MICROBIAL EVOLUTION ANDSYSTEMATICS

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ADVANCE MICROBIOLOGY

Evolution of Earth and Earliest Life Forms

Origin of Earth

• Earth itself is about 4.6 billion years old.

• Our solar system formed when a large, very hot starexploded, generating a new star (sun) and the othercomponents of our solar system.

• The oldest rocks discovered thus far are in the Itsaqgneiss complex, in southwestern Greenland, which dateto about 3.86 billion years ago.

• Ancient rocks are three types: sedimentary, volcanic, andcarbonaceous.

• The formation of sedimentary rocks requires liquid water--------------The presence of liquid water in turn implies thatconditions were compatible with life.

The oldest known stromatolite,found in a rock about 3.5 billionyears old, from the WarrawoonaGroup in Western Australia.

Stromatolites of conical shapefrom 1.6-billion-year-old dolomiterock of the McArthur basin of theNorthern Territory of Australia.

Modern stromatolites in a warm marinebay, Shark Bay, Western Australia.

Modern stromatolites composed ofthermophilic cyanobacteria.

Another view of modern andvery large stromatolitesfrom Shark Bay.

Evolution of Earth and Earliest Life Forms

• Evidence for microbial life in ancient rocks rest on thefossilized remains of cells and the isotopically “light”carbon abundant in these rocks.

• Some ancient rocks contain microfossils that appear verymuch like bacteria, typically as simple rods or cocci.

• Stromatolites:

- Fossilized microbial mats consisting of layers offilamentous prokaryotes and trapped sediment.

- In rocks of 3.5 gigayears (GY) or younger

- Formed by filamentous phototrophic bacteria,perhaps relatives of the green nonsulfur bacteriumChloroflexus.

Evidence for Microbial Life on Early Earth

Evolution of Earth and Earliest Life Forms

Ancient microbial life. Scanning electron micrograph of microfossil prokaryotes from3.45 billion year old rocks of the Barberton Greenstone Belt, South Africa. Note therod-shaped bacteria (arrow) attached to particels of mineral matter. The cells areabout 0.7 µm in diameter.

a

b

Fossil prokaryotes and eukaryotes from morerecent rocks than those shown. (a) show fossilprokaryotic microorganisms from the Bitter SpringsFormation. (b) Microfossils of eukaryotic cells fromthe same rock formation. The cellular structure isremarkably similar to that of certain modern algae,such as Chorella species.

Evolution of Earth and Earliest Life Forms

Evolution of Earth and Earliest Life Forms

Condition on Early Earth: Hot and Anoxic

• The atmospher of early Earth was devoid of oxygen.

• Besides water, a variety of gases were present, the most abundantbeing methane, carbon dioxide, nitrogen, and ammonia.

• Trace amounts of carbon monoxide and hydrogen likely existed, aswell as large reservoirs of sulfide, as mixture of H2S and FeS. It isalso likely that a considerable amount of hydrogen cyanide, HCN,was produced on early Earth when NH3 and CH4 reacted to yieldHCN.

• Most evidence suggests that the early Earth was hotter than it istoday. For its first two hundred million years or so, the surface ofEarth may have exceeded 1000C.

• Early life form were likely quite heat tolerant, and may haveresembled hyperthermophilic prokaryotes in this regard.

Origin of Life• The synthesis of biomolecules can occur spontaneusly if reducing

atmospheres containing the aformentioned gases are subjected tointense energy sources.

• If gaseous mixtures resembling those thought to be present onprimitive Earth are irradiated with UV or subjected to electricdischarges in the laboratory today, a variety of biomolecules form.These include sugars, amino acids, purines, pyrimidines, variousnucleotide, thioester, and fatty acids.

• Under simulated prebiological conditions in laboratory some of thesebiochemical building blocks have been shown to polymerize, leadingthe formation of polypeptides, polynucleotides, and other importantmacromolecules.

• We can therefore imagine that by whatever means, a mixture oforganic compounds eventually accumulated on the primitive Earth,and in so doing, set the stage for the origin of life.

Evolution of Earth and Earliest Life Forms

Catalysis and The Importance of Montmorillonite ClayTo obtain spontaneous synthesis of macromolecules on early Earth, acatalyst would have had to be avilable. Good possibibilities include thesurface of clays or pyrite (FeS2)

Evolution of Earth and Earliest Life Forms

Lipid vesicles made in laboratory from the fatty acid myristic acid and RNA.The vesicle itself stains green and the RNA complexed inside the vesicles stain red.Vesicle synthesis is catalyzed by the surfaces of Montmorillonite clay particles.

RNA Life

• In an RNA world, if it ever existed, various self-replicating RNAswould have carried out the catalytic reactions necessary for theirself-replication.

• Self-replicating RNA life forms may have evolved into the firstcelluar life forms when they become enclosed within lipoproteinvesicles.

• The proper constituents and set of circumtances come together anda primitive enclosed structure arose, capable of self-replication.Although still lacking DNA and proteins, this protocellular life formwould otherwise have resembled a modern cell.

• As primitive organisms become more complex and catalyticreactions more biochemically demanding, proteins would have

replaced RNAs as the cells’ primary biocatalysts.• However, as evolution selected for more and more prices

biochemical catalysts, RNA was eventually replaced almost entirelyby proteins as cellular enzymes. By this point, the modern cell wasclearly on the horizon.

The RNA World and Molecular Coding

Possible scenario for theevolution of cellular lifeforms from RNA life forms.

Self-replacing RNAs couldhave become cellular entitiesby becoming stably integratedinto lipoprotein vesicles. Withtime, proteins replaced thecatalytic functions of RNA, andDNA replaced the codingfunctions of RNA

The Modern Cell: DNA-RNA-Protein

Somewhere in the early stages of microbialevolution the three-part system-DNA, RNA,

Protein-become fixed in cells as the bestsolution to biological information processing.

That this system was an evolutionary successstory obvious, since without exeption, modern

cells contain all three types of thesemacromolecules.

The RNA World and Molecular Coding

Energy and Carbon Metabolism

The Primitive Life

A Possible Energy-generating scheme forprimitive cell.

Formation of pyrite leads toH2 production and S0

reduction, which fuels apimitive ATPase. Note howH2S plays only a catalyticrole; the net subtrates wouldbe FeS and S0. Also notehow few dofferent proteinswould be reqiured. An

alternative source of H2 couldhave been the UV-catalyzedreduction of H+ by Fe2+ asshown.

Major Landmarks in biologicalevolution and Earth’s changinggeochemistry.

Note how the oxygenation of theatmosphere due to cyanobacterialmetabolism was a gradualprocess, occuring over a period ofabout 2 billion years. Althaoughfull (20%) oxygen levels arerequired for animals and mostother higher organism, this is nottrue of prokaryotes, as many arefacultative aerobes ormicroaerophiles. Thus,prokaryotes respiring at reducedO2 levels may have dominatedEarth for the period of a billionyears or so before Earth’satmosphere reached currentlevels of oxygen.

Endosymbiosis

• The modern (organell-containing) eukaryotic cell did notcome about from the gradual partitioning of cellularfuntions into enclosed structures, the organells.

• Instead, organells originated from the stableincorporation of chemoorganotrophic and phototrophicsymbionts from the domain bacteria.

• Endosymbiosis, probably began when an aerobicbacterium established stable residency within the

The Primitive life

cytoplasm of a primitive eukaryote and supplied the cellwith energy in exchange for a protected environment anda ready supply of nutrient. This symbiont was theforerunner of the modern mitochondrion.

The Primitive life

• The endosymbiotic uptake of an oxygenic phototrophconferred photosynthesic properties on a primitiveeukaryote; such a cell was freed from reliance onorganic compounds for energy production and becomephototrophic. The phototrophic endosymbiont was theforerunner of the modern day chloroplast.

• A few eukaryotic cells either never incorporatedendosymbionts or, what appears more likely, had them atone time and then disposed of them, possibly bacausetheir niche was permanently anoxic.

• Microfossil records suggest that endosymbiotic eventsbegan sometime after about 2 gigayear ago.

Origin of the moderneukaryotic cell byendosymbiotic events.

Note how organellsoriginated from Bacteriarather than Archaea. Someprimitive eukaryotes eithernever underwentendosymbiotic events orpermanently lost theirsymbionts, but otherwisemaintained the basicproperties of eukaryoticcells.

Signature SequencesComputer analyses of ribosomal RNA sequence haverevealed so-called signature sequences, shortoligonucleotides unique to certain groups of organism(genus or species can be determined by computerinspection of aligned sequences).

Phylogenetic Probes and FISHRecall that probe is a strand of nucleic acid that can belabeled and used to hybridize to a complementarynucleic acid from a mixture. Probes can be general orspecific.

Signature Sequences, Phylogenetic Probes,and Microbial Community Analyses

Signature Sequences, Phylogenetic Probes, and Microbial Community Analyses

FISH. Flourescent in-situ hybridization; a process in which acell is made flourescent by labeling it with a specific nucleicacid probe that contains an attached flourescent dye.

Same field, cells stained with a yellow-green universal rRNA probe (this probereacts with species from any domain.

Phase contrast photomicrograph (noprobe present) of cells of Bacillusmegaterium (rod, member of theBacteria) and the yeast Saccharomycescerevisiae (oval shaped cells, Eukarya)

Same field, cells stained with aeukaryal probe (only cells ofSaccharomyces cerevisiae react).

Signature Sequences, Phylogenetic Probes, and Microbial Community Analyses

Use of phylogenetic stain to make nitrifying bacteria visible in agranule of activated sewage sludge.

Microbial Community Analysis

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PCR-amplified ribosomal RNA genes do not need to originate frompure culture grown in the laboratory.

A phylogenetic snapshot of a natural microbial community can betaken using PCR to amplify the genes encoding SSU ribosomalRNA from all members of that community.

Such genes can be easily be sorted out, sequenced, and aligned.

From the data, a phylogenetic tree can be generated of“environmental” sequences that show the different ribosomal RNAspresent in community.

From this tree, specific organism can be inferred even though noneof them were actually cultivated or otherwise identified.

Signature Sequences, Phylogenetic Probes, and Microbial Community Analyses

Signature Sequences, Phylogenetic Probes, and Microbial Community Analyses

A bacterial community in a sewage sludge sample. The sample was stainedwith a series of dyes, each of wich stained a different bacterial group.

Microbial Phylogeny

Microbial Phylogeny

Microbial Phylogeny

Characteristics of the Domain of Life

Bacteria Archaea Eukarya

Lack peptidoglycan

(pseudopeptidoglycan,made of polysaccharide,protein or glicoprotein)

Consist of ether-linkedmolecules

Structurally morecomplex than those

bacteria, contain eight ormore polypeptides

70S ribosomes

The initiator tRNA carriesan unmodified

methionine

Lack peptidoglycan

(made of cellulose orchitin)

Synthesize membranelipid with a backbone

consisting of fatty acidbonded in ester linked

to a molecule of glycerol

The major RNApolymerase (there arethree) contains 10-12

polypeptides

80S ribosomes

The initiator tRNAcarries an unmodified

methionine

ina a of

Cell Walls

Lipids

RNARNA Polymerase

Polymerase

Features ofProtein

Synthesis

, , , , ’,’, )

ContainningContainning peptidoglycan

peptidoglycan

Synthesize membranelipid with aabackbone

consisting of fatty acidbonded in ester linked to

to molecule of glycerol

Contain aasingle type ofRNA polymerase (fourpolypeptides, ααββββ σσ)

70S ribosomes

An initiator tRNAcontaining aamodifiedmethionine residue,

formylmethionine

Classical TaxonomyTaxonomy, the science of classification, consist of twomajor subdisciplines, identification and nomenclature.

Taxonomy: relies on phenotypic analyses (what anorganism looks like, its energy metabolism, itsenzyme, and other properties).

Phylogeny: has emerged from genotypic analyses.

GC Ratio ----- One property that is informative in drawingtaxonomic conclusions is an organism’s genomic DNAGC ratio. The GC ratio is defined as the precentage ofguanine plus cytosine in an organism’s DNA.

Microbial Taxonomy

Microbial Taxonomy

Microbial Taxonomy

Ranges of genomic DNA base compotition of various organism.Note that the greatest range of GC ratios exists with bacteria.

Some phenotypic characteristics of taxonomic value

Major Catagory

I. Morphology

II. Motility

III. Nutrient and

Physiology

IV. Other Factors

Components

Shape; size; gram reaction; arrangement offlagella; if present

Motile by flagella; motile by gliding; motile by gasvesicles; nonmotile

Mechanism of energy conservation (phototroph,chemoorganotroph, chemolitotroph); relation tooxygen; temperature; pH; and saltrequirements/tolerances; ability to use variouscarbon; nitrogen; and sulfur sources; growth factorrequirements

Pigments; cell inclusions; or surface layers;pathogenicity; antibiotic sensitivity

Microbial Taxonomy

Example of methods that would be used for identification of a newlyisolated enteric bacterium. This scheme uses classical microbiologicalmethods (the example given shows the procedures that would be used foridentifying Eschericia coli).

Molecular Taxonomy or Chemotaxonomy

Involves molecular analyses of one or more constituentsin the cell. Ribotyping, Genomic DNA:DNA hybridization,multilocus sequence typing, and lipid profiling.

RibotypingRibosomal RNA-based phylogenetic characterizations.Unlike comparative sequencing methods, ribotypingdoes not involve sequencing. Instead, it measures theunique pattern that is generated when DNA from anorganism is digested by a restriction enzyme and thefragments are separated and probed with a ribosomalRNA probe.

Microbial Taxonomy

Microbial Taxonomy

DNA:DNA HybridizationGenomic hybridization measures the degree ofsequence similarity in two DNAs and is useful fordifferentiating very closely related organism where rRNAsequencing may fail to be definitive.

Multilocus Sequence Typing (MLST)MLST involves sequencing fragments of six to seven“houskeeping genes” from an organism and comparingthese with the same gene set from different strains of thesame organism. The comparative sequencing data isthen expressed in a dendogram.

Fatty Acid Analyses: FAMECharacterization of the types and proportions of fattyacids present in cytoplasmic membrane and outermembrane (gram-negative bacteria) lipids of cells.

How Many Prokaryotic Species Are There?

Several thousand prokaryotic species are alreadyknown and several thousands more, perhaps as

many as 100,000 – 1,000,000 in total (or 10times this by some estimates) are suspected toexists. By anyone’s count, the final number ofprokaryotic species will likely be enormous.

Microbial Taxonomy

Taxonomic ranks and numbers of known prokaryotic species

Ranks

Domains

Phyla

Classes

Orders

Families

Genera

Species

Bacteria

1

25

34

78

230

1227

6740

Archaea

1

4*

9

13

23

79

289

Total

2

29

43

91

253

1306

7029

* The phyla category for Archaea includes the Korarchaeota, and theNanoarchaeota, not yet officially recognized phyla.

Microbial Taxonomy

Nomenclature And Bergey’s Manual

• Following the binomial system of nomenclature usedthroughout biology, prokaryotes are given genus namesand species ephitets.

• The nomenclature of prokaryotes, Bacteria as well asArchaea, is regulated by the rules of the BacteriologicalCode-the International Code of Nomenclature ofBacteria.

• International culture collection : American Type CultureCollection (ATCC, Manassas, Virginia, USA), DeutscheSammlung von Microorganismen und Zellkulturen(DSMZ, German Collection for Microorganisms,Braunschweig, Germany).

Microbial Taxonomy

• IJSEM (International Journal of Systematic andEvolutionary Microbiology). In each issue the IJSEMpublishes and approved list of newly created names andserves as the publication of record for research inprokaryotic taxonomy.

• By validating newly proposed names, IJSEM paves theway for their inclusion in Bergey’s Manual of SystematicBacteriology, a major taxonomic treatment ofprokaryotes.

• A second major reference in prokaryotic diversity is TheProkaryotes.

Microbial Taxonomy