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Prokaryotic Microbial Diversity 1 • 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 3 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 www.steve.gb.com/science/transcription.html 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 7 • 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. 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 H2S 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; 2nd 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