Notes to Microbial Evolution

© Erik Gustafsson
[Link]

How did it all begin

The earth was formed about ____ billion years ago!
• From ”The Big Bang!”, ____ billion years ago
o Our sun was formed in the "dust cloud" and begins to undergo nuclear fusion
o Through collisions and gravitation, planets are then formed from residual matter
formed during the supernova

Evolution as a clock
• 00:00: The Earth was formed
• 00:00-03:00: The Earth was bombarded by meteors
• 04:00: Some sort of life exists
• ~05:30: Bacteria definitively existed (fossils)
• ~05:30-06:00: Photosynthetic bacteria have evolved
• 09:44: Cyanobacteria came to life
• 11:00: Great oxidation event starts
• ~12:00: The atmosphere becomes very rich in oxygen
• 13:00: Eukaryotic organisms have evolved
• 13:06: Great oxidation event stops
• ~17:00: First multicellular forms of life have evolved
• 20:00: The first aquatic animals have evolved
• 21:20: The first fishes have evolved
• 21:36: The first land plants come Colin [CC BY-SA 4.0], via Wikimedia Commons

• 22:47: The first dinosaurs see day light

• 23:40:48: The dinosaurs got extinct
• 23:59:08: Homo spp. is formed
• 23:59:56: Modern man

Bacteria have existed for > ___ billion years

The history of modern man is _______ thousand years

The Theory of Evolution

24th of December, 1859
• Charles Darwin published ”On the Origin of Species by Means of Natural Selection”
• Sv. ”Om arters uppkomst”
Evolution
• A change in the hereditary properties in a population over time

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Age of the Earth
The earth is approximately 4.54 ± 50 million years old
• Based on slowly decaying radioactive isotopes (and more)
The oldest rock found on The Earth is ~4.0 billion years old
• The oldest Earth rock (4.46 billion years old) was found on The Moon in 1971 (Apollo 14)
Evidence support that water probably existed 4.3 billion years ago
• Analysis of ancient zircon crystals (impurities and oxygen isotope ratios)
There are strong evidences that bacteria existed ~3.5 billion years ago (fossils)
• Belonged to the phylum Thermotogota

Hadean Eon
The time between 4.54- to 4.0 billion years ago (B.y.a)
• The name "Hadean" comes from Hades and
referrers to the hellish conditions of the early earth
o Surface of molten superheated lava
o Abundance of radioactive elements
o Bombarded by meteors
The impacts from extraterrestrial bodies was large
• Probably persisted for ~500 million years
• The released enormous amounts of heat that likely
prevented much of the rock from solidifying at the surface
• A very early impact with an object by the size of
Jeancarlosptino [CC BY-SA 4.0], via Wikimedia Commons
Mars probably formed the moon
o The collision also resulted in the 23.5° ___________ of the Earth

The Early Earth

The Early Earth had no ozone layer
• _______ were not destroyed and hit the Earth
• Sterile surface due to intense ________
The Earth was also very warm
• Maybe 2000 ºC due to collisions with rocky debris
• During the first 200 million years the temperature was above 100 °C
Anoxygenic environment
• Atmosphere of water (from volcanos), methane, carbon dioxide, nitrogen and ammonia
Liquid water
• Evidence of liquid water 4.3 B.y.a
• Oceans start to form

Origin of life
In 1952 Stanley Miller and Harold Urey showed that synthesis of organic compounds from inorganic
constituents can occur (Miller-Urey-experiment)
• CH4, NH3, H2 and H2O was exposed to electric arc (simulating lightning)
• Five amino acids were formed

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One hypothesis is that life may have originated well below
Earth’s surface on ___________________ on the ocean floor
• Less hostile and more stable conditions
than on the Earth’s surface
• Firm supply of energy in form of reduced
inorganic compounds, e.g. H2 and H2S
Another hypothesis is some of the molecules important to life
was produced outside the Earth (came from meteorites or comets) NOAA [Public domain], via Wikimedia Commons
• The Murchison meteorite (Australia, 1969)
contained dozens of different amino acids
• Samples of asteroid Ryugu (collected during Japan’s
Hayabusa2 mission) contained 20 different types of amino acids

Earliest “evidence” of life

Stromatolite fossils have been found in 3.7 billion-year-old rocks from western Greenland
• Layered sedimentary formations created by microorganisms
2015: Biotic life was maybe found in 4.1 billion-year-old rocks in western Australia?
• Graphite (carbon) inclusions in a zircon (special mineral common in Earths crust)
2017 reports on life from maybe 4.28 B.y.a in the form of fossilized microorganisms came
• Found in hydrothermal vent precipitates in the Nuvvuagittuq Belt of Quebec, Canada
• Tricky to determine correct age but the finding is at least from 3.77 B.y.a)

The “RNA-world” etc.

Before organisms was developed “things” to create the cells from must have been formed
• Biological building blocks must have been available
o Amino acids, nucleoside, sugar etc.
Scientists believe that RNA was the central component of the first self-replicating system
• “_____________”
o RNA can catalyse simple biological reactions and can be self-replicating
o RNA is the backbone of some important molecules (ATP, NADH, coenzyme A)
Proteins synthesized by these RNAs replaced the catalytic role of RNA
• Proteins are more efficient catalysts than RNA
o Can catalyse many reactions RNA can’t
Finally, DNA replaced RNA as coding element
• “_____________”
o DNA is much more stable than RNA and became template for RNA-synthesis
Lipid bilayer (maybe lipid vesicles) as membrane for protection

Energy-generation in primitive cells

The Earth was at this time anoxic
• Anaerobic metabolism of primitive cells
Organic material was largely absent
• The primitive cells must have been chemolithotrophs
o Were probably using H2 and H2S as electron
donors (from volcanos and thermal springs)

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[Link]
 o S0 (elemental sulphur) was likely the first electron
acceptor from H2-oxidation (H2 + S0 → H2S)
• The carbon source was most probable CO2

LUCA
The Last Universal Common Ancestor (LUCA) likely
existed 3.8 to 3.7 B.y.a
• The point when bacteria and archaea diverged
• Proposed by Carl Woese

Classification
Initially, all living organisms were divided
into five different groups
• Plants
• Animals Chiswick Chap [CC BY-SA 4.0], via Wikimedia Commons
• Fungi
• Protists
• Monera
When starting to examine the ribosomes only three different
types was observed and class-division was changed
• ________
• _______
• __________
This work was led by Carl Woese in the 1970s

Evolutionary chronometers
An evolutionary chronometer is a nucleotide sequence
(usually) that can be used to measure the evolutionary
separation of two organisms
• Universally distributed across the group chosen for study
• Functionally __________ in each organism studied
• Conserved _______ for alignments of adequate length
• Must change (______) at ”sufficient” rate
Ribosomal RNA (rRNA) is a “good” evolutionary chronometer!

How to do! Don Hamerman [CC BY 3.0], via Wikimedia Commons

Isolate the DNA

• Obtaining DNA is generally easy when they can be cultivated in laboratory
Amplify 16S gene using PCR
• Standard primers exist
Run PCR-product on agarose gel
• To see if the correct fragment has been amplified
Bands (DNA) from the gel are excised and extracted (not necessary), and also purified
Sequence
Align sequences and generate tree
• Bioinformatics

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Anoxygenic photosynthesis
Stromatolites can be found in old rocks (3.5 B.y.a, 05:30)
• Early conclusive evidence of life on Earth
The first phototrophic organisms were anoxygenic
(O2 is not produced)
• Used H2S as electron donor for CO2 fixation
resulting in S0 as waste product
• Probably used infrared radiation generated
by the heat from hydrothermal vents as
energy source
Paul Harrison [GFDL], via Wikimedia Commons
These microorganisms produce adhesive compounds
• Cemented sand and other rocky materials to
form mineral "microbial mats“ (type of biofilm)
• These are built up in a layer by layer principle,
growing gradually over time
The microbes slowly move upwards to avoid being
smothered by sediment

Oxidation of the Earth

_____________ (oxygenic phototrophs)
• Evolved ~2.7 B.y.a (09:44)
• Oxygen → iron-oxides (also trapped by the oceans), Joydeep [CC BY-SA 3.0], via Wikimedia Commons
i.e. atmosphere was not altered
_____________________
• Happened 2.46-2.06 B.y.a (11:00-13:06)
• Anoxic to oxic atmosphere
o O2 accumulation in atmosphere (required by aerobic organisms)
o O3 accumulation
(protection against UV)
Aerobic/anaerobic organisms could start evolving

Mechanisms of Microbial evolution

Mutations (random changes in the DNA sequence)
• Most are neutral or bad but some can be beneficial
• They take different forms
o _____________ (one nucleotide is replaced by a different nucleotide)
o _________ (the loss of one or more nucleotides)
o __________ (addition of one or more nucleotides)
o ____________ (one or more copies of a DNA segment is produced)
Bacterial _____________
• The physical exchange of DNA between genetic elements
Bacterial homologues recombination
• A type of genetic recombination in which nucleotide sequences are exchanged between two
similar or identical molecules of DNA.

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Homologues recombination
Homologues recombination is the genetic exchange
between homologues DNA-sequences from two different sources
• Is responsible for “_____________” in prokaryotes
• Will happened if the recipient lack something that is beneficial to gain (antibiotic cassette)
DNA fragments are transferred from the donor- to the recipient cell in different ways

Gene transfer in Bacteria

________ gene transfer
• Organisms replicate their genome and provide copies to descendants
__________ gene transfer
• A donor cell contributes parts of genome to a recipient cell
o Bacterial ___________
o ______________
o ____________

Bacterial conjugation
1. Donor cell (F+) attaches to recipient cell (F-) with its pilus.
2. The pilus draws the cells together and the cells contact one another
3. Transfer of one strand from donor- to recipient cell
4. Simultaneously replication of the complementary strand
5. Cells separate

Transformation
A recipient cell takes up DNA from the environment (such as DNA from a dead organism)
Was identified in 1928 by Frederick Griffith
• Tried to develop a vaccine for Streptococcus pnuemoniae
Griffiths experiments
He had two kinds of S. pneumonia strains
• S-strain forming smooth colonies on plates
o Producing a bacterial capsule
R-strain forming rough colonies on plates
• Capsule-deficient strain
o These strains were injected in mice

1. Live cells of strain S (+) → RIP

2. Live cells of strain R (-) → Nothing happened
3. Heat treated dead cells of strain S (+) → Nothing happened
4. A mix of live cells of strain R (-) and heat-treated dead cells of strain S (+) → RIP
Culturing of cells from experiment 4 resulted in smooth colonies
• The R-strain (-) had acquired a capsule!

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Transduction
DNA from a donor cell is transferred to recipient cell via a replicating virus (_____________)

© Erik Gustafsson, 2018

Selection and genetic drift

Mutations and recombination result in “new genes”
with altered function
• Evolution occurs when different genes change in frequency
in a population over a span of many generations
There are different mechanisms responsible for this
• Evolutionary _________
o Is defined on the basis of fitness
(how much “offspring” is produced?)
o Bacteria with beneficial genes will survive
(antibiotic resistance)
• _____________
o Some bacteria will have more “offspring“ by chance
o Most common if a population size is severely _______ [Link]
and a regrowth of the remaining bacteria occur (infections)