bimm 120 final - the new stuff

magnetosomes: how they are made

nano-sized crystals of a magnetic iron mineral that is enveloped by a lipid bilayer

how are magnetosomes organized?

magnetosomes are arranged in a CHAIN within a cell

how do magnetosomes help the organism?

magnetotactic bacteria (MTB) use magnetosomes to move in accordance with the Earth’s magnetic field

what organisms make magnetosomes?

magnetotactic bacteria are motile, diverse prokaryotes that biomineralize magnetosomes

• aquatic prokaryotes which have their motility directed by Earth’s geomagnetic and externally applied magnetic fields

true or false: magnetotactic bacteria often help fix nitrogen

true

• exhibit nitrogenase activity if cultured!

• aka fix atmospheric nitrogen

how do magenetotactic bacteria switch preferred directions?

rapidly!

• lol, trust

what is the origin/spread of magnetotaxis?

magnetosome biomineralization is responsible for magnetotaxis in MTB, we assume that the evolution of genes involved in magnetosome formation reflects the evolution of magnetotaxis

bacterial-eukaryotic symbiosis for spatially separating nitrogen and carbon fixation

basically… cyanobacteria has to spatially segregate nitrogen and carbon fixation because nitrogenase enzyme is sensitive to oxygen (will die)

• circadian rhythm less common in prokaryotes than eukaryotes

• segregation of photosynthesis and nitrogen fixation due to mutually exclusive oxidative environments

spatial separation of nitrogen fixation from carbon fixation in cyanobacteria like Anabaena

Anabaena spatially isolates nitrogen fixation activity, heterocysts

• heterocyst is nitrogen fixation

• heterocysts are at night

• photosynthesis during day with vegetative cell chain between the heterocysts

what species of bacteria does Dr. Golden study?

cyanobacteria: Synechococcus elongatus

• ancestor of chloroplast

• form oxygen during photosynthesis

• convert inert atmospheric nitrogen into an organic form (fix N2)

• closely related to chloroplasts, but not plants

what is the circadian infradian rule?

circadian-infradian rule:

• infradian: cycle time greater than one day (includes tidal, seasonal rhythms)

• doesn’t make sense for cell to have a circadian rhythm if its lifetime is shorter than one day (less than one day for reproduction time)

nitrogen fixation and oxygen production cycles in cyanobacteria

nitrogen fixation at night

oxygen production during the day (photosynthesis needs sun)

what is the gene expression in the circadian rhythms in cyanobacteria?

circadian rhythm gene expression

• class I peaks at dusk

• class II peaks at dawn

basics and functions of the Kai circadian rhythm cycle and mechanism

Kai = cycle

KaiA: dimeric protein

KaiB: tetramer

• inactive = tetramer; active = monomer

KaiC: hexamer

• has ATP-binding motifs

• can autophosphorylate

RpaA: transcription factor that binds upstream of genes

SasA & CikA: histidine kinases that phosphorylate a transcription factor RpaA

• SasA bind to bottom of CI ring, CikA free floats nearby & has job of dephosphorylation

what changes throughout the day for the circadian mechanism?

during the day, SasA binds to CI ring of KaiC during its phosphorylation mode, and upon binding, it autophosphorylates, then transfers its phosphate group to RpaA

• the more CII is phosphorylated, the more SasA phosphorylates RpaA

at night, KaiB monomers begin to bind to the SasA site to knock off SasA

what phenotypes do fimbrial gene knockouts yield?

fimbrial PapF knockout: 20% of WT fimbriation

PapK knockout: fimbrium is %x as long

PapG-H knockout: terminates A-component polymerization and fimbrial assembly

molecular tropism

more than one molecular form can provide different phenotypes

• tropism: p-type pili can present 3 adhesin, each with a different host range

  • allow organism to expand its host range of functions

asymmetric division in Caulobacter crescentus

Caulobacter crescentus life cycle:

• asymmetric division to produce one motile swarm cell and one sessile stalked cell

• 2 poles of division exhibit differential levels of DNA condensation, replication machinery, transcription, assembly and modification of complexes

major morphogens in C. crescentus and what they do

2 morphogenetic proteins:

• TipN: marker protein for spatial and temporal differentiation (tip of NEW pole), localizes to cell division site & stays there until next cell div

  • localization depends on FtsZ and FtsI (septum cell wall synthesis protein)

  • determines asymmetric cell division site

  • if overexpressed: many new poles with cell branching and each pole become flagellum

  • localizaes actin-like MreB

• TipF: c-diGMP phosphodiesterase

  • c-diGMP is a cytoplasmic “alarmon” regulating cell cycle

  • tipF negative mutants cannot make flagella

  • new septum achors TipN, which anchors TipF, which lowers c-diGMP concentration so the flagellum can be made

  • TipF = interacting catalytic protein

what does the MinCDE system do? how does it do that?

cell division in E.coli is controlled by the MinCDE system

• MinC: FtsZ polymerization inhibitor

• MinD: ATPase that interacts with C and E, stimulates MinCD release from membrane

• Mine E: topological specificity determinant

• MinCD shuttle between 2 poles together while MinE shuttles between a pole and the median MinC/D prevents separation at the poles

process prevents separation (cell div) from occurring at poles

persisters

persister cells = superfits = a subgroup of a bacteria population that can tolerate adverse environmental conditions via dormancy

• tolerant, not resistant to traditional antimicrobials

• enter dormancy with reduced metabolic activity

how do persisters form and come back to vegetative states?

• superfit done in response to nutrient deficiencies, not as a result of antibiotic exposure

(p)ppGpp

ppGpp is involved in the relaxed vs. stringent response in amino acid starvation

• the alarmone

• level of ppGpp varies randomly in a growing population of bacteria

• cells with the highest [ppGpp] become slow growers

how do persisters tolerate antibiotics? why do they tolerate antibiotics?

tolerance via phenotypic variation, not heritable

slow growth (highest ppGpp) triggers activation of toxin encoded (results from loss of antitoxin due to proteolysis)

what are bistable systems? how do feedback loops contribute to state-switching?

two stable states

no stable intermediates

• population exists in two stable phenotypes with no long-term intermediates

type II toxin-antitoxin system

toxic (protein) is stable

antitoxin (protein/RNA) is degraded quickly by enzymes

• translatiionally coupled, expressed on same mRNA

• ribosome always translates antitoxin

• ribosome usually translates toxin

• amount of antitoxin > amount of toxin (general)

how to activate toxin? degrade antixtoxin

common quorum sensing signals

AHLs: acyl-homoserine lactones

• bacteria only produce light (luciferase) when the concentration of quorum sensing molecules accumulates in medium

• affinity of receptor for AHL is SUPER high, AHL binds and induces gene transcription

indole: small ring shaped molecule found in tryptophan

• decreases motility in bacteria to cause pathogenicity

• recognize translational coupling

PQS: protein quorum sensors

AI-2: borate-furanosyl

• produced by gram-pos

• sensed by gram-neg

bet-hedging and division of labor in bacterial colonies/communities

division of labor: distinct cell types performing specialized tasks that benefit the population as a whole

• work together, project like biofilm formation and secretion of virulence factors

bet-hedging: singlet motile cells and chains of non-motile cells

• motile cells - foragers, seek new nutrient sources

• maintain diff phenotypes to deal with diff stresses

• non-motile chains take advantage of current niche

public goods v. private goods (in a microbial context)

public goods = quorum sensing regulates collective features that involve production of energetically costly public goods only effective/functional if perfromed by microbial crowd

private goods are not shared, also energetically costly, but the whole population can still benefit

• happens when population has phenotypic heterogeneity but performs distinct and complementary functions

microbial cheating

invading members do not contribute to costly production o public goods but still benefit from them, so they have a fitness advantage

Caulobacter swarmers v. stalked cells

swarmer = motile Caulobacter

• differentiates into stalked cell

• condensed/compact DNA, can’t generally divide

stalked cells: holdfast-producing adherent Caulobacter

• divides to generate new swarmer cells

• stays adhered to substrate

PleC: the deets

obligate differentiation: motile swarmer undergo differentiation into sessile, replicating stalked cells

• initiated by activation of the PleC histidine kinase

• PleC phosphorylates PleD, which causes increase in c-di-GMP, which turns on CtrA transcription factor

• CtrA inhibits replication initiation, and in a pre-differentiated cell, promotes transcription of over 90 genes

early epidemiology

study of distribution and determinants of health-related states. in a population

• pandemic, infectious disease

• took a long time to eliminate viruses because they didn’t even know microbes and viruses existed!

ancient/medieval plagues/outbreaks

Black Death: Yersinia pestis, zoonotic, transmitted from rodents to humans by fleas

smallpox: caused by variola virus, vaccination to build immunity

• eradicted in 1980

1918 influenze pandemic:

• causative agent: H1N! influenza virus

• 10% fatality rate: 100x more lethal than current strain

• notably higher fatality in youth

1918 flu pandemics and who was hit hardest

caused by H1N1 virus

higher fatality for youth

vaccine development/targeting

to make vaccine:

• predict which strains will be common

• adapt virus to somethingunlike humans to weaken it (ie. eggs)

• weaken virus more

germ theory & Koch’s postulates

germ theory: anticipation exist as far back as Thucydides

• disease is caused by disease-causing units (germs)

Koch’s postulates: A given microbe X is both necessary and sufficient to cause disease Y

how to think like a pathogen :)

survival is success

maximize transmissibility

do NOT always maximize virulence

• higher virulence can mean higher contagiousnesss

• lower virulence can enhance transmissibility (but is limited by need for symptoms that allow for transmission)

do NOT maximize lethality, dead hosts don’t spread disease

quorum sensing relationship to bioluminescence

AHLs involved in enabling bioluminescence

• alone = no use for light

• colony = light up

switchable light gland: headlight, doward illumination breaks up silhouette

• LuxI consitutively produces AHLs

• LuxR activates QS-regulated genes oo binding to AHLs (needs HIGH AHLs concentration)

what are other roles for quorum sensing?

biofilm formation

extracellular enzyme

antibiotic/toxin synthesis

nodule formation

clinically relevant biofilms on catheter tubes, in lungs, in intestines

what are sigma factors? what do they do?

sigma factors regulate the broad subset of genes expressed in terms of sporulation in diff comparements

• targets functions of interest by compartment

• bacterial transcription initiation factor, allows RNA polymerase to bind specific promoters

what are the major sigma factors involved in Bacillus sporulation?

sigma A activates sigma H

sigma H activates sigma F (FS)

sigma F activates sigma E (MC)

sigma E activates sigma G

sigma G activates sigma K

endospores and their role in survival

bacterial endospores (spores) are most heat, radiation, starvation, and desiccation-resistant life states/cell types known

• used in biological warfare (anthrax) and can cause food poisoning in repeatedly reheated/unrefrigerated food

what do the mother cell and forespore do?

mother cell contains forespore and lyses to release spore into environment

• spore will be dormant until conditions are appropriate for germination

what are the general stages of germination?

activation: requires spor to be subject to some kind of stress (shock, heat, acid)

germination: hydrate dormant spore, loss of resistance, no protein synthesis

outgrowth: first stage of protein synthesis, restores vegetative state

what are the major steps in sporulation?

vegetative growth

pre-septation: chromosome condensation

septation: asymmetric, split at one pole of cell

engulfment: phagocytosis of forespore by mother cell = 2 membrane layer

coat formation/maturation: COT proteins deposited on outer layer of outer membrane = resistant to environmental stresses

• cortex formed: proteins in space between 2 membranes (proteins formed in forespore)

lysis: mother cell death, release of the mature spore (only release when mom dies)

what are spo, ger, cot, ssp, and out genes? what do they do?

spo: eliminates A stage and named after stage II, where it stops

ger: germination, fail to generate reducing power before protein synthesis

cot: coat proteins (~50%), most made by mother cell & deposited between spore and mother cell

ssp: small acid soluble proteins (in spore core, 10%)

out: outgrowth, prevent new macromolecule synthesis

how were spo, ger, cot, ssp, and out genes discovered?

forward genetics: Spo- stage blocks phenotypes traced to various genotypes

• spo, ger, out

• spo nonessential for vegetative growth, necessary to progress past sporulation stage

reverse genetics: discovery of genes with products incorporated into spores; individually nonessential

• cot, ssp

what is SpoIIIE and what does it do?

Spo = nonessential for vegatative growth, essential for sporulation

III = locus deletion prevents progressing past stage III

E = fifth discovered gene to block III to IV

• pumps DNA strand across membrane into forespore

what is the phosphorelay? what does it do (overall)?

phosphorelay: responsive to environment, includes cell cycle and metabolic signals

• leads to sporulation via phosphorylation pass on

what is partner switching?

phosphorylation/dephosphorylation can dissociate or trigger binding

what are 0A boxes? what is their role in kicking off sporulation?

0A boxes are areas of the genome, Spo0A binds to the 0A boxes, potentially causing sporulation, competence, cannibalism

• upregulates/downregulates genes

recognize thermotaxis, electrotaxis, phototaxis, thigmotaxis

thermotaxis: temperature

electrotaxis: electrical fields

phototaxis: light

thigmotaxis: touch

S-motility and A-motility in Myxococcus

S-motility: requires high cell density to proceed

• social

• exclusively associated with coordinating group movement

• ATP-powered extension/retraction of type IV pili, twitching

A-motility: similar to gliding in Flavobacteria

• adventurous

• pmf-powered adhesion engine, gliding

• movement of single cells near colony edge

scouts v. loners. v. swarm

scout: small group, 1-20 cells, isolated from main colony in front of swarm

loners: lie close to other cells in colony front

swarm: large, closely packed group of cells

how do cells move/spread when they are growing on a plate?

A motility required for collective movement

S motility strengthens cohesion

what are biofilms made of?

extracellular DNA (eDNA)

protein

lipids

polysaccharides

how are biofilms arranged?

planktonic bacteria, dead cells, lipid, polysaccharide, eDNA, protein, water, AHL, etc.

• polysaccharide: alginate (agar), Psl, Pel (hydration, structure, ion-binding)

• eDNA: structural support, ion binding, horizontal gene transfer

• proteins: CdrA, Fap, lectins (carb-binding for adhesion, matrix integrity)

• lipids/surfactants: rhamnolipids maintain water channels and trigger dispersal

how do biofilms form?

stages:

1) reversible attachment, weak electrostatic forces

2) irreversible attachment (adhesin, pili)

3) microcolony formation (cell div and extracellular polysaccharide)

4) maturation, 3D architecture, water channels

5) dispersal, enzymatic matrix degradation

what do biofilms do?

• infections

• bioremediation

• wastewater treatment

• bioenergy production

• agricultural applications

directed mutation

organisms respond to environmental stresses by reorganizing their genes purposefully

• mutations that arise occur with higher frequency if they relieve the stress that causes them

• non-random, accelerated course of evolution

• silence OR activate gene

IS elements: transposons, transposases, and insertion sequences

transposons: DNA segment, jumping genes that move to distal locations on a chromosome

transposases: enzyme that allows genes to jump

insertion sequences: homologous for large scale genomic rearrangements (transposase gene + inverted repeats)

is evolution predictive?

no.

what is flh, bgl, glp?

flh: flagellar biosynthesis gene

glp: glycerol utilization gene

bgl: beta-glucoside utilization gene

what is H-NS in the context of transposons?

the nucleoid protein in bacteria

• if lost, drastically reduces transposon hopping

• reason for keeping certain operons silenced until the transposition event with one of the IS elements occurs