Micr 3070 Final Exam Study Guide

How does diversity originate within microbial populations?

mutations, reassortment, selection, genetic drift (bottleneck events)

What role does selection play in microbial evolution?

fitness/ reproductive sucess

random selection/ differential success

what role does genetic drift play in microbial evolution?

bottleneck events - separation of populations

What does rapid trait change plus slow speciation mean for microbial genomes within a species?

?

What is a core genome?

genes similar across all strains

what is a pan genome?

entire set of genes within a species including core genomes

what is the current definition for a microbial species?

group of strains with shared characteristics and a common ancestor

what two methods are commonly used to characterize a new species?

compare SSU rRNA gene sequences -- %

assess genomic similarity

Describe SSU rRNA to characterize a new species\*\*

use sanger sequencing

Describe assessing genomic similarities to characterize a new species

DNA-DNA hybridization: two organisms, probe DNA and target DNA; mix DNA on a nylon membrane, adding probe DNA in excess;

if 100% hybridization=same strain; 25%=organisms 1 and 2 likely different genera

What cut-offs are used for 16S sequencing ?

at least 97% similarity

What cut-offs are used for DNA-DNA hybridization?

at least 70% similarity

What are the taxonomic methods of gene ,multigene, and genome sequencing?

multiple genes: 16s, gyrB, recA, comparing several instead of just 1

more possible to find strains

what is MLST?

multilocus sequence typing

how does MLST work?

Isolate DNA from a new isolate or clinical sample, amplify 6-7 target genes, sequence, analyze alleles, compare with other strains, and generate a tree

What is a type of phenotypic analysis?

FAME (fatty acid methyl ester) analysis

How does FAME work?

Extract fatty acids from bacterial culture, derivative them to form methyl esters, gas chromatography, peaks from various fatty acid methyl esters, compare patterns of peaks with patterns in databases and identify organisms.

Any problems with FAME?

yes, microbes change amount of fatty acids depending on environmental conditions, so have to be meticulous when growing them

Vertical vs Horizontal transmission

vertical: transmitted via mother to child (hereditary)

horizontal: transmitted to members of same generation (not hereditary)

Name the mechanisms of Horizontal gene transfer.

transformation, transduction, and conjugation

Describe transformation

uptake of naked DNA by a competent cell followed by incorporation of the DNA into the recipient cells genome

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binding DNA, uptake ss DNA, recA-mediated

homologous recombination → transformed recipient cell

describe transduction

generalized: associated with phages (virulent or temperate phages that enter lytic cycle) any piece of host genome can be transduced, random

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specialized: only for lysogenic cycle temperature phages, only certain parts of genome

describe conjugation

contact mechanism, bacterial cells have to be touching

F+, retracts to F- cell, cell pairs are stabilized, F plasmid nicked in one strand, transfer of one strand from F+ to F- cell

What other mechanisms exist for HGT?

membrane vesicles : phospholipid bilayer vesicles

nanotubles- smaller, membranes

phage-like gene transfer agents (GTAs): lost genes over time, protein expression, genomic replication

What are the possible outcomes of recombination?

Donor DNa enters recipient chromosome → partially diploid recipient cell

integration of donor DNA → reproduction → stable recombinant

donor DNA self-replicates → reproduction → stable recombinant

donor DNA cannot self replicate → recipient reproduces, donor DNA doesn’t → no stable recombinants

Host restriction (attacks) → no stable recombinant

How are HGT events detected?

look for gene not found in relatives

differences in GC content

phylogenetic discordance

How often does HGT occur between related organisms?

very difficult to determine but occurs across domains

Do rates of HGT vary across organisms?

yes, depends on similarity between recipient and donor organism

What sorts of things impact rates of HGT?

rate is dependent on

* amount of free DNA; rate of transformation, number of phages, number of cells with F plasmid
* amount of benefit/selective advantage

What is the overall impact of HGT on phylogeny?

unknown: bigger contributor to early evolutionary history, BIG contributor to diversity (speciation events), possible makes phylogenetic relationships between prokaryotes meaningless

What is the great plate count anomaly?

difference between number of microbes observed by microscopy vs number of colonies that form from the same sample

what are some proposed reasons for discrepancy in the great plate count anomaly?

microscopic counts include dead cells, colony counts do not

mostly use standard media and conditions, where vast majority don’t grow under these conditions

Why is cultivation of microbes important?

phenotypic reasons (visualization)

can show efficiency and rates in media

Explain the idea behind enrichment cultures.

targeting a microbe of interest from a mixed community

set growth conditions to encourage growth of target organisms while inhibiting growth of other organisms

Why is GFP not a useful tool for natural populations?

GFP is not naturally produced

Why is GFP not a useful tool for anaerobic populations?

GFP requires oxygen to work/ for fluorescence to work, so it will not work on anaerobic bacteria

Describe FISH

Fluorescent In Situ Hybridization

* nucleic acid probe hybridizes to a complementary sequence in a target gene or RNA
* probe has fluorescent dye attached
* hybridization of target + probe = fluorescence
* probes complement ribosomal RNA
* whole cells fluoresce
* can be general or specific
* small individual response

Describe CARD-FISH

Catalyzed Reporter Deposition FISH

* used for gene expression on slow growing microbes
* highly sensitive to RNA content, microbes living in environments with low nutrients (oligotrophs) grow slowly and have low RNA contnet that CARD-FISH can detect
* horseradish peroxidase-probe
* few targets

When would you use CARD-FISH instead of FISH?

slow growing microbial communities?

What are some common problems with PCR-based techniques?

introduce bias

* amplifying wrong target
* cell lyses
* sample impurities

How would you use PCR and sequencing to determine the active portion of a community?

dual analysis PCR

* DNA: use 16s rRNA to identify microbes present
* RNA - 16s rRNA (transcriptionally active automatically)

Define metagenomics

sequence and analyze microbial genomes from an environment- functional, nonfunctional, dead/alive

* targeting any and all genes
* huge amounts of data (high throughput sequencing is necessary)
* used for finding info that cannot be found through culturing
* tells potential of community

define metatranscriptomics

only investigates genes actually being expressed

* investigating gene expression of entire community
* uses same basic methods as metagenomics
* 1. reverse transcriptase to convert mRNA → cDNA
* RNA- not made if organisms not alive
* gives better idea of what is actually occurring, finds activity
* mRNA: smaller quantities in enviro sample (unstable)

Define metaproteomics

protein expression of entire community

* gives snapshot of what is occurring at a given time

How do metagenomic studies link function and diversity?

produces direct link between genes and function without necessarily needing to culture a sample → links genes to phenotypes

whole picture of community

what challenges are there to studying the metatranscriptome of a sample/ environment?

smaller amounts of mRNA, can’t amplify the mRNA without introducing bias, and mRNA is unstable

Why would one want to study the metatranscriptome or the metaproteome rather than metagenome?

to see what is actually alive and active

explain the basic procedure for microautoradiography.

1. incubate cells with substrate containing radioactive isotope
2. fix cells
3. dip slide in photographic emulsion and incubate in dark
4. silver grains are produced as substrate decays


1. silver grains form black dots in and around cells

Why would combining microautoradiography with FISH be useful?

tells which cells use a particular substrate, FISH identifies the cell

How would stable isotope probing allow you to identify which organisms in a particular environment are utilizing a substrate of interest?

DNA separates based on the incorporation of heavier or lighter isotopes, isolates and analyzes heavy DNA

tag the substrate with heavier isotope, so will be able to see the microbe that used the substrate

what are some potential issues with stable isotope probing?

* have to be able to culture
* can it use glucose? this does not mean this is efficient or preferred by the microbe
* microbes use and share end products
* product is labeled and then labels second microbe that doesn’t actually use substrate
* sometimes modification happens outside of the cell so cells that can’t use substrate are labelled

What is meant by assimilative processes?

\-consumes energy

\-products end up assimilating into cellular material

\-do just enough to build new cells, nothing more

What is meant by dissimilative processes?

\-a large amount of electron acceptor must be reduced to guarantee generation of enough energy (anaerobic respiration)

\-captured energy doesnt end up in the cell (excreted)

\-do this ALOT bc it gets energy

What is autotrophy?

CO2 is used as a carbon source (phototrophs and chemolithotrophs)

\-almost always have to gain energy from another source

What is phototrophy?

energy from the sun is captured and converted into chemical energy in the form of ATP

what is the purpose of the light reactions in photosynthesis?

to trap light energy to produce ATP and NADPH

What is the purpose of the light-independent/ dark reactions in photosynthesis?

use products from the light reactions, ATP and NADPH, to make glucose

What is the difference between oxygenic and anoxygenic photosynthesis?

oxygenic gets reducing power from H2O (produces O2), uses CO2 for carbon source, ADP→ATP, and is used by cyanobacteria

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anoxygenic: reducing power from H2S, CO2 for carbon source, ADP→ATP, and is used by purple and green sulfur bacteria, does NOT produce O2

What drives the difference between oxygenic and anoxygenic photosynthesis?

where would you find photosynthetic machinery in prokaryotic phototrophs?

membrane invaginations and chlorosomes

\-not in chloroplasts

mostly in plasma membranes

Describe anoxygenic photosynthesis in terms of electron flow.

cyclical - no net gain or loss of electrons

how is ATP generated by photosynthesis?

by PMF as electron follow cyclic phosphorylation

how is NADH generated in photosynthesis?

reducing power from reverse electron flow

What is meant by cyclic photophosphorylation?

electrons expelled by photosystem I, feed back into the system

What is meant by reverse electron flow?

light enters → electrons go up electron tower → electrons in excited state → fall down, circle around back to beginning

Why is reverse electron flow necessary?

aerobic lithotrophs need to reduce NADH

Describe oxygenic photosynthesis in terms of electron flow.

z scheme

How is ATP generated in oxygenic photosynthesis?

first photosystem

how is NADH generated in oxygenic photosynthesis?

second photosystem - reducing power

What happens when oxygenic phototrophs perform anoxygenic photosynthesis?

\-oxygenic not always beneficial

\-photosystem II is shut off

\-use different donor

How does iron oxidation differ in an acidic vs a neutral environment? why?

acidic:

\-oxidation occurs more slowly, not spontaneous

\-does it themselves, not getting a lot of energy out of the process,

\-have to oxidize tons of iron, short ETC, need to keep intercellullar pH neutral

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neutral:

\- spontaneous and occurs rapidly

\-maintains own PMF, makes ATP, iron is better e- donor

\-location specific due to oxic/anoxic interface,

\-take O2 for aerobic respiration, hopefully grab iron before it spontaneously oxidizes, converts Fe2+→Fe3+ (soluble to insoluble), before oxygen does

Why does Iron oxidation differ between acidic and neutral environments?

iron(III) ions have low solubility, so it is less bioavailable at neutral pH than at low pH

What are the impacts of the differences between acidic and neutral environments for iron oxidation?

in acidic: stressful, working harder to keep neutral pH and make ATP

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in neutral: don’t have the same problems, but have to be careful of location (oxic/anoxic interface)

What happens to the ETC and PMF for iron oxidizers?

ETC is short

must oxidize huge amounts of iron to generate enough proton motive force to make ATP

Does production of ATP “cost” energy for iron oxidizers?

yes, they have to maintain proton motive force for ATP production

What is nitrification?

two-step process of converting ammonia to nitrate

What are the two steps of nitrification?

1) ammonia to nitrite via ammonia oxidizers

2) nitrite to nitrate via nitrite oxidation

Do microbes generally perform one or both steps of nitrification?

usually do one step, but Nitrospira can do both

How are acetogenesis and methanogenesis similar

\-anoxic environment

\-reducing CO2

\-H2 is e- donor

\-use ion motive force to make ATP (sodium or protons)

How do acetogens generate ATP?

substrate-level oxidation phosphorylation

redox→energy→ion motive force→ATP synthesis

how do methanogens generate ATP?

oxidative phosphorylation

what is electron bifurcation?

\-splitting of electrons into two paths-

normally, electrons come in to reduce NAD+→ NADH

* thing you are trying to reduce isn’t easily reduced
* would be good e-donor, needs to be acceptor
* endergonic process
* force it to accept electrons, pair it with exergonic process

ex. reduction of ferredoxin (endergonic) with reduction of NADH (exergonic) = overall exergonic

what types of substrates can methanogens use to make methane?

CO2 + H2

\- various methyl compounds

\- acetate

how does energy conservation typically occur during fermentation?

\-reduction of NADH

\-reduction of pyruvate at the end of cycle

\- SLP to create ATP from energy rich compounds

what is the importance of organic compounds with energy rich-phosphate bonds or acetyl-CoA molecules?

have required -32 kj to create ATP molecule

ATP production in metabolism

what is syntrophy?

a pair of microbes together can break down something they couldn’t break down individually

What is the link between the organisms in many cases?

transfer of H2

one produces and one consumes

why does syntrophy work?

ability to change the concentration of H2 and change between endergonic and exergonic (H2 doesn’t accumulate in the cell, so cell is able to ferment)

what is secondary fermentation?

taking fermented product from another microbe and undergoing fermentation again

what is functional diversity?

type of biodiversity that covers the range of function traits in microbes in an ecosystem (what they do)

what does functional diversity include?

phototrophic bacteria, sulfur cycle, nitrogen cycle, other functional groups

what is phylogenetic diversity?

type of biodiversity that covers differences in phylogenetic traits

how do functional and phylogenetic diversity correlate?

functional traits often based on phylogenetic diversity BUT sometimes wont line up due to HGT, gene loss (non-advantageous gene mutations→smaller genome), convergent evolution

how do different nitrogen fixing organisms protect their enzymes from O2?

obligate aerobes

heterocysts

capsules/slimes (slow O2 diffusion)

some do photosynthesis during day, Nitrogen fix at night

what are the problems associated with dissimilative iron-reduction and iron oxidation?

Fe3+ is insoluble

Fe2+ is soluble

how do dissimilative iron-reducers and oxidizers solve the problems associated with iron solubility?

Reducers:

\- electrons transferred outside cell via nanowires

\-cytochromes to outer portion of envelope

\-to transport soluble iron out

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Oxidizers:

\- may form sheaths, so it “sheds” and product is soluble

explain the mechanism through which Dienococcus is thought to be able to survive very high levels of radiation.

accumulate high levels of manganese

\-interact withnormal repair proteins, repairs DNA very quickly

Mollicutes lack a cell wall. what implications does this have for their shape? why are sterols potentially important for these organisms?

more sensitive to osmostic stress

are pleomorphic (can change shape)

Briefly explain the two mechanisms of gliding motility used by Mollicutes.

Proteins similar to legs- flip back and forth as ATP is hydrolyzed

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Proteins adhere to surface-extend parts of cell, adheres to surface and pulls itself

What are the versions of chalmydiae?

elementary bodies: infectious form, survive outside of host (inactive)

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reticulate bodies: active inside of cell, reproduce