Midterm Exam #2
MB 451 : Microbial Diversity
Honor pledge: “I have neither given nor received unauthorized aid on this test.”
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Date : __________________________
Key
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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
E. none 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
E. none of the above
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 βproteobacterium
C. member of the Bacteriodes group
D. sequence derived from different organisms at
each end
E. none of the above
6. __ C__ The hydroxypropionate pathway is used by
Chloroflexus to … (2pts)
A. synthesize hydroxypropionate
B. fix nitrogen
C. fix carbon
D. perform cyclic photophosphorylation
E. none of the above
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
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8. __ A__ The prevalent genus of bacteria in feces is …
(2pts)
A. Clostridium
14. __ B__ The root of the universal phylogenetic tree
separates the three Domains into … (2pts)
B. Bacteroides
A. Archaea vs Bacteria & Eukarya
C. Escherichia
B. Bacteria vs Archaea & Eukarya
D. Lactobacillus
C. Eukarya vs Archaea & Bacteria
E. Verrucomicrobium
D. Archaea vs Bacteria vs Eukarya
E. none of the above
9. __ E__ Which of the following phylogenetic groups
contains no cultivated members? (2pts)
A. γ-proteobacteria
15.__ D__ The transfer of genes from one phylogenetic
group to another is called … (2pts)
B. Coprothermobacter
A. endosymbiosis
C. Acidobacterium & relatives
B. Southern hybridization
D. Verricomicrobia
C. vertical transfer
E. none of the above
D. horizontal transfer
E. inheritance
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
E. none of the above
11. __ B__ Which of the following is not a phenotype
common to the δ-proteobacteria? (2pts)
A. bacterial intracellular parasites
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
E. none of the above
17. __ C__ Cultivated ε-proteobacteria are … (2pts)
B. anaerobic phototrophs
A. anaerobic phototrophs
C. gliding rods that form fruiting bodies
B. thermophiles
D. sulfate reducers
C. parasites & normal flora of the GI tract
E. all of these are common in δ-proteobacteria
D. sulfate reducers
E. all of the above
12. __ A__ Proteorhodopsin ... (2pts)
A. pumps protons
18. __ A__ A FISH probe is … (2pts)
B. pumps chloride
A. a fluorescently-labeled oligonucleotide
C. signals the presence of light
B. a radioactively-labeled organic compound
D. synthesizes retinal
C. a heavy-isotope labeled growth substrate
D. a microscope/infrared laser combination
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
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
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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
Major phylogenetic group it belongs to
Escherichia
GAMMA PROTEOBACTERIA
Thermus
THERMUS/DEINOCOCCUS GROUP
Treponema
SPIROCHAETES
Thermocrinus
AQUIFEX & RELATIVES
Bacteroides
BACTEROIDS
Chlorobium
GREEN SULFUR BACTERIA
Roseiflexus
GREEN NON-SULFUR BACTERIA
Pseudomonas
GAMMA PROTEOBACTERIA
Campylobacter
EPSILON PROTEOBACTERIA
Desulfovibrio
DELTA PROTEOBACTERIA
Deinococcus
THERMUS/DEINOCOCCUS GROUP
Aquifex
AQUIFEX & RELATIVES
Neisseria
BETA PROTEOBACTERIA
Borellia
SPIROCHAETES
Flavobacterium
BACTEROIDS
Chloroflexus
GREEN NON-SULFUR BACTERIA
Bordetella
BETA PROTEOBACTERIA
Myxococcus
DELTA PROTEOBACTERIA
Cytophaga
BACTEROIDS
Bdellovibrio
DELTA PROTEOBACTERIA
Beggiatoa
GAMMA PROTEOBACTERIA
Thauera
BETA PROTEOBACTERIA
Chromatium
GAMMA PROTEOBACTERIA
Salmonella
GAMMA PROTEOBACTERIA
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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 PERIPLASMIC 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 DENATURATION 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.
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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 DENATURATION 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 DENATURATION 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 SSURDNA 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.
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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
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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. DESCRIPTION OF EITHER OF THESE APPROACHES WOULD BE ACCEPTABLE. OTHER ANSWERS MAY ALSO BE POSSIBLE
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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.
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