Micr222 Labs 1-4

2 chemicals needed in a winogradsky column

carbon and sulfur

carbon in winogradsky column comes from

cellulose in our column and newspaper in NASA column

sulfur in winogradsky column comes from

calcium sulfate in our column and egg yolk in NASA column

microbes in winogradsky column

clostridium, desulfovibrio, chlorobium, chromatium, rhodomicrobium, beggiatoa and cyanobacteria (algae)

clostridium function in winogradsky column

anaerobic, breaks down cellulose into glucose subunits then ferments the glucose to gain energy. produces ethanol and organic acids as by-products

desulfovibrio function in winogradsky column

uses organic molecules produced by clostridium as carbon sources. uses sulfate as a final electron acceptor in respiration producing H2S. producing a H2S gradient (high at bottom and low at top)

chlorobium and chromatium function in winogradsky column

green sulfur bacteria and purple sulfur bacteria. GSB can tolerate higher H2S than PSB. both are photosynthetic so use light as an energy source, use CO2 from calcium carbonate as a carbon source and use H2S from desulfovibrio as an electron donor

rhodomicrobium function in winogradsky column

purple non-sulfur bacteria. low sulfur and oxygen levels. presence of light so photosynthesis. use organic acid or ethanol as electron donors (rather than H2S)

beggiatoa function in winogradsky column

uses H2S as an energy source and oxidises it to sulfuric acid. the energy released by this process is used to fix CO2 to produce organic molecules

cyanobacteria (and algae) function in winogradsky column

in pond water, aerobic photosynthetic. light as an energy source and release O2 as a by-product, which helps to maintain the O2 gradient (high at top and low at bottom). fix CO2 and produce organic molecules.

autotrophs

capable of generating new organic compounds from CO2

heterotrophs

use carbon already fixed by autotrophs. degrade organic matter. recycle carbon, nitrogen, sulfur and phosphorus which is essential for biological growth

energy in an ecosystem

enters through sunlight and is trapped in the bonds of molecules and then is used to power other processes

chemotrophs

gain energy from chemical sources. most microbes are like this

phototrophs

gain energy from light source

carbon source definition of microbes

autotrophs or heterotrophs

energy sources definition of microbes

chemotrophs or phototrophs

photoautotrophs

carbon source \= inorganic CO2
energy source \= light
e.g. photosynthetic bacteria (green sulfur, purple sulfur, cyanobacteria), algae and green plants

chemoautotrophs

carbons source \= inorganic CO2
energy source \= chemical compounds
e.g. iron bacteria, sulfur bacteria, hydrogen bacteria, nitrifying bacteria and some archea

chemoheterotrophs

carbo source \= organic compounds
energy source \= chemical compounds
e.g. most bacteria, all protozoa, all fungi and all animals

photoheterotrophs

carbon source \= organic compounds
energy source \= light
e.g. purple non-sulfur bacteria and green non-sulfur bacteria

chemoautotrophs bacteria

SHIN (sulfur, hydrogen, iron and nitrifying)

microcosm

miniaturised real world systems

the gradient of O2 in the wonogradsky column allows for

both aerobic and anaerobic life

photosynthetic pigments absorb

infrared radiation

photosynthetic bacteria inhabit

mud and anaerobic water regions where light can penetrate and where organic matter and sulfide are present

the sources of carbon in the winogradsky column

organic \= cellulose and inorganic \= carbonate

at bottom of the column

carbon and sulfur are present but low O2

cellulose decomposing bacteria

hydrolyse cellulose into glucose

glucose is

fermented and acids are released

CO2 is produced by

the acids reacting with the carbonate

the CO2 is used by

the photosynthetic bacteria

3 decomposition by products

citrate, pyruvate and acetate

source of soil in the lab

dunedin harbour

what type of plant have a mutualistic relationship with rhizobia

legumes

examples of legumes

soybeans, clover, alfalfa, beans and peas

important symbiosis in NZ's pastoral agriculture

white clover rhizobium leguminosarum biovar trifolii

when roots infected with specific strains of rhizobia

a nitrogen fixing nodule forms which leads to increased levels of nitrogen

relationship between legume and rhizobia

is specific only one strain of rhizobia can infect one species of legume

cross-inoculation group

a group of rhizobia that infect a group of related legumes

the specificity of the rhizobia-legume relationship depends on

both the rhizobia and the plant

rhizobia infection

enter root hair, grow within an infection thread, spread though root hair into the root

rhizobia stimulate

tetraploid cells within the root to divide and form a nodule

bacteroids

swollen, misshapen and branched rhizobia cells that have been budded off from the infection thread into the cytoplasm of tetraploid cells and surrounded by a membrane of plant origin. they fix nitrogen but are incapable of cell division

releasing rhizobia

the nodule deteriorates and the small number of untransformed rhizobia cells within the nodule will grow and be released into the soil to infect other roots or live freely in the soil

colour of nodules and why

red-brown due to the presence of leghaemoglobin

leghaemoglobin

produced by the plant. a specific protein unique to legume root nodules. it acts as an oxygen buffer supplying oxygen to the bacteroids for ATP production and at the same time protecting the oxygen sensitive nitrogenase system

rhizobium colony morphology

creamy white, circular, entire, viscous, 2mm and convex

rhizobium gram reaction

gram negative

rhizobium cell morphlogy

rod, regular, straight rod axis, pairs, 2 micrometers by 1 micrometer and irregular staining due to internal storage granules

enzymes that fungi produce that break down organic material

cellulases and ligninases

type of relationship between plants and fungi

specific, close, mutualistic relationships

benefits for the plant and fungus

fungi receives fixed carbohydrates from the plant and the plant receives minerals and water from the fungi

preferred medium used when isolating fungi

sabouraud dextrose agar as has a low pH 5.6 which selects for fungi and inhibits bacteria

add what antibacterials to sabouraud dextrose agar when isolating fungi

penicillin and streptomycin to inhibit the growth of soil bacteria that can grow at low pH such as Pseudomona
so the media is double selective with low pH and antibiotic pressure

what type of bacteria produce endospores

gram positive rods such as Bacillus and Clostridium

endospores

heat-resistant, dormant structure

under what conditions are endospores produced

when environmental conditions have become detrimental to normal vegetative growth, such as in the presence of heat (leading to desiccation), UV radiation and toxic chemicals

during endospore formation

all biochemical activities of the vegetative cell become centred on endospore synthesis

when endospore formation is complete

the cell ruptures and liberates the endospore that contains the species genome

what makes the outer wall of the endospore so strong

the high content of the AA cysteine that binds the proteins together

what are the features that make the endospore able to survive harsh environmental conditions

impervious outer wall, dry core, presence of dipicolinic acid and small acid soluble proteins in the cortex

what are some of the harsh conditions that an endospore can survive in

heat, desiccation, UV radiation and toxic chemicals

what temperature kills most vegetative cells

65 degrees celsius

what type of microbe would survive 80 degrees celsius for 20 mins

extreme thermophile (not likely to be found in a soil sample)

heating at 80 degrees celsius for 20 mins is a treatment that

selects for endospores

competition for available nutrients

depends on rates of nutrient uptake, inherent metabolic rates and growth rates

antagonistic interaction

advantage to one organism and disadvantage to the other

commensalism

one organism benefits (typically symbiont) and the other one has no benefit nor disadvantage (typically the host)

bacteriocins

inhibitory molecules that are fundamentally different from other inhibitory molecules as they are ribosomally translated products; the information encoding their primary structure is directly encoded in the DNA of the producer strain

what bacterium produces bacteriocin

Streptococcus equi subsp. zooepidemicus

plate used in antagonism experiment

columbia agar base + 0.1% calcium carbonate (CAB + Ca) plate

2 types of broths used in the competition experiment

todd hewitt broth (THB) and minimal salts broth (MSB)

microbes used in the competition experiment

E. coli and Streptococcus equi subsp. zooepidemicus

dilution of competition experiment broths then plated onto

eosine methylene blue plates (EMB) and blood agar plates containing streptomycin so the plate is selective and differential

microbes used in the antagonism experiment

Streptococcus equi subsp. zooepidemicus

examples of commensalism

microbial production of extracellular depolymerases that are released and degrade macromolecules which then provides substrate for the microbe that produced them and for other microbes in the same niche

removal of oxygen from an environment by a faculatiev anaerobe produces an anaerobic environment which would then sustain growth of strict anaerobes e.g. E. coli in the human gut

commensalism experiment plate used

todd hewitt agar + ampicillin (THA + amp) plate

microbe used in commensalism experiment

E. coli XL1 - blue/pbluescript it is resistant to ampicillin as it produces beta-lactamase enzymes that degrade beta-lactam rings in antibiotics destroying them this enzyme is exported from the cell

plasmid that confers ampicillin resistanct

XL1 - blue/pbluescript

Bacillus stearothermophilus

hot pools - thermophile

Bacillus subtilis

soil - increased tolerance to UV

E. coli

lower gut - mesophile, non-halophile and likes neutral pH

Listonella anguillarum

marine - psychrotroph, moderate halophile and alkaliphile

Staphylococcus epidermidis inhabits

skin - extreme halophile

Lactobacillus

upper gut - acidophile

denitrifier

pseudomonas fluroescens, Bacillus and alcaligenes

microbes able to carry out ammonification

pseudomonas fluroescens, bacillus cereus and alcaligenes faecalis

nitrosomonas carries out

nitrification, it oxidises NH4+ to NO2-

nitrobacter carries out

nitrification, it oxidises NO2- to NO3

E. coli and UV rad

can't tolerate long periods of exposure as inhabit the human gut so it is not exposed to UV rad and so is not adapted to cope with it

bacillus subtilis and UV rad

can tolerate longer periods of exposure to UV rad as it is a soil bacteria that is exposed to UV rad so has adapted to cope with it, it is an endospore former

listonella anguillarum and temperature

can grow at 37 degrees and best at 12 degrees as it inhabits marine environments which are generally cold so it is a psychrophile

E.coli and temperature

can grow at 12 degrees but grows best at 37 degrees as it inhabits the human gut so it is a mesophile

bacillus stearothermophilus

only grows at 70 degrees as inhabits hot pools, it is a thermophile

E.coli and salinity

can only grow at low NaCl conc. as it is a non-halophile as it inhabits the human gut

Listonella anguillarum and salinity

can grow at moderate NaCl conc. as it is a moderate halophile as it inhabits marine environments

Staphylococcus epidermidis and salinity

can grow at all NaCl concs as it is an extreme halophile as it inhabits the skin

what enzyme does E. coli XL1 - Blue/pBluescript produce?

beta-lactamase so that it is resistant to ampicillin (and any other beta-lactam ring antibiotics) this also means that bacteria such as M. lutens that are sensitive to ampicillin can grow where the beta-lactamase has diffused --\> commensal relationship

what is the compound that Streptococcus equi subsp. zooepidemicus produces to inhibit the growth of closely related species?

bacteriocin and it inhibits the growth of streptococcus pyogenes but not streptococcus uberis or streptococcus dysgalactiae as they are not similar enough to inhabit the same niche