MicrobiologyBrock 13th edition: chapters 1, 16

a. Evolution of life

b. Microbial evolution

c. Examples for universal importance of bacteria in biology, environment and health

Why study Microbiology ?

Figure 1.6a

Mammals Humans

Vascularplants

Shellyinvertebrates

Algaldiversity

Origin ofcellular life

Moderneukaryotes

Origin ofcyanobacteria

20% O2

O2

AnoxicEarth

Earthis slowlyoxygenated

Present

Mic

rob ial life for ms only

Origin of Earth(4.6 bya)

1bya

2bya

3bya

4bya

Anoxygenicphototrophicbacteria

a. Evolution of life

Figure 16.4

EarlyBacteria

EarlyArchaea

Mound:precipitates of clay,metal sulfides, silica,and carbonates

Ocean water(20°C, containingmetals, CO2 andPO4

2)

Flow of substancesup through mound

Ocean crust

Nutrients in hothydrothermal vent water

Aminoacids

Sugars

Nitrogenbases

Evolution of life

Figure 16.4

Evolution of life

Tim

e

(0.

3 to

0.5

bil

lio

n y

ears

)

1. Prebiotic chemistry

2. “RNA world”

3. proteolipid membrane

4. LUCA (last universal common ancestor)

5. diversification, interaction

6. Dispersal (other habitats)

Figure 16.7

In

OutPrimitivehydrogenase

PrimitiveATPase

Cytoplasmicmembrane

S0 reductase

LUCA’s energy metabolism

S + H2 H2S ΔG0’ = -20,6 kJ

e--acceptor e--donor

pyrite H+ gradient

LUCA’s C-metabolism

S + H2 H2S ΔG0’ = -20,6 kJ

CO2 fixation organic compounds (i.e. acetate)

Chemoorganotrophic bacteria“metabolic diversification”

accumulate

4-4.3 x109 years BC

Metabolic diversification

S + H2 H2S ΔG0’ = -20,6 kJ

CO2 fixation organic compounds

Bacteria

accumulate

4-4.3 x109 years BC

Archaea

3.7 x109 years BCH2, CO2 acetate 4 H2 + CO2 CH4 + 2H2O

methanogenesis

H3CCOO- + H2O CH4 + HCO3-

Bacteria: evolve phototrophy

S + H2 H2S ΔG0’ = -20,6 kJ

CO2 fixation organic compounds

Bacteria

accumulate

4-4.3 x109 years BC

Archaea

3.7 x109 years BCH2, CO2 acetate 4 H2 + CO2 CH4 + 2H2O

methanogenesis

H3CCOO- + H2O CH4 + HCO3-

3.3 x109 years BC Anaerobic phototrophy (H2S S)

2.7 x109 years BC Oxygen generating phototrophy (H2O O2)

CO2 fixation

CO2 fixation

Figure 16.6 and 8

Metabolichighlights

O2level

BYAEon Organisms,events

Phanaerozoic

Proterozoic

Archaean

Hadean

Cambrian

Precambrian

Anoxic

Extinction of the dinosaurs

Early animals

Multicellulareukaryotes

First eukaryoteswith organelles

Ozone shield

Great oxidation event

Cyanobacteria

Purple and green bacteria

Bacteria/Archaeadivergence

First cellular life; LUCA

Formation ofcrust and oceans

Formation of Earth4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0

20%

10%

1%

0.1%

Endosymbiosis?

Aerobic respiration

Oxygenic photosynthesis(2H2O O2 4H)

Sulfate reductionFe3 reduction

Anoxygenic photosynthesis

Acetogenesis

Methanogenesis

Sterile Earth

First: Fe-oxidation

Precambrian Fe3+ sediments

O2 toxicityNew metabolic pathways: - sulfate reduction - nitrification - chemolithotrophy - O2 respiration (grow fast)

Figure 16.9

Bacteria BacteriaEukarya EukaryaArchaea Archaea

cyanobacterium

Ancestor ofmitochondrion(Bacteria)

Animals Plants PlantsAnimals

Archaeonwith nucleus

nucleus formed

cyanobacterium

Engulfment of aH2-producing cellof Bacteria by aH2-consuming cellof Archaea

Endosymbiont theory of Eukaryote evolution

a) From nucleated Archaeon b) Hydrogen hypothesis

three take home messages

1. Bacteria are the ancient form of life. All other organisms evolved

from this. LUCA existed probably 4.3 Bio years ago.

2. All forms of life had extreme effects on their environment... and mediated dramatic change

3. Intense interactions. All organisms have interacted with each other

(directly or indirectly). E.g. Eukaryotes have always been interacting

intensely with bacteria throughout their evolution. The evolution and

function of one cannot be understood in the absence of the other.

b. Bacteria and Archaea evolution

Very high rate of evolution!!

1. Haploid genomes

2. Rapid growth

3. Large populations

Figure 16.10

Wild-type cellPigment mutantsLightDark

Mutantslost inlight

Mutantselectedin dark

Cell populations

Pigment mutants

Wildtype

Subculture number

Bac

teri

och

loro

ph

yll

a/m

l o

f cu

ltu

re 15

10

5

5 10 15 20

54321

Bacteria/ Archaea evolution

Rhodobacter capsulatus

16S rRNA

Phylogenetic analysis of Bacteria, Archaea

Ribosomal RNA genes

Va

ria

ble

……

.co

ns

erv

ed

Base position in 16S RNA gene

16S (bacteria), 18S (Eukaryotes)• Ubiquitous and essential• Ancient• Easy RNA isolation• Conserved and variable regions• Sufficiently long

Small ribosomal subunit RNA sequence: “long distance” relationships

Phylogenetic analysis of Bacteria, Archaea

conserved protein-coding genes: “long distance” and strain differentiation

EF-Tu (protein biosynth.)

Hsp60

aatRNA synthetases

…

Figure 16.12, 13

Kilo-bases 1 2 3 4 5

3.0–

2.0–

1.5–

1.0–

0.5–

16 S gene

Ancestralcell

Distinctspecies

Distinctspecies

Align sequences;generate tree

Sequence

Run on agarosegel; check forcorrect size

Amplify 16Sgene by PCR

Isolate DNA

A C G G T

Phylogenetic analysis

via 16S rDNA

Beforealignment

Afteralignment

Species 1

Species 1

Species 2

Species 2

Nonidentities

Gaps

9

15

Figure 16.14

Unrooted tree

Rooted trees

node

Relative relationships Defines unique paths of evolutionEmploys “outgroup”

Display phylogenetic relationshipCladistics = grouping by common features (absent in more distant relatives)Parsimony = assumes least number of steps

Figure 16.16

Bacteria Archaea Eukarya

PROKARYOTES EUKARYOTES

LUCA

Flavobacteria

Thermotoga

Thermodesulfobacterium

Aquifex

Cyanobacteria

Chloroplast

Proteobacteria

Mitochondrion

Gram-positivebacteria

Green nonsulfurbacteria

Crenarchaeota

Euryarchaeota

Thermoproteus

Pyrodictium

Thermococcus

MarineCrenarchaeota

Methano-bacterium

Methano-coccus

Pyrolobus

Methanosarcina

Thermoplasma

Methanopyrus

Extremehalophiles

Entamoebae Slimemolds

Animals (7.7 Mio species)

Fungi (0.6 Mio species)

Plants (0.3 Mio spec.)

Ciliates

Flagellates

Trichomonads

Microsporidia

Diplomonads(Giardia)

Universal phylogenetic tree

3 “domains”

Extensive geneticexchange??

> 80 phyla> 10 Mio species??

2 major phyla 8 Mio species ?

c. Current topics of interest

Methods: for analyzing microorganisms

Philosophy: the bacterial species problem

health: effects of the microbiota

environment: metabolic effects on C, N, P, S… cycles

Industry/safety: genetic engineering

health: what is a pathogen? How to kill bacteria?

Analyzing Bacteria and Archea

Physiology: << 5 % of all bacteria have been cultured

phenotype (motility, morphology, metabolism…)

FISH (Fluorecence in situ hybridization): DNA-oligo binding rRNA

Bacillus

Yeast

DNA sequencing (fast evolving field!!):

- 16S “community sequencing”

- “metagenome sequencing”

predict genes/metabolism

predict physiology

Universal probe eukaryal probe

Costs of DNA sequencing

Figure 22.16

Communitysampling approach

Environmentalgenomics approach

Outcomes

Single-gene phylogenetic tree Total gene pool of the community

1. Identification of all gene categories2. Discovery of new genes3. Linking of genes to phylotypes

Phylogenetic snapshotof most members of the community

1.

Identification of novelphylotypes

2.

Amplify single gene,for example, geneencoding 16S rRNA

Restriction digest total DNA andthen shotgun sequence, ORsequence directly (withoutcloning) using a “next generation”DNA sequencer

Extract totalcommunity DNA

Microbialcommunity

Sequence andgenerate tree Assembly and

annotation

DNA

Partialgenomes

DNA sequencing for microbial community analysis

Example 1: The gut microbiotaRole in health and diseases

Animal evolution

=>>

Animal evolution

A long history of co-evolution

Example 1: The gut microbiotaRole in health and diseases

Energy: acetate, butyrate

Vitamins: K, B12, C, niacin,panthotenic acid, biotin,folic acid

GALT: maturation

Colonization resistance

Innate defense: priming

Inflammatory bowel disease: stimulus

Mucosa: maturation

Th17 immune responses: stimulus

Host metabolism: stimulus

Microbiota enzyme/function MAMP signaling/innate immunity

Disease

Health

Example 1: The gut microbiotaRole in health and diseases

cancer: stimulus ?

Dethlefsen, 2008, Nature 448, pp. 811ff

Example 1: The gut microbiotaRole in health and diseases

Example 2: The “bacterial species problem”

Plants/animals: cross fertile offspring

Bacteria, Archaea: ??

Example 2: The “bacterial species problem”

The “bacterial species problem”

Phylogenetic tree181 genomes

proteobacteriales

Dagan et al., 2008, PNAS 105, pp. 10039 ff.

Phylogenomic tree≥5 genes exchanged by «horizontal gene transfer»

Streptococcus

Fraser et al., 2009, Science 323, pp. 741ff

The “bacterial species problem”

Bacteria, Archaea: - no sexual cycle - “long distance” gene exchange phylogenetic species concept niche occupation: “ecotype”

16S rRNA Gene Tree Multigene Tree

ATCC 11040T

ATCC 51760T

BAA-1194T

50 changes

Photobacterium damselae

Photobacterium leiognathi

Photobacterium mandapamensis

Photobacterium angustum

Photobacterium phosphoreum

Photobacterium iliopiscarium

Photobacterium kishitanii

Photobacteriumphosphoreum

Photobacteriumiliopiscarium

Photobacteriumkishitanii

FS-2.1FS-4.2FS-3.1FS-5.1FS-2.2

FS-5.2

ATCC 51761NCIMB 13476

NCIMB 13478NCIMB 13481

chubb.1.1ckamo.3.1canat.1.2

hstri.1.1calba.1.1

apros.2.1ckamo.1.1

vlong.3.1

The “bacterial species problem”

Figure 16.25

One microbial habitat

Ecotype I Ecotype II

Ecotype III

New speciesof Ecotype III

Cell containingan adaptivemutation

Populationof mutantEcotype III

Periodic selection

Adaptive mutantsurvives. OriginalEcotype III wild-typecells out competed

Repeat processmany times

The “bacterial species problem” ecotypes

Classification: traditional approach

Taxonomic systems:

Bergey’s Manual of Systematic Bacteriology

The Prokaryotes

International Committee on Systematics of Prokaryotes

Example 3: global Carbon-cycle

Humanactivities

Biological pump

Death andmineralization

Respiration

CO2

CO2

CO2

Landplants

Aquaticplants and

phyto-plankton

Animals andmicroorganisms

Fossilfuels

Humus

Soil formation

Earth’s crust Rock formation

Aquaticanimals

Figure 24.1

CH4

CH4, a greenhouse gas

CH4

Atmosphere 0.003

Example 3: global Carbon-cycle

Figure 24.2

Organic matter

Organic matter

Syntrophassisted

Oxygenic photosynthesis

Chemolithotrophy

Respiration

Methanotrophy

Methanogenesis

Acetogenesis

Anoxygenicphotosynthesis

OxicAnoxic

Anaerobicrespirationandfermentation

(CH2O)n

CO2

(CH2O)n

Example 3: anaerobic methane oxidation

Figure 14.28

Methanotrophic Archaea(ANME-types)Sulfate-reducing Bacteria

Organiccompounds

Marine sediments

c. Current topics of interest

Methods: for analyzing microorganisms

Philosophy: the bacterial species problem

health: effects of the microbiota

environment: metabolic effects on C, N, P, S… cycles

Industry/safety: genetic engineering

health: what is a pathogen? How to kill bacteria?