1 Prokaryotic Microbial Diversity Early attempts at taxonomy: all plants and animals Whitaker scheme (late 20th century): Five kingdoms –Animalia, Plantae, Fungi, Protista, and Monera –Monera comprised of prokaryotes Classification of bacteria difficult –Plants, animals can be distinguished from each other by physical characteristics; backed up by DNA –Bacteria look very similar –Convergent evolution a problem Unrelated bacteria develop similar physical and biochemical traits

Goal: Evolutionary classification In order to understand relatedness, organisms must be viewed at the DNA level –Similar sequences, mutations should give clues –Which genes? Bacteria readily swap genes around. Carl Woese and 16S RNA –Ribosomal RNA genes cannot afford to mutate much Changes would interfere with protein synthesis –Change in rRNA genes over time very gradual Useful for looking at large differences among organisms 2

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Domains Sequencing rRNA genes reveal differences –Bacteria, Archaea, Eukarya all different from each other Despite Bacteria, Archaea both being prokaryotes Differences in Bacteria, Archaea backed up –Aspects of molecular biology –Membrane lipid chemistry –Cell wall chemistry –Extreme environments 4

What’s a Species? Eukaryotes: plants and animals –Generally, 2 organisms are of same species if they can successfully interbreed –Definition based on sexual reproduction –Bacteria don’t reproduce sexually Bacterial species: a group of strains that are more closely related to each other than to another group. –Members of a species have DNA that can hybridize –Because of gene exchange, mutation, phase variation, there are no sharp boundaries between species. 5

Viable, non-culturable bacteria Many bacteria present in environments: –Do not grow when placed in conventional media –include known pathogenic bacteria –Include bacteria previously unknown –Do not appear to multiply, but many can be shown to be metabolically active We just don’t know how to grow them? –Pathogenic forms can resume growing in infection –Others…? –Detected and studied using molecular techniques 6

Metagenomics Mixed population studies –Using molecular techniques (PCR, sequencing) we find various unique DNA sequences –Most of these bacteria have not/ cannot be cultured –Using molecular techniques to classify unculturable bacteria is called metagenomics Identification techniques can be molecular or traditional –Traditional techniques require isolation into pure culture, biochemical tests, sometimes serological tests. 7

Classical and MolecularTaxonomy Identification by phenotypic analysis –Shape, size, Gram stain –Basic metabolism (aerobic, fermentative, autotrophic) –Motility, pigments, metabolic products, usable carbon sources, temperature range ID by examining chemical features –Fatty acid composition (FAME) –DNA-DNA hybridization –Ribotyping (specific rRNA sequences; uses PCR) –Multilocus sequence typing (sequences from several conserved genes; uses PCR) 8

Major groups of Bacteria-1 Hyperthermophilic Gram negatives –Grow at >70 degrees Green Sulfur and Green Non-sulfur bacteria –Photosynthetic, anoxygenic –Sulfur bacteria use H 2 S as electron donor Deinococcus and relatives –Highly radiation resistant; great DNA repair –Gram negative or positive? Odd mixture of traits Cyanobacteria –“blue-green algae”; oxygenic photosynthesis 9

Major groups of Bacteria-2 Proteobacteria –Largest group of Gram negative bacteria –Enteric bacteria (E.coli, Salmonella, Shigella) –Vibrio (related to enterics; V. cholerae; curved rods) –Pseudomonads (strictly respiratory) –Various groups affecting N and sulfur cycles –Purple sulfur and Purple non-sulfur anoxygenic phototrophs –Rickettsia: obligate intracellular parasites Bacteroides and Cytophaga –First is strict anaerobic; 2 nd aerobic and gliding 10

Major groups of Bacteria-3 Gram Positive bacteria –Endospore formers (Bacillus, Clostridium) –Cocci (Staph, Strep, Micrococcus) –Other rods (Mycobacteria, Listeria, etc.) –Actinomycetes (filamentous, antibiotic producers) –Mycoplasma (DNA says G+, but no cell wall) Spirochetes –Tight spirals, internal flagella, G- Chlamydia –Obligate intracellular parasites; 2 stage life cycle 11

Archaea Methanogens and Halophiles –Methanogens strict anaerobes, make methane –Halophiles need at least 1.5 M salt Mostly hyperthermophiles –Growth from 80 upwards to 120 degrees C Third major group has one species! 12