(27) Regulation of Phenotypes

lecture 27

development and differentiation refer to the

expression of different genes in different cells → leads to differences in morphology and function/activity of the different cell types

because most bacteria and archaea grow as single cells, few

show differentiation

however a few prokaryotes display the basic principle of differentiation with differences in gene expression in gentically identical descendants:

• endospore formation in the gram-positive soil bacterium Bacillus

• formation of two cell types (motile and stationary) in the gram-negative aquatic bacterium Caulobacter

• Heterocyst formation in the nitrogen-fixing cyanobacterium Anabaena

sporulation is carries out in response to

adverse growth conditions: starvation, desiccation, or inhibitory temperature

endospore =

spore formed inside a mother cell; requires that the cell divides asymmetrically and the smaller cell develops into the endospore

Bacillus uses 5 sensor kinases to monitor environment; kinase function via a

phosphotransfer system similar to but more complex Han two-component regulatory systems

multiple adverse conditions result in the

phosphorylation of sporulation factor proteins, eventually leading to the phosphorylation of Spo0A

When Spo0A is highly phosphorylated,

sporulation proceeds

Spo0A controls the expression of

several sporulation-specific proteins, including SpoIIE

SpoIIE removes the phosphate from

SpoIIAA → triggers SpoIIAA to remove the anti-sigma factor SpoIIAB → liberates sigma E

sigma F binds RNAP and promotes

the expression of proteins, including sigma G and proteins that cross into the mother cell and activate sigma E

• eventually spore coat is formed and the mature endospore is released

sporulating cell cannibalism cells of their own species as

a source of nutrients

bacillus cannibalism - cells in which So0A has become activated (phosphorylated) secrete

a toxic protein that lyses nearby Bacillus cells lose Spo0A has not yet been activated

bacillus cannibalism - cells already committed to sporulation make an

antitoxin to protect themselves from their own toxic proteins

bacillus cannibalism - shortage of key nutrients, particularly phosphate increases

the expression of the toxic protein in the sporulating cell

caulobacter

genus of Alphaproteobacteria that is common on oligotrophic (nutrient-poor) aquatic environments

caulobacter are

free-swimming swarmer cells alternate with cells that lack flagella and are attached to surfaces by a stalk with a holdfast

the role of swarmer cells is

strictly dispersal; cannot replace to make new cells

the tole of stalked cells is

strictly reproduction

to divide, swarmer cells must

differentiate into stalked cells

to swim, stalked cells must first

producer swarmer cells

caulobacter life cycle is controlled by

three major regulatory proteins whose concentration oscillate in succession

two of the proteins involved in caulobacter life cycle are

transcriptional regulators GcrA and CtrA

the third protein in caulobacter life cycle is DnaA

functions in its normal role to imitate DNA replication (binds ori) and as a transcriptional regulator

each of the regulators in caulobacter life cycle is active at a

specific stage in the cell cycle and each controls many other genes needed at that stage

CtrA is activated by

phosphorylation in emerging swarmer cells in reponse to external signals

phosphorylated CtrA activates genes encoding

flagellum synthesis and other swarmer-specific functions

CtrA-P also represses synthesis of

GcrA and inhibits of DNA replication by blocking origin of replication

as the cell cycle proceeds, CtrA-P is

degrades and DnaA levels rise

absence of CtrA-P allows

DnaA access to the ori → triggers DNA replication

• DnaA also activates expression of other proteins needed for chromosomal replication

the level of DnaA then falls due to

protease degradation

the level of GcrA rises; GcrA is a

regulatory protein that promotes chromosome replication, cell division and the growth of the stalk in the immobile daughter cell

eventually GcrA levels fall and CtrA

reappears in the daughter cell destined to swim away, repeating in the cell cycle

cell cycle resembles eukaryotic cells in many respects

no mitosis, ut analogs of G1, G2, and S are apparent

cyanobacteria are

oxygenic phototrophs that yield oxygen and fix nitrogen

nitrogen fixation is an

energy-demanding process catalyzed by nitrogenase

nitrogenases are extremely

oxygen-sensitive

some filamentous cyanobacteria form specialized cells called heterocyst that

are dedicated to nitrogen fixation

because heterocyst lack PSII, they are

anoxic cells → provide a good environment for nitrogenase and nitrogen fixation

photosystem II

the pigment-protein complex that produces O2 during oxygenic photosynthesis

heterocysts arise from

differentiation of phototrophic vegetative cells and typically develop in a pattern along a filament

• separates incompatible metabolic processes while allowing for nutrient exchange and growth

heterocyst formation = changes include the

formation of a thickened cell wall to prevent O2 diffusion into the cell, inactivation of PSII, expression of nitrogenase

nutrients can be exchanged between heterocyst and

adjacent vegetative cells from developing into heterocyst

heterocyst formation - the cascade is triggered by

a limitation of fixed nitrogen (nitrate, ammonia) that is sensed as an elevation in a-ketoglutarate, the acceptor molecule for formation of the amino acid glutamate

heterocyst formation - when the cell is starved for nitrogen,

a-ketoglutarate accumulates and activates the global transcriptional regulator NtcA

heterocyst formation - activated NtcA activates expression of

HetR

• major transcription regulator controlling heterocyst formation

heterocyst formation - HetR activates a

cascade of proteins needed for heterocyst differentiation, expression of cytochrome c oxidase to remove traces of O2, and expression of the nif operon for the synthesis and regulation of nitrogenase

heterocyst formation - only specific cells along the filament form

heterocyst in controlled manner

heterocyst formation - intracellular connections between cells in the filament allow

vegetative cells to provide fixed carbon to the heterocyst as an electron donor (for N2 reduction by nitrogenase) in exchange for ammonia produced in the heterocyst

• cell connections also allow intercellular communication

heterocyst formation - differentiating cells produce PatS (small peptide) →

diffuse away fro the developing heterocyst and inhibits differentiation in vegetative cells by preventing HetR rom activating the expression of proteins needed for heterocyst formation

biofilms are

polysaccharides matrices that are attached to surfaces and contain embedded bacterial cells

biofilm formation is a type of development cycle with four basic stages

attachment → colonization → development→ active dispersal

biofilm formation - cell attachment is facilitated by

structures such as flagella and pili or by cell surface proteins

biofilm formation - attachment is a single for

expression of biofilm specific proteins → produce intercellular signaling molecules and extracellular polysaccharides that imitate matrix formation

biofilm formation - once committed to biofilm formation, cell loses

flagella and becomes nonmotile; however, cells can be released form the biofilm through active dispersal

biofilm formation - signals guide bacteria in transitioning from

planktonic growth to life in a semisolid matrix

biofilm formation - trigged by cellular accumulation of the

regulatory molecule cyclic di-guanosine monophosphae (c-di-GMP)

biofilm formation - synthesis and degradation of c-di-GMP depends on

environmental and cellular cues

• triggers physiological events

pseudomonas aeruginosa is a classic

opportunistic pathogen that forms a tenacious biofilm containing polysaccharides that increase pathogenicity and prevent antibiotic penetration

biofilm formation - pseudomonas aeruginosa = form its primary reservoir of soil, it can

infect the blood, lungs urinary tract, ears, skin, and other tissues

biofilm formation - major symptom of cystic fibrosis are caused by

thick pseudomonas aeruginosa biofilms that develop in the lungs

biofilm formation - intracellular communication by quorum sensing is also necessary for

the development and maintenance of pseudomonas aeruinosa biofilms

biofilm formation - high cell density and accumulation of

acyl homoserine lactones (AHLs) signal to adjacent cells that the population is growing

biofilm formation - production of AHL also triggers

expression of protein needed for production of extracellular polysaccharides and the synthesis of c-di-GMP

biofilm formation - the final architecture of the biofilm is determined by

multiple factors in addition to signaling molecules

• nutritional factors, local flow environment, etc.

biofilm formation: Vibrio Cholerae - quorum sensing acts in an

opposite manner from that in Pseudomonas aerugingsa

biofilm formation: Vibrio Cholerae - accumulation of quorum signaling molecules represses

biofilm formation genes and activates flagellar and virulence genes

biofilm formation: Vibrio Cholerae - biofilm formation is triggered by

low cell densities, and is repressed by high cell densities

biofilm formation: Vibrio Cholerae - biofilm formation is more likely to occur when

it is found in its natural marine environment where nutrients are typically scarce than in the intestinal environment where nutrients are more plentiful

biofilm formation allows cells to

attach to marine surfaces for better access to nutrients and protection

antibiotics =

antimicrobial agents naturally produced by microorganisms; kill or inhibit the growth of bacteria by targeting essential molecular processes

antibiotics often target enzymes involved with

DNA replication, transcription, and translation

antibiotics - quinolones target

DNA gyrase and topoisomerase IV (DNA replication)

antibiotics - rifampin and actinomycin prevent

RNA synthesis (transcription)

antibiotics - puromycin inducing polypeptide chain

termination (translation)

antibiotics - aminoglycoside antibiotics (stretomycin) target

16S rRNA of the small ribosomal subunit → cause mRNA misreading (translation)

antibiotics - daptomycin specifically binds to

residues of the bacterial cytoplasmic membrane, leading to pore formation, depolarization, and death

antibiotics - polymyxins are cyclic peptides whose long tails target the

LPS layer of gram-negative outer membranes

antibiotics - B0lactams (penicillin, cephalosporin) interfere with

transpeptidation (formation of cross-links in peptidoglycan)

antibiotics - bacitracin binds to

bactoprenol preventing new peptidoglycan precursors from reaching the site of peptidoglycan synthesis

in order for microbes to survive, they need to have resistance mechanism that them to either

• modify the antibiotic

• inactivate the antibiotic with enzymes

• remove the antibiotic from the cell (efflux pumps)

• change cellular molecules so they’re not affected

random chromosomal mutations can lead to resistance

spontaneous E. coli mutants resistant to rifampin can be obtained by exposing a large population to the drug; mutants produce an altered RNAP unaffected by rifampin

resistance genes can exist on

mobile genetic elements and be transferred by horizontal gene flow

many mobile resistance genes encode enzymes that

inactivate the antibiotic by altering its structure

• B-lactamase cleaves a ring structure of B-lactams; an activating enzymes adds acetyl groups to chloramphenicol

efflux pumps are ubiquitous in gram-negative bacteria and transport

various molecules, including antibiotics, out of the cell

efflux lowers the

intracellular concentration of an antibiotic, allowing the cell to survive at higher external concentrations

many efflux pumps act promiscuously and transport

different classes of antibiotics outside cell thereby contributing to multi drug resistance

AcrAB-TolC of E. coli is one the best characterized

efflux pumps; pumps out rifampicin, chloramphenicol, and floroquinolones

biofilm growth leads to

increased antibiotic resistance; makes infections by biofilm-forming bacteria difficult to treat

antibiotic resistance also occurs when

the target of the antibiotic is no longer essential to the cell’s survival

• methicillin ia a B-lactam antibiotic that targets penicillin-binding proteins

• methicillin is resistance to B-lactamase cleavage

• MRSA strains encode alternative penicillin-binding proteins not recognized by methicillin, so are methicillin-resistant

persistance occurs when

a population antibiotic-sensitive bacteria produces rare cells that are transiently tolerant to multiple antibiotics

persisters are genetically identical to

their antibiotic-sensitive siblings, but are dormant (viable but do not grow)

because antibiotics target active processes, dormancy prevents

the antiobiotic from killing the cell

when the antibiotic treatment is stopped, cell emerge from

dormancy and grow

persistance uses chromosomal encoded

toxin-antitoxin modules, the stringent response, and phenotypic heterogeneity

toxin-antitoxin modules =

genetic locations that encode a toxin and an antitoxin

expression of the toxin-antitoxin genes provides

protection from the toxin but also stalls ribosomes, leading to the activation of the stringent response

strident responses decreases

rRNA and tRNA synthesis, protein synthesis, DNA replication, and cell division → leads to cell dormancy

E. coli hipAB genes encode a toxin-antitoxin (TA) modules that triggers resistance

• hipA = toxin that inhibits translation

• hipB = antitoxin

• normally HipA and HipB form a stable complex

• under antibiotic stress, some cells produce the alarming ppGpp

  • in these cells, increase ppGpp levels inhibit polyphosphatase → results in higher levels of polyphosphate (polyP)

E. coli hipAB toxin-antitoxin (TA) module that triggers persistence:

• polyP activates Lon

• activated Lon degrades HipB, freeing HipA

• free HipA targets glutamyl-tRNA synthetase (gltX) → prevents tRNA charging

• uncharged tRNA enter the ribosome leads to ribosome stalling

  • translation is inhibited which leads to arrested cell growth