BIMM 120 - final part 1 (saier)

magnetotactic bacteria (MTB)

motile aquatic prokaryotes, biomineralize magnetosomes, motility = directed by Earth’s geomagnetic and externally applied magnetic field

when cultured, exhibit nitrogenase activity —> fix atmospheric nitrogen/denitrify

depend on oxicanoxic interface (OAI)

where are magnetotactic bacteria found

present in sediments of freshwater, brackish, marine, and hypersaline habitats

magnetosomes

enveloped by a lipid bilayer, responsible for a cell’s magnetotactic behavior (magnetotaxis)

MTB swimming

hypothesized have 1 of 2 magnetic poles: north/south seeking polarities = based on preferred swimming direction of cells in oxic conditions

north-seeking cell swims down in N. hemis

south-seeking cell swims down in S. hemis

use cell polarity to swim down along magnetic field lines —> microaerobic/anaerobic environments, away from high, toxic [O2]

magnetosomes arrangement

in 1 or more chains parallel to long axis of cell, or in dispersed clusters

magnetotaxis evolution

assume evolution of genes involved in magnetosome formation —> magnetotaxis formation

origin/spread of magnetotaxis

2 hypotheses:

• HGT - supports MTB’s vast biodiversity + wide phylogenetic distribution, evidence = found putative genomic islands that enclose genes involved in magnetosome formation

• VGT - evolution and divergence of proteins and organisms’ 16S rRNA genes occurred similarly

2 methods of spatially separating carbon and nitrogen fixation w/ examples

bacterial-eukaryotic symbiosis: candidarus atelocyanobacteria (UCYN-A) has complete nif genes and fixes N2, fixed N goes directly to B. bigelowii cell which fixes CO2 into metabolites, go back to UCYN-A

anabaena cyanobacteria: spatially isolates nitrogen fixation to heterocysts, carbon fixation in vegetative cells

circadian-infradian rule

“infradian” = cycle time > 1 day

nonsensical for a cell to have a circadian rhythm if its lifetime (reproduction time) is shorter than 1 day

has been disproven w cyanobacteria work

nitrogen fixation and oxygen production cycles in cyanobacteria

N2 fixation depends on anoxic conditions bc nitrogenase enzyme = very sensitive to O2

separated temporally: O2 made in the day, N2 made @ night

Dr. Golden’s study

Synechococcus elongatus - type of cyanobacteria, has reporter protein: luciferase w robust clock-driven gene expression

despite continuous light conditions, see changes in gene expression following a clock pattern

all S. elongatus genes = rhythmic

Kai circadian rhythm components

KaiA, KaiB, KaiC = oscillator —> has output signaling using a 2-component system: RpA, CikA, SasA

Kai circadian rhythm - KaiA

dimeric protein

binds to exposed A-loop on CII —> promotes CII phosphorylation of Ser431 and Thr432 by localizing ATP and disturbing active site

binds in the day

Kai circadian rhythm - KaiB

inactive tetramer, active monomer

binds to P-KaiC at B-loops in CI, are exposed upon full phosphorylation of Ser431 and Thre432

binds in the night

Kai circadian rhythm - KaiC

hexamer, has internal duplication —> double donut form and ATP binding motifs

consists of CII (at top, w/ A-loops) and CI (at bottom, w B-loops)

CI ATPase activity oscillates with unphosphorylated form, consume more ATP than phosphorylated form

autodephosphorylation at night to dawn —> creates ATP

autophosphorylation at day to dusk

Kai circadian rhythm - RpA

response regulator (RR), governs transcription from a locus that controls global gene expression in circadian rhythms

regulated by 2 HKs —> phosphorylation of Asp53 on RpA —> TF to bind to DNA

Kai circadian rhythm - CikA

sensor histidine kinase (HK), reverses SasA’s activation of RpA

dephosphorylates RpA

play integral role in time-keeping

Kai circadian rhythm - SasA

HK, competes w KaiB for B-loops in CI of P-KaiC

SasA binding = faster bc KaiB needs KaiC to refold

fimbrial gene KO - F KO

20% of WT fimbriation

fimbrial gene KO - K KO

fimbrium = 5x longer

fimbrial gene KO - G-H KO

terminates A-component polymerization and fimbrial assembly

fimbrial gene KO - overproduce K

fimbrium = 5x shorter

molecular tropism

more than 1 molecular form can provide different phenotypes

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

caulobacter crescentus cell division

divides asymmetrically into 2 cell states: motile swarmers and sessile stalks

caulobacter crescentus cell cycle progression

controlled by sequentially acting regulatory proteins that must localize to specific poles in the cell

coupled to morphological events, if cell division = blocked at an early stage, flagella and pili can’t be assembled

can still assemble if blocked at later stage

division scar - markers for the new pole

caulobacter crescentus - asymmetric division process

mediated by polarity morphogens: TipN and TipF

caulobacter crescentus - asymmetric division process - TipN

“tip of the New pole”, marker protein for spatial and temporal differentiation, localizes to new pole, determines polarity

TipN localization depends on FtsZ (forms septum) and FtsI (septum cell wall synthesis protein), localizes actin-like protein MreB —> may determine cell shape by dictating cell wall growth size

determines asymmetric cell division site and localizes some regulatory proteins

w/o it: lose polarity and random pole switching

if overexpressed: many new poles w cell branching and each pole can assemble a flagellum, extra new poles form

caulobacter crescentus - asymmetric division process - septum formation

can form at either left/right pole or middle

caulobacter crescentus - asymmetric division process - TipF

“For interacting with TipN”, is a c-diGMP phosphodiesterase

breaks down c-diGMP —> direct flagellar biosynthesis to TipN and TipF complexes

new septum anchors TipN thus TipF —> lower [c-diGMP] —> flagellum made

w/o it: no flagella can be made

c-diGMP

a cyotplasmic alarmone that regulates cell cycle

ubiquitous, suppresses flagellar biosynthesis

also involved in triggering biofilm formation in other bacteria via pilZ receptor

also involved in nanowire-producing bacteria

caulobacter - swarmer cells

motile caulobacter, differentiates to stalked cells

condensed/compact DNA, can’t divide

NOT same as swarming motility

caulobacter - stalked cels

holdfast-producing adherent caulobacter, divide to make new swarmer cells

MinCDE system

in E. Coli, forms “MINICELLS”, prevents division at the poles, has a cyclic system

MinCDE system - MinC

a FtsZ polymerization inhibitor

shuttles btwn 2 poles w MinD —> prevents separation at the poles

MinCDE system - MinD

an ATPase that interacts w C and E, stimulates MinC/D release from membrane

shuttles btwn 2 poles w MinC —> prevents separation at the poles

MinCDE system - MinE

a topological specificity determinant

shuttles btwn pole and the median

persister cells

aka “superfits”, tolerant to traditional antimicrobials, NOT resistant —> equally susceptible to stressor after waking

enter dormancy w decreased metabolic activity

superfit formation is not bc of antibiotic exposure but a common response to a lack of nutrients

aren’t mutants, formation can be induced by exogenous factors (ie. QS signals)

tolerance via phenotypic variation is not heritable

vegetative cells

“normal”, actively growing form of bacteria

genetically identical bacteria (vegetative and persister cells) aren’t phenotypically identical

type II toxin/antitoxin (TA) system

translationally coupled, expressed on same mRNA

toxin - protein = stable

antitoxin - protein/RNA = unstable, degraded quickly by enzymes, regulates TA operon at promoter

antitoxin degradation —> activates toxin

active toxin —> RNA degradation, low protein synthesis —> low growth —> persistence, creates persister cells

hipA/B system

causes persistence in E. Coli

hipA toxin phosphorylates Glu-tRNA ligase —> inactivates it —> accumulation of uncharged tRNA(glu) —> activates RelA —> inhibits protein synthesis —> dormancy

hipA/B system - RelA/SpoT

RelA - generates (p)ppGpp

SpoT - degrades (p)ppGpp

(p)ppGpp

(p)ppGpp - high levels —> high Lon protease activity —> decreasing polyphosphate breakdown —> antitoxin degradation —> triggers toxin releases form other TA systems, upregulates genes that inactivate ribosomes

how antibiotics work

many antibiotics target ATP-dependent pathways —> require active cells to work

bacteria in stationary phase (persisters) have low ATP lvls

after resuscitation/waking, persister cells grow same as log phase cells

persister → vegetative cell

resuscitation/waking of persisters = triggered by presence of nutrients (ie. amino acids like alanine), detected via chemotaxis MCPs

quorum sensing

bacteria communication via autoinducers, cell-density-dependent regulatory principle

autoinducers = small, diffusible organic molecules

intra- & interspecies signaling, trans-kingdom communication, and self-sensing = all possible

quorum sensing molecules

AI-2, AHLs, indole, PQS

quorum sensing molecule - AI-2

boron-containing autoinducer-2, increases biofilm

quorum sensing molecule - AHLs

N-acyl-homoserine lactone

quorum sensing molecule - indole

decreases biofilm, decreases virulence and QS

ways QS increase overall fitness

bet-hedging and division of labor

bet-hedging

maintain different phenotypes to help deal with different stresses

allows bacterial population to optimally adapt to consecutive, rapid and frequent changes in environment without need to respond to external signals

can cause differential production of flagella

ex: e. coli —> expression of different metabolic capacities

division of labor

occurs when individuals in a clonal population interact with each other and together express distinct but complementary traits

ex. biofilm formation, s. typhimurium —> production of energetically costly type III secretory system

QS and autoinducers regulation

regulated by positive feedback loops

QS - public/private goods

regulates:

public goods - a collective features, energetically costly, are most effective/only functional if done by a microbial crowd

private goods - energetically costly, not shared but whole pop can still benefit —> occurs when pop has phenotypic heterogeneity and is lacking private good but performs distinct and complementary functions

microbial cheaters

invaders, don’t help w costly production of public goods, just benefit from them —> fitness advantage

bistable systems

defining characteristics = 2 stable states, no stable intermediates

macro ex: toggle switches can’t be balanced

microbial ex: bacteria pop exists in 2 stable phenotypes w no long-term intermediates

first record of immunity

thycydides on the plague of athens

black death

14th century

cause = yersinia pestis, zoonotic

transmitted from rodents —> fleas —> humans

smallpox

cause = variola virus

used to be regularly occurring to Old World

variolation = live smallpox, vaccination: live cowpox to build immunity

officially eradicated in 1980

1918 influenza pandemic

“spanish flu” aka “swine flu”

cause = H1N1 influenza virus, >100x more lethal than most current strains

higher fatality in youth (20s)

H = hemagglutinin, surface protein that helps virus bind to and enter host cells

N = neuraminidase, surface protein that helps virus exit infected host cells and spread

H5N1 = avian/bird flu, most deadly one to humans, spread bird-humans not human-human

COVID-19

cause = SARS-CoV-2

bats —> pangolins —> humans

thinking like a pathogen

goal = survive/reproduce = success

maxxing transmissibility = correlated with maxxing virulence & lethality

virulence: higher = more contagious but then ppl take precautions, lower = can enhance transmissibility but need symptoms for transmission

lethality: rarely done bc dead hosts usually aren’t good at transmitting

germ theory

disease = caused by disease-causing agents (germs)

koch’s postulates (4)

given microbe X is both necessary and sufficient to cause disease Y

1) must be found in all diseased organisms and not in healthy ones

2) must be isolated from a diseased organism and grown in pure culture

3) cultured form should cause disease when intro.ed to a healthy organism

4) reisolated from the inoculated, diseased organism host and identifies as the original causative agent

vaccine dvlpt process + shortcomings

process: predict which strains will be common —> adapt virus to something like humans to weaken it (ie. eggs)

shortcomings: strain predictions don’t always match emerging ones, adapting eggs can make strains that don’t match wild virus well, wild viruses mutate

QS and bioluminescence

1st discovered QS because of bacterial luciferase (bioluminescence)

LuxI: constitutively produces AHLs (QS signal)

LUXR: activates QS-regulated genes on binding to AHLs

other examples of QS

cellulose synthesis —> biofilm formation, extracellular enzymes, antibiotic/toxin synthesis, nodule formation in rhizobial bacteria, clinically relevant biofilms (ie. in lungs/intestines, catheters)

notation: SpoIIIE vs spoIIIE

SpoIIIE = protein, spoIIIE = gene

spo = gene = nonessential for vegetative growth, essential for sporulation

III = locus deletion prevents progression past stage III

E = 5th discovered gene to block III to IV

loss of function

something stops doing what it’s supposed to do when it’s supposed to do it

ex: bacillus strains fail to progress past sporulation phase due to mutations

gain of function

something starts doing something it shouldn’t when it shouldn’t

ex: bacillus strains w a nonfunctional sporulation gene mutates —> sporulation capacity

sporulation

vegetative cells —> spores

occurs asymmetrically and close to 1 pole —> different sized daughter cells

bacterial endospores (spores)

most heat, radiation, starvation, and desiccation-resistant life states/cells known

spore-state = reversible by necessity, if not = dead

when bacillus subtillus are spores, have practically no energy requirements to survive

sigma factors

regulate which broad subsets of genes are expressed —> useful for targeting functions of interest by compartment

bacterial transcription initiation factor, allows RNA pol to bind specific promoters

SpoIIIE

motor protein that pumps DNA into forespore

loss of function mutations —> stalled forespore chromosome localization

found to be a FtsZ ortholog: membrane-anchored, large c-terminal cytoplasmic motor domain, DNA-dependent ATPase activity

forms translocation complex at septal midpoint during early sporulation

sporulation steps - stage 0

vegetative growth, normal cell

sporulation steps - stage I

chromosome condensation, DNA replicates and spans long axis of cell, division sites shift to polar positions

sporulation steps - stage II

asymmetric septation, 1 division site forms a septum —> larger mother cell + smaller forespore

polar septum traps forespore chromosome (transported by SpoIIIE)

sporulation steps - engulfment

membrane of mother cell migrates around forespore

sporulation steps - stage III

forespore is enclosed w/in mother cell —> 2 membranes

sporulation steps - stage IV

cortex formation, cortex made of peptidoglycan from between the 2 forespore membranes

small acid soluble proteins (SASPs) build onto chromosomes

sporulation steps - stage V

coat formation, coat assembled, forespore chromosome = compact and saturated w SASPs —> protect DNA

sporulation steps - stage VI

maturation, dipicolinic acid (DPA) made in mother cell = added to forespore

forespore core = partially dehydrated —> heat resistance

sporulation steps - stage VII

lysis, mother cell lyses —> release spore

genes for sporulation

spo, ger, cot, ssp, out

most sporulation genes = expressed in 1 of 2 cells needed to form a spore (mother cell/forespore)

forespore-essential genes can’t be rescued by transforming the mother chromosome and vice versa

genes for sporulation - spo

loci = nonessential to vegetative growth but necessary to progress past a sporulation stage, designed by stage blockage (whichever # is whatever stage blocked)

genes for sporulation - ger

loci = necessary for germination

genes for sporulation - cot

coat protein, most = made by mother cell

gene product = added into spores

genes for sporulation - ssp

spore-specific protein/small acid-soluble proteins (SASPs), in spore core

gene product = added into spores

genes for sporulation - out

outgrowth —> prevent new macromolecular synthesis

germination

brings cell back to vegetative state (state 0), completing full cycle

germination - step I

activation - shock, heat, acid, etc

germination - step II

germination - hydration, loss of resistance, no protein synthesis

germination - step III

outgrowth - longer than 1 hr, protein synthesis and more sequential steps

phosphorelay

chain of transferring phosphate groups —> sporulation

Spo0A

part of phosphorelay, up/downregulates genes

Spo0A binds —> 0A boxes (areas of the genome) —> sporulation, competence, cannibalism

sigma factors in b. subtilis

activation of different sporulation sigma factors follow hierarchal order —> sequentially activated in alternating cellular components (cascade)

most important sigma factors in sporulation + know which compartment

sigmaH, sigmaF-G, sigmaE-K

know which compartment bc of phosphorylation

across time: early sporulation phase after polar septation: sigmaF —> sigmaE —> late sporulation phase after engulfment: sigmaG —> sigmaK

most important sigma factors in sporulation - sigmaH

(grand)mother —> i’m Hungry

most important sigma factors in sporulation - sigmaF-G

Fuck, time to sporulate

sigmaF - starts sporulation gene regulation cascade

most important sigma factors in sporulation - sigmaE-K

Eeek, i’m going to die!

sigmaE (aka sigma29) = 1st sporulation-specific factor