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73 Cards in this Set

  • Front
  • Back

what are nutrients?

taken from the environment and used as:


- sources of major bioelements


- sources of minor bioelements


- sources of electrons


-sources of energy or components of reactions that yield energy

what are the trophs ?

energy source: light=photo, chemical=chemo


electron source: organic=organo, inorg=litho


carbon source: org=hetero, inorg=auto

what are major and minor bioelements?

major: C, N, P, S, O, H - required to build organic cellular molecules




minor: Fe, Ca, Mg, Zn, Cu - required for enzyme function, electron carriers, membrane stabilization, they are everywhere

what are vitamins? what are some examples?

small organic molecules compose of major bioelements, have different roles (electron carrier, enzyme active sites), most synthesized by plants, some microbes can synthesize


eg thiamine, niacin, riboflavin

explain the calvin cycle

reduces CO2 to glyceraldehyde 3 phosphate for biosynthesis, uses 3co2+3h2o and takes 6ATP/6NADPH

what is assimilative nitrate/nitrite reduction? what is biological nitrogen fixation?

No3 or NO2 to NH4 with NADPH, N2 to NH4 with APT and NADPH then to glutamine

assimilative sulphate reduction?

SO4 to H2S with ATP and NADPH then to cysteine

describe respiration

done by some chemotrophs. use two nutrients in a redox process to obtain energy, first is energy source, second is TEA


nutrients are different chemicals

fermentation

same nutrient as the electron donor and acceptor, both called energy source

phototrophy

electron donor and acceptor are not nutrients, they are molecules synthesized inside cell

describe all the types of "trophy" that purple bacteria can do

can use light or CHX as energy source, can use CO2 or CHX as C source, can use a variety of electron sources to make NADPH

what conditions allow phototrophy?

darkness and anaerobic, can grow photohetero or photoautotrophically

explain the "great plate count anomaly"

serial dilution of soil suspension plated yields 10^6 CFU/g oven dried soil, where as microscopic field stained with DAPI gives 10^9

what is the shot-gun approach?

direct plating - plate sample on solid nutrient medium and characterize colonies that form


- survey of who's there


- remember all media is selective

what factors affect the shot-gun approach?

- all nutrient media and conditions are selective


- absolute abundance (a small community component may not be able to form a colony)


- sample prep/dilution can kill some organisms


- relative abundance and relative growth rates: some microbes have very slow growth rates, so will have low abundance


- colonial growth: some do not form hemispherical colonies


- antibiosis: antibiotics diffuse from one colony, inhibiting the growth of others


- disruption of microbial interactions: some are dependent on presence of others



explain the targeted approach

uses enrichment - anything a microbiologist does to deliberately favour the reproduction of one organism over another



explain enrichment culture

manipulate media and incubation conditions to favour the reproduction of an organism with particular characteristics


manipulates:


- ecological resources = nutrients


- ecological conditions = not consumed by the organism

what makes up "enrichment conditions"?

incubation conditions and the enrichment medium

explain solid enrichment culture

less commonly used, direct plating method

why is growth in liquid culture different from solid?

in liquid, all organisms interact together


- they also interact on solid by not to the same degree

explain why the most fit organism for the initial conditions is not necessarily the dominant one

often a succession of organisms occurs, the one that dominates first changes the conditions to favour the dominance of another


- enrichment method provides NO information about the proportion of the desired organism in the community


- can detect presence of a minor component

what are "weed species"

microorganisms successfully isolated using liquid enrichment culture - because they take over and become dominant


- a lot of the cultured organisms are weed species


- now called the "rare biosphere"

what are some modifications we can make to try to culture the uncultured?

1. avoid plastics - inhibitory compounds can leach from plasticware used to make media solutions


2. replace agar with other media solidifying agents - agar inhibits growth of some orgs


3.dont autoclave things together - heat and pressure can destroy labile molecules, facilitate chemical reactions creating toxic byproducts or precipitates


4.make more natural - use plant/soil extracts, seawater, add humid acids


5. use low nutrients - most environments are low [nutrients], some orgs are inhibited by high [nutrients]


6. be patient - slow growers

explain how the diffusion chamber works

a metal washer is the chamber between the lower membrane (with the environmental sample) and an upper microporous membrane


this is placed into "natural" environments and colonies grow


can be repeatedly subcultured

what is domestication?

being able to grow/culture previously uncultivable organisms


not really understood how


eg gram negative bacterium from soil produces new antibiotic "teixobactin" with no observed resistance

what is problematic about species definition for prokaryotes?

many things, this is referring to the fact that prokaryotes reproduce asexually, haploid, and can have horizontal gene transfer

what are phenetic and polyphasic?

phenetic = observable properties


polyphasic = phenotypic and genotypic

what are some things described by phenotypic characterization?

gram reaction, shape, motility, uni/multicellular, types and proportion of fatty acids, effect of temp/o2/ph/salt, kinds of enzyme made, energy and electron sources used, habitats

what are some genotypic characterizations?

low resolution: GC content, varies from 20-80%


high resolution: multi locus sequence typing


- nucleotide sequences of "housekeeping"


- SSU rRNA


- amino acid sequences of house proteins

what is the 70 and 25 % rule?

70% nucleotide sequence identity = same species


69-25% identity = same genus, different species


24-0% = different genus


- can about from DNA:DNA hybridization

explain DNA:DNA hybridization.

DNA from unknown(excess) is made ss and mixed with ss DNA(radiolabeled from known), ... too long to write all out just make sure you can explain it

where does the 70% rule come from?

early species designations were primarily based on phenotypic characters


microbiology establishment didn't want to re-do all that


70% was established because it didn't disrupt these designations


means that docile e coli and O157:h7 are in the same species even though bad one has 20% more DNA

can you identify an organism down to the species level with using the sequence of the 16S rRNA gene?

NO!


- many in different genus have very similar genes


- many in same species have very different genes

what "unit" is used when looking at 16S rRNA gene?

operational taxonomic unit = a 16S rRNA gene sequence identity value deemed to represent the same taxonomic unit


e.g. sharing 97-98% identity would say they are in the same OTU

what is the downside of the "repositories of information"?

managed by different organizations, do not really have any error checking, lots of redundancy


- contain many inaccuracies

what do you do if you think you have discovered a new species?

have to do DNA:DNA hybridization, have to have type strain culture, proposal almost always has the gram reaction result

explain community sampling analysis vs whole community analysis

community sampling analysis:


- what we do in 401


- take DNA and sequence a single gene


- compare gene to database


- find the species in the community


whole community analysis:


- sequence all DNA


- compare all DNA to database


- find new genes in the community

explain "colony PCR"

pick a colony form a plate, mix with 50uL water and heat lyse, spin down cell debris, use some supernatant in PCR

what are some problems with extracting DNA from environmental samples?

- microbial biomass is a very small portion of the total biomass


- non-microbial portion contains inhibitors

what are humic acids?

large molecular weight, recalcitrant, organic molecules, resulting from the decomposition of organic matter


- abundant in sewage, soil and water


- especially important issue when tracking pathogens in water samples

what are the basic principles of DNA extraction?

- differential solubility or differential binding

dielectric constant?

refers to the solvents relative permittivity = ratio of the permittivity of the solvent to its permittivity in a vacuum

what are chaotropes?

chemicals that disrupt biological structure but not covalent bonds


- alter solubility properties


- eg SDS, NaI, Phenol

what are some examples of physical cell lysis techniques?

bead beating, ultrasonication, freeze thaw cycles, grinding in liquid N2

what are examples of chemical lysis methods?

1. chaotropic detergents (SDS)


2. chaotropic salts (guanidinium)


3. enzymes:


- PG wall use: lysozyme, mutanolysin, lysostaphin


- proteinaceous walls: proteinase K


kits usually do not have enzymes, some have proteinase K

what are two methods for removing cellular debris after lysis?

1. differential solubility in PC


- dsDNA is water soluble (not denatured by PC)


- PC does denature proteins (some soluble some not)


- lipids are soluble in PC


2. differential solubility in high salt


- use non-chaotropic salt


- raising [salt] causes proteins to precipitate out while DNA remains in solution


kits: do not use PC, use salting out

what are some methods for collecting DNA and washing it?

1. alcohol-salt/centrifugation:


- DNA is less soluble in alc/salt than water (precipitates)


- wash precipitated DNA with alcohol based wash solution with a lower [salt]


2. binding to silica:


- both DNA/silica remain (-) charged over broad range of pH


- both are hydrated by water


- need: low pH, high chaotropic [salt]

for DNA binding to silica: what conditions promote binding?

1. low pH


- partially protonates O- -> OH so less electro-repulsion


2. high chaotropic [salt]


- screens/bridges (-) between silica and DNA

for DNA binding to silica: what conditions are needed to wash bound DNA?

- want to lower [salt/impurities]


1. low [salt], alcohol based solution


- because using alcohol based solution, salt can be lowered without DNA solubilizing



for DNA binding to silica: what conditions are used to elute DNA from silica?

- now we want to solubilize DNA


- water based (low alcohol) solution


- no chaotropic salt


- low salt


- high pH (O will be deprotonated again)

What are 3 methods of dealing with nucleases when extracting DNA?

1. use chaotropes:


- denature nucleases


eg SDS and guanidinium salts


2. use proteinase K to degrade nucleases


- fungal enzyme


- not denatured by 1% SDS at 65'c


3. EDTA in alkaline pH


- binds to Mg2+ which is a nuclease cofactor

what are the similarities between DNA and humic acids?

- aromatic rings


- negatively charged at neutral pH


- heterogenous molecular weight




differences:


- no consistent structure

name 6 different HA removal methods

1. sequestering agents, 2. gel electrophoresis, size exclusion (gel filtration), anion exchange, washing DNA bound to silica, CsCl buoyant density gradient centrifugation

how does anion exchange chromatography work?

anion exchange beads are positively charged, DNA and humic acids bind in low [salt]


as [salt] is raised, the anion competes for the positive charge and displaces DNA while humic acids remain bound

how does CsCl buoyant density work?

add sample to a pre-set density/concentration gradient and swing the mixture on a rotor


add EtBr to visualize DNA and dispense fractions into collection tubes

what are some of the principles of buoyant density?

sedimentation rate increases with particle size, it is proportional to the difference in density between particle and medium, increases with centrifugal force, decreases with viscosity

why is there a large band of DNA stained with EtBr after CsCl buoyant density centrifugation?

density of DNA depends on base composition, since DNA is from a community there is a range

how can we evaluate the success of HA removal?

observe the clearness of the solution, find the a260/230 ratio


- note peptide bonds also absorb strongly at A230

define representativeness

the degree to which DNA of every organism in the DNA preparation is present in the same proportion as it is in the original environmental sample


- this is the only thing that matters for trying to find out about microbial diversity (DNA yield doesn't matter)


- can never really tell if we get a representative DNA bec this is what we are trying to determine

what is the most important determining factor in DNA extraction that effects the representativeness?

cell lysis


- microbes vary in their susceptibilities


- chemical lysis is clearly bias (PG vs protein cell wall)


- physical lysis is less selective but leads to DNA shearing

what is major evidence that our methods are failing to produce representative DNA extracts?

paper in 2012 compared 6 methods on a mock community


- none of the methods worked - none yielded a DNA sample that accurately reflected the composition of the mock community

explain the "brute force approach"

1. isolate total community DNA


2. amplify the 16S gene (community sampling analysis)


3. clone these PCR products into plasmids


4. sequence amplicons to assess diversity


- pick white colonies, preparing plasmid DNA and individually sequencing each one


problems: time consuming, expensive

name and explain the 3 dyes that can be used for epic-fluorescence microscopy

1. FITC = fluorescein isothiocyanate


- at alkaline pH FITC reacts with surface primary amines/SH groups mostly found in proteins


- good for prokaryotes, protozoa


2. aniline blue, calcofluor:


- stains surface chitin or cellulose


- goof for fungi


3. DAPI


- stand ds nucleic acids


- planar so intercalates between AT base pairs

what things affect what you see in epifluorescence (or really any) microscopy?

1. sample preparation - could blend/lyse organisms


2. stain used and duration of application


3. observer judgement - different observers make different calls

what is an indirect method of counting organisms?

Q PCR

what is wrong with using a gene as an indicator for number ?

many cells have more than 1 copy but regardless of this do we assume:


1 gene copy = 1 genome copy = 1 cell = 1 organism?

what dye is used during Q PCR?

SYBR Green I dye - binds ds DNA

what are direct methods of measuring biomass?

physically separate microbes from rest of environmental sample and weigh them


- done for faces

what is a method of indirectly calculating biomass?




why is this usually impractical?

use the density of prokaryotes and estimate the volume of all the prokaryotes


multiply to find the mass




usually impractical bec most sample have very low microbial biomass

what are some examples of universal and groups-specific biomass indicators?

universal: ATP, membrane fatty acids




specific: chlorophylls, photopigments, ergosterol (membrane steroid), LPS, muramic acid

what are criteria of a good biomass indicator?

1. must be present in only living cells and not accumulate in the environmental sample if it is released by dead cells (short half life)


2. must be quantitatively (=100%) extractable from the environment (or extraction efficiency must be known)


3. conversion factor from lab grown organisms must be known and must apply to the entire community in the environment

how did researchers measure the effect of raising CO2 on microbes?

used FACE (free-air carbon dioxide enrichment)


- estimated soil prokaryotic biomass from prokaryotic biovolume by direct microscopic observation stained by DAPI


- estimated fungal through staining with calcofluor and looking for hyphae

explain the CFI/E method:

1. fumigate soil with CHCl3 gas - boil, soil is maintained at 55% field capacity


2.place fumigated soil in gas tight container with CO2 trap (NaOH)


3. some surviving microbes decompose dead ones producing CO2


4. amount of CO2 produced used to estimate the biomass in the soil sample