Midterm Exam #2. MB 451 : Microbial Diversity. 1. What are the 3 primary evolutionary branches of life? (5pts) ARCHAEA, BACTERIA, EUKARYA

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1 Midterm Exam #2 MB 451 : Microbial Diversity Honor pledge: I have neither given nor received unauthorized aid on this test. Signed : Date : Name : Key 1. What are the 3 primary evolutionary branches of life? (5pts) ARCHAEA, BACTERIA, EUKARYA 2. A The generation of phylogenetic trees containing all kinds of organisms lead to the discovery of (2pts) A. Archaea B. horizontal transfer C. microbial species D. all of the above 3. B The root of the universal tree can be determined by phylogenetic analysis of (2pts) A. small subunit ribosomal RNA B. ancient duplicated genes C. horizontal transfer D. microbial populations 4. B Which of the following is not a form of bacterial motility? (2pts) A. flagellar rotation B. ciliary beating C. gliding D. twitching E. whatever spirochaetes do F. all are forms of bacterial motility 5. D In a molecular phylogenetic analysis of a microbial community, a chimera is a (2pts) A. single organism with two or more very different rrna gene sequences B. symbiosis between Chlorobium and a β- proteobacterium C. member of the Bacteriodes group D. sequence derived from different organisms at each end 6. C The hydroxypropionate pathway is used by Chloroflexus to (2pts) A. synthesize hydroxypropionate B. fix nitrogen C. fix carbon D. perform cyclic photophosphorylation 7. E The election transport chain (2pts) A. pumps protons from one side of the membrane to the other B. separates oxidation/reduction reactions into their constituent half-reactions C. can be run in reverse to generate NADH or NADPH D. creates a chemical (H + ) and electrical gradient E. all of the above Page 1 of 8

2 8. A The prevalent genus of bacteria in feces is (2pts) A. Clostridium B. Bacteroides C. Escherichia D. Lactobacillus E. Verrucomicrobium 9. E Which of the following phylogenetic groups contains no cultivated members? (2pts) A. γ-proteobacteria B. Coprothermobacter C. Acidobacterium & relatives D. Verricomicrobia 10. C In a denaturing gradient gel (DGGE), rdnas are separated by (2pts) A. size B. charge/mass ratio C. denaturation point D. hydrodynamic radius 11. B Which of the following is not a phenotype common to the δ-proteobacteria? (2pts) A. bacterial intracellular parasites B. anaerobic phototrophs C. gliding rods that form fruiting bodies D. sulfate reducers E. all of these are common in δ-proteobacteria 12. A Proteorhodopsin... (2pts) A. pumps protons B. pumps chloride C. signals the presence of light D. synthesizes retinal 13. E Which of the following are not based on the electron transport chain? (2pts) A. Sulfate reduction B. heterotrophy C. photosynthesis D. sulfur oxidation E. all of these use the electron transport chain 14. B The root of the universal phylogenetic tree separates the three Domains into (2pts) A. Archaea vs Bacteria & Eukarya B. Bacteria vs Archaea & Eukarya C. Eukarya vs Archaea & Bacteria D. Archaea vs Bacteria vs Eukarya 15. D The transfer of genes from one phylogenetic group to another is called (2pts) A. endosymbiosis B. Southern hybridization C. vertical transfer D. horizontal transfer E. inheritance 16. A The most commonly seen organisms (sequences, really) in molecular phylogenetic analyses of microbial populations are (2pts) A. proteobacteria B. Gram-positive Bacteria C. cyanobacteria D. members of groups with no cultured members 17. C Cultivated ε-proteobacteria are (2pts) A. anaerobic phototrophs B. thermophiles C. parasites & normal flora of the GI tract D. sulfate reducers E. all of the above 18. A A FISH probe is (2pts) A. a fluorescently-labeled oligonucleotide B. a radioactively-labeled organic compound C. a heavy-isotope labeled growth substrate D. a microscope/infrared laser combination E. a way to measure O2 concentration in situ F. a computer program to detect chimeras G. a machine for high-throughput sequencing H. all of the above I. none of the above J. a sonar for your bass boat Page 2 of 8

3 19. For each genus, give the major phylogenetic group to which it belongs. Answer 21 out of the 24; if you answer more, only the first 21 will be graded. (1pt each) A list of these major phylogenetic groups is given on the last page of the exam. Genus Escherichia Thermus Treponema Thermocrinus Bacteroides Chlorobium Roseiflexus Pseudomonas Campylobacter Desulfovibrio Deinococcus Aquifex Neisseria Borellia Flavobacterium Chloroflexus Bordetella Myxococcus Cytophaga Bdellovibrio Beggiatoa Thauera Chromatium Salmonella Major phylogenetic group it belongs to THERMUS/DEINOCOCCUS GROUP SPIROCHAETES AQUIFEX & RELATIVES BACTEROIDS GREEN SULFUR BACTERIA GREEN NON-SULFUR BACTERIA EPSILON PROTEOBACTERIA DELTA PROTEOBACTERIA THERMUS/DEINOCOCCUS GROUP AQUIFEX & RELATIVES BETA PROTEOBACTERIA SPIROCHAETES BACTEROIDS GREEN NON-SULFUR BACTERIA BETA PROTEOBACTERIA DELTA PROTEOBACTERIA BACTEROIDS DELTA PROTEOBACTERIA BETA PROTEOBACTERIA Page 3 of 8

4 20. Describe an organism (species or genus) we ve talked about in class. (5 pts) EXAMPLE ANSWER: BEGGIOTOA : MICROAEROPHILIC FILAMENTOUS GLIDERS THAT OXIDIZE H2S TO SULFUR (STORED 'FOR LATER' IN PERI- PLASMIC GLOBULES)AND EVENTUALLY TO SULFATE. THEY ARE FOUND IN SULFUR SPRINGS, MARINE SEDIMENTS, DEEP SEA HYDROTHERMAL VENTS, AND ABOUT ANYWHERE THERE'S SULFIDE AND A LITTLE OXYGEN. MOST HAVE NEVER BEEN GROWN IN PURE CULTURE. 21. Describe another organism, from a different major phylogenetic group. (5 pts) EXAMPLE ANSWER: THERMUS AQUATICUS: THERMOPHILIC HETEROTROPHIC RODS FOUND IN MOST THERMAL ENVIRONMENTS AROUND THE WORLD (EVEN HOUSEHOLD WATER HEATERS), IT WAS FIRST ISOLATED FROM MUSHROOM SPRING, OCTOPUS SPRING, AND OTHER SPRINGS IN THE WHITE CREEK AREA OF YELLOWSTONE NATIONAL PARK. THERMUS AQUATICUS IS THE SOURCE OF TAQ POLYMERASE USED IN PCR - SINCE THE THERMUS DNA POLYMERASE IS THERMOSTABLE, IT SURVIVES THE DENATURA- TION STEP & DOESN'T HAVE TO BE ADDED IN EACH ROUND, ALLOWING AUTOMATION OF THE PROCEDURE. IT WAS THE USE OF TAQ POLYMERASE THAT LEAD TO THE WIDESPREAD USE OF PCR, AND DEMONSTRATED THE GENERAL UTILITY OF THERMOPHILIC ENZYMES (OTHER THAN LAUNDRY DETERGENT PROTEASES), NOW A HUGE INDUSTRY. Page 4 of 8

5 22. Describe in detail one method used to perform an ssu-rrna molecular phylogenetic analysis of a microbial environment. (10pts) Do not describe the same technique in your answer to question 24! EXAMPLE ANSWER: DGGE STARTS OUT LIKE ALMOST MOLECULAR PHYLOGENETIC ANALYSIS DOES THESE DAYS; BY THE ISOLATION OF DNA FROM ENVIRONMENTAL SAMPLES, FOLLOWED BY PCR OF SSU-RRNA GENES. RATHER THAN CLONING AND SEQUENCING FROM THIS POOL OF GENES, HOWEVER, THEY ARE FIRST SEPARATED INTO UNIQUE SEQUENCES BASED ON THEIR DENATU- RATION PROPERTIES. DGGE IS CARRIED OUT IN POLYACRYLAMIDE GELS IN WHICH THE CONCENTRATION OF UREA AND FORMAMIDE INCREASES FROM TOP TO BOTTOM IN THE GEL; I.E. THE GEL CONTAINS A GRADIENT OF DENATURANTS. (REMEMBER THAT DENATU- RATION OF DNA MEANS SEPARATION OF THE TWO STRANDS.) THE PCR-AMPLIFIED SSU-RDNA IS LOADED IN WELLS AT THE TOP OF THE GEL, WHERE THE CONCENTRATION OF UREA/FORMAMIDE IS TOO LOW TO DENATURE THE DNA. AS THE SSU- RDNA MIGRATES DOWN THE GEL DURING ELECTROPHORESIS, THE CONCENTRATION OF UREA/FORMAMIDE INCREASES UNTIL, AT SOME POINT, IT IS HIGH ENOUGH TO DENATURE THE DNA. AT THIS POINT, THE SSU-RDNA BAND ESSENTIALLY STOPS MOVING (IT SLOWS WAY DOWN). BECAUSE EVERY SSU-RDNA SEQUENCE WILL HAVE A DIFFERENT DENATURATION POINT, THEY WILL DENATURE AT DIFFERENT LEVELS OF THE GEL AND SEPARATE INTO DISTINCT BANDS DESPITE THE FACT THAT THE SSU-RDNAS IN ALL OF THE BANDS ARE ALL THE SAME SIZE. Page 5 of 8

6 23. Summarize the question/problem, approach, results and conclusion of any one of the papers discussed in class. (10 pts) A list of these papers can be found on the last page of the exam. EXAMPLE ANSWER: MICROBIAL POPULATION STRUCTURES IN THE DEEP MARINE BIOSPHERE. QUESTIONS: PROOF OF PRINCIPLE FOR THE METHOD HOW BIG IS MICROBIAL DIVERSITY? WHAT IS THE COMMUNITY COMPOSITION OF THESE ENVIRONMENTS? HOW SIMILAR ARE THE MICROBIAL COMMUNITIES IN THESE TWO NEARBY SIMILAR ENVIRONMENTS? APPROACH: THEY USED THE TRADITIONAL 16S RRNA POPULATION ANALYSIS APPROACH, EXCEPT THAT THEY AMPLIFIED ONLY A SMALL VARIABLE REGION, SEQUENCED THESE BY PRYOSEQUENCING (454 SEQUENCING) SO THAT THEY COULD GET HUNDREDS OF THOUSANDS OF SEQUENCES AND AVOID THE CLONING STEP, AND ANALYZED THE RESULTING SEQUENCES BY BINNING RATHER THAN GENERATING PHYLOGENETIC TREES. RESULTS: THEY OBTAINED ALMOST A MILLION SEQUENCES! THESE FELL INTO ABOUT 36,000 UNIQUE SEQUENCES FROM A WIDE RANGE OF ORGANISMS. THESE SEQUENCES COULD BE READILY BINNED INTO PHYLOGENETIC GROUPS CONCLUSIONS: THIS APPROACH WORKS MICROBIAL DIVERSITY (AT LEAST IN THESE SAMPLES) IS ENORMOUS AT ALL PHYLOGENETIC LEVELS THESE POPULATIONS ARE PREDOMINANTLY PROTEOBACTERIA, ESPECIALLY EPSILON PROTEOBACTERIA THESE TWO ENVIRONMENTS CONTAINED VERY DIFFERENT POPULATIONS, WITH ALMOST NO OVERLAP Page 6 of 8

7 24. In an ssu-rrna molecular survey of a dark bacterial mat on an Icelandic glacier surface, you get a predominance of sequences of two kinds of organisms you presume to be phototrophic; Cryoflexus (which is filamentous) and Fridgobacter (which is rod-shaped). Given that this environment is otherwise devoid of organic material, you presume that one of these organisms is the primary producer for this community; in other words, is fixing carbon from CO2. What approach would you use to determine which of these organisms is fixing CO2? Describe the method in detail. (10 pts) THE PAPERS WE VE DISCUSSED IN CLASS SUGGEST TWO POSSIBLE APPROACHES TO THIS PROBLEM: STABLE-ISOTOPE PROBING OR COMBINED FISH/AUTORADIOGRAPHY. IN EITHER CASE THE LABELED SUBSTRATE WOULD BE CO2. DESCRIP- TION OF EITHER OF THESE APPROACHES WOULD BE ACCEPTABLE. OTHER ANSWERS MAY ALSO BE POSSIBLE Page 7 of 8

8 E X A M I N F O R M A T I O N S H E E T Major phylogenetc groups of Bacteria we ve talked about so far: Aquifex & relatives Deinococcus/Thermus group Green non-sulfur Bacteria Green sulfur Bacteria Spirochaetes Bacteroids β-proteobacteria γ-proteobacteria δ-proteobacteria ε-proteobacteria Note: α = alpha β = beta γ = gamma δ = delta ε = epsilon List of Papers: Phylogenetic analysis of the hyperthermophilic pink filament community in Octopus Spring, Yellowstone National Park. Thermocrinus ruber, gen. nov., sp. nov., a pink-filament-forming hyperthemophilic bacterium isolated from Yellowstone National Park. Direct analysis of genes encoding 16S rrna from complex communities reveals many novel molecular species within the human gut. Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. Microscopic Examination of Distribution and Phenotypic Properties of Phylogenetically Diverse ChloroflexaceaeRelated Bacteria in Hot Spring Microbial Mats Highly diverse community structure in a remote central Tibetan geothermal spring does not display monotonic variation to thermal stress. Changes in oral microbial profiles after peridontal treatment as determined by molecular analysis of 16S rrna genes. Microbial population structures in the deep marine biosphere. Bacterial rhodopsin: Evidence for a new type of phototrophy in the sea. RNA stable isotope probing, a novel means of linking microbial community function to phylogeny. Page 8 of 8