isolating heavy isotope–enriched DNA
and determining conditions that induce exposed photographic emulsion and analyzing the genes
the expression of specific genes Microbial ecology the study of the interac- Winogradsky column a glass column
Fundamental niche the range of environ- tion of microorganisms with each other packed with mud and overlaid with wa-
ments in which a species will be sustained and their environment ter to mimic an aquatic environment, in
when it is not resource-limited, such as Microfluidic devices miniaturized systems which various bacteria develop over a
may result from competition with other for fluid handling that are increasingly period of months
species used for high-throughput culturing of
Green fluorescent protein (GFP) a pro- microorganisms
tein that fluoresces green and is widely Microsensor a small glass sensor or
used in genetic analysis electrode for measuring pH or specific