1. MICROBIAL EVOLUTION AND SYSTEMATICS 4 ADVANCE MICROBIOLOGY

2. 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 star exploded, generating a new star (sun) and the other components of our solar system. The oldest rocks discovered thus far are in the Itsaq gneiss complex, in southwestern Greenland, which date to about 3.86 billion years ago. Ancient rocks are three types: sedimentary, volcanic, and carbonaceous. The formation of sedimentary rocks requires liquid water- -------------The presence of liquid water in turn implies that conditions were compatible with life.

3. The oldest known stromatolite, found in a rock about 3.5 billion years old, from the Warrawoona Group in Western Australia. Stromatolites of conical shape from 1.6-billion-year-old dolomite rock of the McArthur basin of the Northern Territory of Australia.

4. Modern stromatolites in a warm marine bay, Shark Bay, Western Australia. Modern stromatolites composed of thermophilic cyanobacteria. Another view of modern and very large stromatolites from Shark Bay.

5. Evolution of Earth and Earliest Life Forms Evidence for microbial life in ancient rocks rest on the fossilized remains of cells and the isotopically “light” carbon abundant in these rocks. Some ancient rocks contain microfossils that appear very much like bacteria, typically as simple rods or cocci. Stromatolites: - Fossilized microbial mats consisting of layers of filamentous prokaryotes and trapped sediment. - In rocks of 3.5 gigayears (GY) or younger - Formed by filamentous phototrophic bacteria, perhaps relatives of the green nonsulfur bacterium Chloroflexus. Evidence for Microbial Life on Early Earth

6. Evolution of Earth and Earliest Life Forms Ancient microbial life. Scanning electron micrograph of microfossil prokaryotes from 3.45 billion year old rocks of the Barberton Greenstone Belt, South Africa. Note the rod-shaped bacteria (arrow) attached to particels of mineral matter. The cells are about 0.7 µm in diameter.

7. a b Fossil prokaryotes and eukaryotes from more recent rocks than those shown. (a) show fossil prokaryotic microorganisms from the Bitter Springs Formation. (b) Microfossils of eukaryotic cells from the same rock formation. The cellular structure is remarkably similar to that of certain modern algae, such as Chorella species. Evolution of Earth and Earliest Life Forms

8. 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 abundant being methane, carbon dioxide, nitrogen, and ammonia. Trace amounts of carbon monoxide and hydrogen likely existed, as well as large reservoirs of sulfide, as mixture of H 2 S and FeS. It is also likely that a considerable amount of hydrogen cyanide, HCN, was produced on early Earth when NH 3 and CH 4 reacted to yield HCN. Most evidence suggests that the early Earth was hotter than it is today. For its first two hundred million years or so, the surface of Earth may have exceeded 100 0 C. Early life form were likely quite heat tolerant, and may have resembled hyperthermophilic prokaryotes in this regard.

9. Origin of Life The synthesis of biomolecules can occur spontaneusly if reducing atmospheres containing the aformentioned gases are subjected to intense energy sources. If gaseous mixtures resembling those thought to be present on primitive Earth are irradiated with UV or subjected to electric discharges in the laboratory today, a variety of biomolecules form. These include sugars, amino acids, purines, pyrimidines, various nucleotide, thioester, and fatty acids. Under simulated prebiological conditions in laboratory some of these biochemical building blocks have been shown to polymerize, leading the formation of polypeptides, polynucleotides, and other important macromolecules. We can therefore imagine that by whatever means, a mixture of organic 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

10. Catalysis and The Importance of Montmorillonite Clay To obtain spontaneous synthesis of macromolecules on early Earth, a catalyst would have had to be avilable. Good possibibilities include the surface of clays or pyrite (FeS 2 ) 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.

11. RNA Life In an RNA world, if it ever existed, various self-replicating RNAs would have carried out the catalytic reactions necessary for their self-replication. Self-replicating RNA life forms may have evolved into the first celluar life forms when they become enclosed within lipoprotein vesicles. The proper constituents and set of circumtances come together and a primitive enclosed structure arose, capable of self-replication. Although still lacking DNA and proteins, this protocellular life form would otherwise have resembled a modern cell. As primitive organisms become more complex and catalytic reactions 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 entirely by proteins as cellular enzymes. By this point, the modern cell was clearly on the horizon. The RNA World and Molecular Coding

12. Possible scenario for the evolution of cellular life forms from RNA life forms. Self-replacing RNAs could have become cellular entities by becoming stably integrated into lipoprotein vesicles. With time, proteins replaced the catalytic functions of RNA, and DNA replaced the coding functions of RNA

13. The Modern Cell: DNA-RNA-Protein Somewhere in the early stages of microbial evolution the three-part system-DNA, RNA, Protein-become fixed in cells as the best solution to biological information processing. That this system was an evolutionary success story obvious, since without exeption, modern cells contain all three types of these macromolecules. The RNA World and Molecular Coding

14. Energy and Carbon Metabolism The Primitive Life A Possible Energy- generating scheme for primitive cell. Formation of pyrite leads to H 2 production and S 0 reduction, which fuels a pimitive ATPase. Note how H 2 S plays only a catalytic role; the net subtrates would be FeS and S 0. Also note how few dofferent proteins would be reqiured. An alternative source of H 2 could have been the UV-catalyzed reduction of H + by Fe 2+ as shown.

15. Major Landmarks in biological evolution and Earth’s changing geochemistry. Note how the oxygenation of the atmosphere due to cyanobacterial metabolism was a gradual process, occuring over a period of about 2 billion years. Althaough full (20%) oxygen levels are required for animals and most other higher organism, this is not true of prokaryotes, as many are facultative aerobes or microaerophiles. Thus, prokaryotes respiring at reduced O 2 levels may have dominated Earth for the period of a billion years or so before Earth’s atmosphere reached current levels of oxygen.

16. Endosymbiosis The modern (organell-containing) eukaryotic cell did not come about from the gradual partitioning of cellular funtions into enclosed structures, the organells. Instead, organells originated from the stable incorporation of chemoorganotrophic and phototrophic symbionts from the domain bacteria. Endosymbiosis, probably began when an aerobic bacterium established stable residency within the The Primitive life cytoplasm of a primitive eukaryote and supplied the cell with energy in exchange for a protected environment and a ready supply of nutrient. This symbiont was the forerunner of the modern mitochondrion.

17. The Primitive life The endosymbiotic uptake of an oxygenic phototroph conferred photosynthesic properties on a primitive eukaryote; such a cell was freed from reliance on organic compounds for energy production and become phototrophic. The phototrophic endosymbiont was the forerunner of the modern day chloroplast. A few eukaryotic cells either never incorporated endosymbionts or, what appears more likely, had them at one time and then disposed of them, possibly bacause their niche was permanently anoxic. Microfossil records suggest that endosymbiotic events began sometime after about 2 gigayear ago.

18. Origin of the modern eukaryotic cell by endosymbiotic events. Note how organells originated from Bacteria rather than Archaea. Some primitive eukaryotes either never underwent endosymbiotic events or permanently lost their symbionts, but otherwise maintained the basic properties of eukaryotic cells.

19. Signature Sequences Computer analyses of ribosomal RNA sequence have revealed so-called signature sequences, short oligonucleotides unique to certain groups of organism (genus or species can be determined by computer inspection of aligned sequences). Phylogenetic Probes and FISH Recall that probe is a strand of nucleic acid that can be labeled and used to hybridize to a complementary nucleic acid from a mixture. Probes can be general or specific. Signature Sequences, Phylogenetic Probes, and Microbial Community Analyses Signature Sequences, Phylogenetic Probes, and Microbial Community Analyses

20. FISH. Flourescent in-situ hybridization; a process in which a cell is made flourescent by labeling it with a specific nucleic acid probe that contains an attached flourescent dye. Same field, cells stained with a yellow- green universal rRNA probe (this probe reacts with species from any domain. Phase contrast photomicrograph (no probe present) of cells of Bacillus megaterium (rod, member of the Bacteria) and the yeast Saccharomyces cerevisiae (oval shaped cells, Eukarya) Same field, cells stained with a eukaryal probe (only cells of Saccharomyces cerevisiae react).

21. Signature Sequences, Phylogenetic Probes, and Microbial Community Analyses Use of phylogenetic stain to make nitrifying bacteria visible in a granule of activated sewage sludge.

22. Microbial Community Analysis PCR-amplified ribosomal RNA genes do not need to originate from pure culture grown in the laboratory. A phylogenetic snapshot of a natural microbial community can be taken using PCR to amplify the genes encoding SSU ribosomal RNA 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 RNAs present in community. From this tree, specific organism can be inferred even though none of them were actually cultivated or otherwise identified. Signature Sequences, Phylogenetic Probes, and Microbial Community Analyses

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

24. Microbial Phylogeny

25. Characteristics of the Domain of Life BacteriaArchaeaEukarya Lack peptidoglycan (pseudopeptidoglycan, made of polysaccharide, protein or glicoprotein) Consist of ether-linked molecules Structurally more complex than those bacteria, contain eight or more polypeptides 70S ribosomes The initiator tRNA carries an unmodified methionine Lack peptidoglycan (made of cellulose or chitin) Synthesize membrane lipid with a backbone consisting of fatty acid bonded in ester linked to a molecule of glycerol The major RNA polymerase (there are three) contains 10-12 polypeptides 80S ribosomes The initiator tRNA carries an unmodified methionine in a a of Cell Walls Lipids RNA RNA Polymerase Polymerase Features of Protein Synthesis,,,, ’,’, ) Containning Containning peptidoglycan peptidoglycan Synthesize membrane lipid with aabackbone consisting of fatty acid bonded in ester linked to to molecule of glycerol Contain aasingle type of RNA polymerase (four polypeptides, ααββββ σσ) 70S ribosomes An initiator tRNA containing aamodified methionine residue, formylmethionine

26. Classical Taxonomy Taxonomy, the science of classification, consist of two major subdisciplines, identification and nomenclature. Taxonomy: relies on phenotypic analyses (what an organism looks like, its energy metabolism, its enzyme, and other properties). Phylogeny: has emerged from genotypic analyses. GC Ratio ----- One property that is informative in drawing taxonomic conclusions is an organism’s genomic DNA GC ratio. The GC ratio is defined as the precentage of guanine plus cytosine in an organism’s DNA. Microbial Taxonomy

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

28. 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 of flagella; if present Motile by flagella; motile by gliding; motile by gas vesicles; nonmotile Mechanism of energy conservation (phototroph, chemoorganotroph, chemolitotroph); relation to oxygen; temperature; pH; and salt requirements/tolerances; ability to use various carbon; nitrogen; and sulfur sources; growth factor requirements Pigments; cell inclusions; or surface layers; pathogenicity; antibiotic sensitivity

29. Microbial Taxonomy Example of methods that would be used for identification of a newly isolated enteric bacterium. This scheme uses classical microbiological methods (the example given shows the procedures that would be used for identifying Eschericia coli).

30. Molecular Taxonomy or Chemotaxonomy Involves molecular analyses of one or more constituents in the cell. Ribotyping, Genomic DNA:DNA hybridization, multilocus sequence typing, and lipid profiling. Ribotyping Ribosomal RNA-based phylogenetic characterizations. Unlike comparative sequencing methods, ribotyping does not involve sequencing. Instead, it measures the unique pattern that is generated when DNA from an organism is digested by a restriction enzyme and the fragments are separated and probed with a ribosomal RNA probe. Microbial Taxonomy

31. DNA:DNA Hybridization Genomic hybridization measures the degree of sequence similarity in two DNAs and is useful for differentiating very closely related organism where rRNA sequencing may fail to be definitive. Multilocus Sequence Typing (MLST) MLST involves sequencing fragments of six to seven “houskeeping genes” from an organism and comparing these with the same gene set from different strains of the same organism. The comparative sequencing data is then expressed in a dendogram. Fatty Acid Analyses: FAME Characterization of the types and proportions of fatty acids present in cytoplasmic membrane and outer membrane (gram-negative bacteria) lipids of cells.

32. How Many Prokaryotic Species Are There? Several thousand prokaryotic species are already known and several thousands more, perhaps as many as 100,000 – 1,000,000 in total (or 10 times this by some estimates) are suspected to exists. By anyone’s count, the final number of prokaryotic species will likely be enormous. Microbial Taxonomy

33. 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 the Nanoarchaeota, not yet officially recognized phyla. Microbial Taxonomy

34. Nomenclature And Bergey’s Manual Following the binomial system of nomenclature used throughout biology, prokaryotes are given genus names and species ephitets. The nomenclature of prokaryotes, Bacteria as well as Archaea, is regulated by the rules of the Bacteriological Code-the International Code of Nomenclature of Bacteria. International culture collection : American Type Culture Collection (ATCC, Manassas, Virginia, USA), Deutsche Sammlung von Microorganismen und Zellkulturen (DSMZ, German Collection for Microorganisms, Braunschweig, Germany). Microbial Taxonomy

35. IJSEM (International Journal of Systematic and Evolutionary Microbiology). In each issue the IJSEM publishes and approved list of newly created names and serves as the publication of record for research in prokaryotic taxonomy. By validating newly proposed names, IJSEM paves the way for their inclusion in Bergey’s Manual of Systematic Bacteriology, a major taxonomic treatment of prokaryotes. A second major reference in prokaryotic diversity is The Prokaryotes. Microbial Taxonomy