PowerPoint® Lecture

Presentations prepared by
John Zamora
Middle Tennessee State
University

CHAPTER 12
Microbial
Evolution and
Systematics

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 12.1 Formation and Early History of Earth

• The Earth is ~4.5 billion years

old
• First evidence for microbial life
can be found in rocks ~3.86
billion years old (Figure 12.2)
• Early Earth was anoxic and
much hotter than present day
• First biochemical compounds
were made by abiotic systems
that set the stage for the origin
of life
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 Eventually, lipid bilayers took the place of mineral
compartments, allowing the first cells to disperse
to new habitats.

Subsurface origin hypothesis

• Life originated at hydrothermal
springs on ocean floor Compartments
allow coupling of
energetic
reactions to
• Conditions would have been molecular
replication.
Mound:
precipitates of clay,
metal sulfides, silica,
and carbonates
more stable
Ocean water
(< 20ºC, containing
• Steady and abundant supply Mineral pores form
metals, CO2, and
PO42–)
first biological
compartments.
of energy (e.g., H2 and H2S) Flow of substances
up through mound

may have been available at Amino

acids
Nitrogen
bases

Sugar
these sites s Ocean crust

Nutrients in hot
hydrothermal water

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 12.1 Formation and Early History of Earth

• Prebiotic chemistry of early Earth set stage for

self-replicating systems

• First self-replicating systems may have been

RNA-based (RNA world theory; Figure 12.4)
• RNA can bind small molecules (e.g., ATP, other
nucleotides)

• RNA has catalytic activity; may have catalyzed its own

synthesis

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 RNA world theory
Prebiotic Precellular life Early cellular LUCA Evolutionary
chemistry life diversification

4.3–3.8 bya 3.8–3.7 bya

RNA A
T A
G C
A T

U C G

DNA
G
C

C G G

Protein G C
A U
T A A
C
T
C G
A U
G
T
Bacteria
A U T
C G G
G C C
T A A
G C C
A U T
C G G
T A

mRNA T
C
C G
G Archaea
G G G A G G A G C
U U C C U G G AG
A C G
G A C U G A C C
U G C C T A

C G
A T
G C

HGT between cells

CG

Biological RNA world Protein synthesis DNA Lipid bilayers Divergence of Bacteria and Archaea
building blocks
- Catalytic RNA - RNA-templated - Replication - Cellular - Components of DNA replication,
- Amino acids - Self-replicating RNA translation - Transcription compartments transcription, and translation all
- Nucleosides - Early cells likely in place
- Sugars had high rates
of HGT

© Pearson Education Limited 2015 Figure 12.4

 12.1 Formation and Early History of Earth

• DNA, a more stable molecule, eventually became

the genetic repository

• Three-part systems (DNA, RNA, and protein)

evolved and became universal among cells

• Other important steps in emergence of cellular life

• Buildup of lipids

• Synthesis of phospholipid membrane vesicles that

enclosed the cell's biochemical and replication machinery
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 12.1 Formation and Early History of Earth

• Last universal common ancestor (LUCA)

• Population of early cells from which cellular life may
have diverged into ancestors of modern-day Bacteria
and Archaea

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 12.1 Formation and Early History of Earth

• Because early Earth was anoxic, energy-generating

metabolism of primitive cells was exclusively
anaerobic and likely chemolithotrophic
• Obtained carbon from CO2

• Obtained energy from H2; likely generated by H2S

reacting with H2S or UV light (Figure 12.5)

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 Alternative source of H2
UV Proton motive
force drives energy
conservation.

Primitive
ATPase
Primitive Out
hydrogenase

Cytoplasmic ATP In
membrane

S0 reductase

© Pearson Education Limited 2015 Figure 12.5

 12.1 Formation and Early History of Earth

• Early forms of chemolithotrophic metabolism

would have supported production of large amounts
of organic compounds

• Organic material provided an abundant, diverse,

and continually renewed source of reduced
organic carbon, stimulating evolution of various
chemoorganotrophic metabolisms

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 12.1 Formation and Early History of Earth

• Molecular evidence suggests ancestors of Bacteria

and Archaea diverged ~4 billion years ago

• As lineages diverged, distinct metabolisms

developed

• Development of oxygenic photosynthesis

dramatically changed course of evolution

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 12.2 Photosynthesis and the Oxidation of Earth
Stromatolites
• Fossilized microbial mats of filamentous
prokaryotes and trapped sediment (Figure 12.6)

• Found in rocks 3.5 billion years old or younger

• Comparisons of ancient and modern

stromatolites
• Anoxygenic phototrophic filamentous bacteria
formed ancient stromatolites

• Oxygenic phototrophic cyanobacteria dominate

modern stromatolites
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 12.2 Photosynthesis and the Oxidation of Earth
• ~2.7 billion years ago, cyanobacteria developed a
photosystem that could use H2O instead of H2S,
generating O2

• By 2.4 billion years ago, O2 concentrations raised to 1

part per million; initiation of the Great Oxidation Event

• O2 could not accumulate until it reacted with abundant

reduced materials in the oceans (e.g., FeS, FeS2)
• Banded iron formations: laminated sedimentary rocks;
prominent feature in geological record (Figure 12.8)

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 12.2 Photosynthesis and the Oxidation of Earth

• Development of oxic atmosphere led to evolution

of new metabolic pathways that yielded more
energy than anaerobic metabolisms

• Consequence of O2 for the evolution of life

• Formation of ozone layer that provides a barrier against
UV radiation
• Without this ozone shield, life would have continued only
beneath ocean surface and in protected terrestrial
environments
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 12.3 Endosymbiotic Origin of Eukaryotes

• Oxygen also spurred evolution of organelle-

containing eukaryotic microorganisms
• Oldest eukaryotic microfossils ~2 billion years old

• Fossils of multicellular and more complex eukaryotes

are found in rocks 1.9 to 1.4 billion years old

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 12.3 Endosymbiotic Origin of Eukaryotes

• Endosymbiosis
• Well-supported hypothesis for origin of eukaryotic cells

• Contends that mitochondria and chloroplasts arose from

symbiotic association of prokaryotes within another type
of cell

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 12.3 Endosymbiotic Origin of Eukaryotes

• Two hypotheses exist to explain the formation of

the eukaryotic cell:
1. Eukaryotes began as nucleus-bearing lineage that
later acquired mitochondria and chloroplasts by
endosymbiosis (Figure 12.9a)

2. Eukaryotic cell arose from intracellular association

between O2-consuming bacterium (the symbiont),
which gave rise to mitochondria, and an archaeal
host (Figure 12.9b)
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 Bacteria Eukarya Archaea Bacteria Eukarya Archaea

Animals Plants Animals Plants

Ancestor of Nucleus Ancestor of Nucleus

chloroplast formed chloroplast formed

Ancestor of
mitochondrion
Engulfment of a
H2-producing cell
of Bacteria by a
H2-consuming cell
of Archaea

© Pearson Education Limited 2015 Figure 12.9

 12.3 Endosymbiotic Origin of Eukaryotes

• Both hypotheses suggest eukaryotic cell is chimeric

• This is supported by several features:

• Eukaryotes have lipids and energy metabolisms similar
to those of Bacteria

• Eukaryotes have transcription and translational

machinery most similar to those of Archaea

• Please see Figure 12.10

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 II. Living Fossils: DNA Records the History of
Life
• 12.4 Molecular Phylogeny and the Tree of Life

• 12.5 Molecular Phylogeny: Making Sense of

Molecular Sequences

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 12.4 Molecular Phylogeny and the Tree of Life

• Carl Woese
• Pioneered the use of rRNA for phylogenetic studies in
the 1970s

• Established the presence of three domains of life:

• Bacteria, Archaea, and Eukarya

• Provided a unified phylogenetic framework for Bacteria

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 12.4 Molecular Phylogeny and the Tree of Life

The most widely used rRNAs are small

subunit ribosomal RNA (SSU rRNA) genes
• Found in all domains of life
• 16S rRNA in prokaryotes and 18S rRNA in
eukaryotes

• Functionally constant

• Sufficiently conserved (change slowly)

• Sufficient length

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 12.4 Molecular Phylogeny and the Tree of Life

• Phylogeny
• Evolutionary history of a group of organisms

• Inferred indirectly from nucleotide sequence data

• The universal phylogenetic tree based on SSU

rRNA genes is a genealogy of all life on Earth
(Figure 12.13)

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 ARCHAEA
BACTERIA

Crenarchaeota Thaumarchaeota
Tenericutes
Fusobacteria Firmicutes Euryarchaeota
Gemmatimonadetes Actinobacteria

Lentisphaerae Bacteroidetes
Korarchaeota
Fibrobacteres Acidobacteria

Verrucomicrobia Nitrospira Nanoarchaeota

Chlamydiae
Gammaproteobacteria EUKARYA
Betaproteobacteria
Alphaproteobacteria
Planctomycetes Plants
Mitochondria
Cyanobacteria Deltaproteobacteria Cercozoans
Epsilonproteobacteria
Plastids Stramenopiles

Alveolates
Chlorobi
Parabasalids
Spirochaetes
Thermodesulfobacteria Diplomonads
Thermotogae
Chloroflexi Aquificae Euglenozoa
Deinococcus– Amoebozoa
Thermus
Fungi

LUCA Animals

Origin of life

© Pearson Education Limited 2015 Figure 12.13

© Pearson Education Limited 2015
 Figure 1 | A current view of
the tree of life, encompassing
the total diversity represented
by sequenced genomes. The
tree includes 92 named
bacterial phyla, 26 archaeal
phyla and all five of the
Eukaryotic supergroups..
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 12.4 Molecular Phylogeny and the Tree of Life

• Domain Bacteria
• Contains at least 90 major evolutionary groups (phyla)

• Many groups are defined from environmental

sequences alone—i.e., there are no cultured
representatives

• Many groups are phenotypically diverse—i.e.,

physiology and phylogeny are not necessarily linked

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 12.4 Molecular Phylogeny and the Tree of Life

• Domain Archaea consists of seven major phyla:

• Crenarchaeota

• Euryarchaeota

• Nanoarchaeota

• Korarchaeota

• Thaumarchaeota

• Two phyla do not contain cultivatable species

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 12.4 Molecular Phylogeny and the Tree of Life

• Domain Eukarya
• Eukaryotic organelles originated within Bacteria
• Mitochondria arose from Proteobacteria

• Chloroplasts arose from Cyanobacteria

• Each of the three domains of life can be

characterized by various phenotypic properties

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 12.5 Molecular Phylogeny: Making Sense of
Molecular Sequences
• Comparative rRNA sequencing is a routine
procedure that involves the following:
• Amplification of the gene encoding SSU rRNA

• Sequencing of the amplified gene

• Analysis of sequence in reference to other

sequences

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 12.5 Molecular Phylogeny: Making Sense of
Molecular Sequences
• PCR can be used to amplify SSU rRNA genes
from members of a microbial community
• Genes can be sorted out, sequenced, and analyzed

• Such approaches have revealed key features of

microbial community structure and microbial interactions

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 12.5 Molecular Phylogeny: Making Sense of
Molecular Sequences
• The first step in sequence analysis involves aligning the sequence of
interest with sequences from homologous (orthologous) genes from
other strains or species

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 12.5 Molecular Phylogeny: Making Sense of
Molecular Sequences
• Phylogenetic tree
• Graphic illustration of the relationships among
sequences (Figure 12.16)

• Composed of nodes and branches

• Branches define the order of descent and ancestry of

the nodes

• Branch length represents the number of changes that

have occurred along that branch

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 1 Rotating about a node does
4 not change tree topology.
2
1 2 3

3 2 3 2
5
Unrooted tree
3 1 1
1
1 4 6 7
2
2 5 7 6
3
3
Node 6 4 5
4
4 7 5 4
Lineage
Branches The positions of lineages 1 and
7 have been rotated between
5 5
the three trees, but the
Rooted trees branching pattern is unchanged.

© Pearson Education Limited 2015 Figure 12.16

 12.5 Molecular Phylogeny: Making Sense of
Molecular Sequences
• Evolutionary analysis uses character-state
methods (cladistics) for tree reconstruction

• Cladistic methods
• Define phylogenetic relationships by examining changes
in nucleotides at individual positions in the sequence

• Use those characters that are phylogenetically

informative and define monophyletic groups

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 12.5 Molecular Phylogeny: Making Sense of
Molecular Sequences
• Common cladistic methods:
• Algorithms: programmed series of steps (Figure 12.17)
• Unweighted pair group method with arithmetic mean

• Neighbor joining methods

• Optimality criteria: pick the best of many possible trees

• Parsimony

• Maximum likelihood

• Bayesian analysis

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© Pearson Education Limited 2015 Figure 12.17
 III. Microbial Evolution

• 12.6 The Evolutionary Process

• 12.7 The Evolution of Microbial Genomes

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 12.6 The Evolutionary Process

• Mutations
• Changes in the nucleotide sequence of an organism's
genome
• Occur because of errors in replication, UV radiation, and
other factors
• Adaptative mutations improve fitness of an organism,
increasing its survival

• Other genetic changes include gene duplication,

horizontal gene transfer, and gene loss
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 12.6 The Evolutionary Process

• Recombination
• Physical exchange of DNA between genetic elements

• Selection
• Based on fitness
• The ability to produce progeny and contribute to genetic makeup of
future generations

• Genetic drift
• Random process that can cause gene frequencies to change over
time (Figure 12.20)

• New traits can evolve quickly

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© Pearson Education Limited 2015 Figure 12.20
 12.6 The Evolutionary Process

• Speciation of microorganisms
• Species can posses a variety of individuals with
different traits

• Molecular clocks (chronometers)

• Certain genes and proteins that are measures of
evolutionary change

• Major assumptions of this approach are that nucleotide

changes occur at a constant rate, are generally neutral,
and are random
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 12.7 The Evolution of Microbial Genomes

• Dynamic nature of the microbial genome

• Genome sequencing of Escherichia coli (Figure 12.23)
• Core genes
• Genes shared by all members of a species

• Pan genes
• Core genes plus genes not shared by other members of
species

• Deletions play an important role in microbial

genome dynamics
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 Genome 1 Genome 2

Pan
Core

Genome 3

Pan genome

Core genome

© Pearson Education Limited 2015 Figure 12.23

 IV. Microbial Systematics

• 12.8 The Species Concept in Microbiology

• 12.9 Taxonomic Methods in Systematics

• 12.10 Classification and Nomenclature

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 12.8 The Species Concept in Microbiology

• Biological species concept not meaningful,

because prokaryotes are haploid and do not
undergo sexual reproduction

• Phylogenetic species concept is an alternative

• Prokaryotic species is a group of strains that, based on
DNA sequences of multiple genes, cluster closely with
others phylogenetically and are distinct from other
groups of strains

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 12.8 The Species Concept in Microbiology

• Current definition of prokaryotic species

• Collection of strains sharing a high degree of similarity
in several independent traits
• Most important traits include 70% or greater DNA–DNA
hybridization and 97% or greater 16S rRNA gene
sequence identity

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 12.8 The Species Concept in Microbiology

• Phylogenetic analysis
• 16S rRNA gene sequences are useful in taxonomy;
serve as "gold standard" for the identification and
description of new species
• Proposed that a bacterium should be considered a new
species if its 16S rRNA gene sequence differs by more
than 3% from any named strain, and a new genus if it
differs by more than 5%

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 Organisms to Organism 1 Organism 2
be compared:
Genomic DNA Genomic DNA

DNA–DNA DNA
Shear and label ( –P ) Shear DNA
preparation
P P P P
P P

hybridization Heat to
P P

form P P P P
single
• Genomes of two
P P

strands. P P

Probe DNA Target DNA

organisms are Hybridization experiment:

Mix DNA on a nylon membrane, adding probe DNA in excess:

hybridized to examine 1x1

(Control)
P

P
P
P

Hybridization is 100%.

Hybridized DNA
proportion of P
1x2 Hybridization is 25%.
(Experiment)
similarities in their Hybridized DNA

gene sequences Results and interpretation:

Same genus, 1x1 1x2
Same but different Different
100% 25%
(Figure 12.24) species species genera

Same strain 1 and 2 are likely

different genera

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 12.8 The Species Concept in Microbiology

• DNA–DNA hybridization
• Provides rough index of similarity between two
organisms

• Useful complement to SSU rRNA gene sequencing

• Useful for differentiating very similar organisms

• Hybridization values of 70% or higher suggest strains

belong to the same species
• Values of at least 25% suggest same genus

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 12.8 The Species Concept in Microbiology

• No firm estimate on the number of prokaryotic

species

• Nearly 7000 species of Bacteria and Archaea are

currently known

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 12.9 Taxonomic Methods Used in Systematics

• Taxonomy
• The science of identification, classification, and
nomenclature

• Systematics
• The study of the diversity of organisms and their
relationships

• Links phylogeny with taxonomy

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 12.9 Taxonomic Methods Used in Systematics

• Bacterial taxonomy incorporates multiple methods

for identifying and describing new species

• The polyphasic approach to taxonomy uses three

methods:
1. Phylogenetic analysis

2. Genotypic analysis

3. Phenotypic analysis

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 12.9 Taxonomic Methods Used in Systematics

• Phylogenetic analysis
• The lack of divergence of the 16S rRNA gene limits its
effectiveness in discriminating between bacteria at the
species level; thus, a multigene approach can be used
(Figure 12.26)

• Multigene sequence analysis uses complete

sequences, and comparisons are made using
cladistic methods

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© Pearson Education Limited 2015 Figure 12.26
 • Multilocus sequence typing (MLST)
• Method in which several different "housekeeping genes"
from an organism are sequenced (Figure 12.27)

• Has sufficient resolving power to distinguish between

very closely related strains
Bacterial Various "house-
chromosome keeping" genes Analyze alleles.
Strains
New isolate or 1–5
clinical sample New strain
Strain 6
Isolate DNA. Amplify 6–7 Sequence. Compare with
target genes. other strains
and generate Strain 7
tree.

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 12.9 Taxonomic Methods Used in Systematics
Genome fingerprinting

• Ribotyping: Method of identifying microbes from analyzing

DNA fragments generated from restriction enzyme
digestion of genes encoding SSU rRNA
• Highly specific and rapid

• Used in bacterial identification in clinical diagnostics and microbial analyses

of food, water, and beverages

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 12.9 Taxonomic Methods Used in Systematics

• Genome fingerprinting
• Several methods can be used to generate DNA
fragment patterns for analysis of genotypic similarity
among strains, including
• Ribotyping: focuses on a single gene

• Repetitive extragenic palindromic PCR (rep-PCR)

(Figure 12.29) and amplified fragment length
polymorphism (AFLP): focus on many genes located
randomly throughout genome

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© Pearson Education Limited 2015 Figure 12.29
 12.9 Taxonomic Methods Used in Systematics

• Whole genome sequence analyses are becoming

more common
• Genome structure: size and number of chromosomes,
GC ratio, etc.

• Gene content

• Gene order

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 12.9 Taxonomic Methods Used in Systematics

• Phenotypic analysis examines the morphological,

metabolic, physiological, and chemical characters
of the cell

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 12.9 Taxonomic Methods Used in Systematics

• Fatty acid analysis (FAME: fatty acid methyl ester)

• Relies on variation in type and proportion of fatty acids
present in membrane lipids for specific prokaryotic
groups (Figure 12.30a and b)

• Requires rigid standardization because FAME profiles

can vary as a function of temperature, growth phase,
and growth medium

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© Pearson Education Limited 2015 Figure 12.30a
 IDENTIFY ORGANISM

Compare pattern of peaks

with patterns in database
Bacterial culture

Extract fatty acids Peaks from various fatty

acid methyl esters

Derivatize to form
methyl esters

Amount
Gas chromatography

© Pearson Education Limited 2015 Figure 12.30b

 12.10 Classification and Nomenclature

• Classification
• Organization of organisms into progressively more
inclusive groups on the basis of either phenotypic
similarity or evolutionary relationship

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 12.10 Classification and Nomenclature

• Prokaryotes are given descriptive genus names

and species epithets following the binomial system
of nomenclature used throughout biology

• Assignment of names for species and higher

groups of prokaryotes is regulated by the
International Code of Nomenclature of Bacteria

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 12.10 Classification and Nomenclature

• Major references in bacterial diversity:

• Bergey's Manual of Systematic Bacteriology

• The Prokaryotes

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 12.10 Classification and Nomenclature

• Formal recognition of a new prokaryotic species requires:

• Deposition of a sample of the organism in two culture collections

• Official publication of the new species name and description in the

International Journal of Systematic and Evolutionary Microbiology
(IJSEM)

• The International Committee on Systematics of

Prokaryotes (ICSP) is responsible for overseeing
nomenclature and taxonomy of Bacteria and Archaea

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