Qazvin university of medical science Journal Club & MSc ...file.qums.ac.ir/repository/sm/گروه آموزشی میکروب شناسی...The HPK has two domains. The input domain is

Signal integration in bacterial

two-component regulatory

systems

PRESENTED BY: SEPIDEH BENVARI

SUPERVISED BY: DR. ASLANIMEHR

Qazvin university of medical scienceJournal Club & MSc Seminar

Free-living organisms modulate their gene expression patterns in response to environmental cues.

This modulation requires sensors to detect chemical and/or physical signals, and regulators to

bring about changes in the levels of gene products.

Adaptability is a crucial characteristic of bacteria that are able to prosper in a wide variety of

environmental conditions.

produced by: sepideh benvari supervised by: Dr. Aslanimehr

Regulation of Gene

Expression in bacteria

1. Gene copy number

2. Transcriptional control

2.1. Promoters

2.2. Terminators, attenuators and anti-terminators

2.3. Induction and repression: regulatory proteins

2.4. Two-component regulatory systems

2.5. Global regulatory systems

2.6. Quorum sensing

3. Translational control

3.1. Ribosome binding

3.2. Codon usage

3.3. Stringent response

3.4. Regulatory RNA

4. Phase variation


In bacteria, extracellular signals are transduced into the cell predominantly by two-

component systems.

Although two-component signaling systems are found in all domains of life, they are most

common by far in bacteria, particularly in Gram-negative and cyanobacteria.

They are much less common in archaea and eukaryotes; although they do appear in yeasts,

filamentous fungi, and slime molds, and are common in plants.


The number of two-component systems present in a bacterial genome is highly

correlated with genome size as well as ecological niche; bacteria that occupy niches with

frequent environmental fluctuations possess more histidine kinases and response

regulators.

New two-component systems may arise by gene duplication or by lateral gene transfer,

and the relative rates of each process vary dramatically across bacterial species. In

most cases, response regulator genes are located in the same operon as their cognate

histidine kinase.


The average number of two-component systems in a bacterial genome has been estimated as

around 30, or about 1-2% of a prokaryote's genome. A few bacteria have none at all -

typically endosymbionts and pathogens - and others contain over 200.

Two-component signal transduction systems enable bacteria to respond to a wide variety of

stimuli, including nutrients, cellular redox state, changes in osmolarity, quorum signals,

antibiotics, temperature, chemoattractants, pH and more.


two-component systems

In general, these systems comprise an integral membrane protein called a histidine

protein kinase (HPK) and a separate cytoplasmic protein called a response regulator

(RR).

The HPK has two domains. The input domain is usually found on the outside of the

cell, in an ideal position to detect environmental signals. In contrast, the transmitter

domain is located on the cytoplasmic face of the cell membrane, positioned to interact

with the RR.


When a stimulus causes a conformational change in HPK,

the HPK autophosphorylates at a conserved histidine

residue and subsequently transfers this phospho- group

to the response regulator. In this form the RR is able to

bind to DNA to regulate transcription of the target genes.

The RR also consists of two domains: a receiver domain

containing an aspartate residue which accepts the

phospho- group, and an output domain which can bind to

DNA.




Phosphorelays

Phosphorelays are a more complex version of the TCS in which a sensor kinase first

transfers the phosphoryl group to a response regulator possessing the domain with

the conserved aspartate but no output domain.

The response regulator subsequently transfers the phosphoryl group to a histidine-

containing phosphotransfer protein, and it is the latter protein that serves as a

phosphodonor to the terminal response regulator, which possesses an output domain

mediating a cellular response.



The vast majority of response regulators are active

only when phosphorylated. Therefore, any condition

or product that affects the phosphorylated state of a

response regulator will impact its ability to exert its

biological functions.

Consequently, the output of a response regulator is

determined not only by the presence of the specific

signals sensed by its cognate sensor kinase but also

by gene products that stimulate or inhibit its

phosphorylation.



REGULATORY MECHANISMS

The sole purpose of two-component signal transduction systems is to allow for

regulation; the signaling pathway merely provides steps at which the flow of

information can be modulated.

A great diversity of regulatory mechanisms has been overlaid on the central

phosphotransfer/phosphorelay pathways, allowing optimization of signal

transmission for the specific needs of each system.


Some systems output a graded response, such as

the EnvZ-OmpR system that mediates the

differential expression of porin genes ompF and

ompC. Others, such as the B. subtilis Spo system

that controls commitment to sporulation, output

an all-or-nothing response.

Regardless of the output, both types of systems

can involve a significant amount of regulation

and often involve a number of auxiliary protein

(TCS connectors) components. The primary

targets for regulation are the activities of the

HK and dephosphorylation of the RR.


TCS connectors (which for the sake of brevity will also be called connectors) are an emerging group

of proteins that modulate the activity of sensor kinases and response regulators at the post-

translational level.

Because connector proteins are typically synthesized in response to signals that are different from

those sensed by the cognate sensor, they often establish regulatory links between otherwise

independent signal transduction pathways (in other words, they “connect” a TCS to the signal(s)

controlling a different regulatory system).

TCS connectors


TCS connectors use a variety of mechanisms to alter response regulator output:

1. Inhibiting sensor kinase autophosphorylation

2. Promoting dephosphorylation of phosphorylated response regulators and His-containing

phosphotransfer proteins

3. Inhibiting response regulator dephosphorylation

4. Activating a sensor kinase

5. Inhibiting DNA binding by a response regulator

6. Inhibiting recruitment of RNA polymerase to promoters

7. Sequestering adaptor proteins that promote protein degradation


Inhibiting sensor kinase

autophosphorylation

TCS connectors can inhibit sensor autophosphorylation or promote dephosphorylation of

phosphorylated response regulators.

In the B. subtilis phosphorelay, the sensor kinase KinA is activated by an unknown signal,

which results in autophosphorylation from ATP and subsequent phosphotransfer to the

response regulator Spo0F. Spo0F passes on the phosphoryl group to the His-containing

protein Spo0B, which in turn transfers it to the terminal acceptor, the response

regulator Spo0A.


The phosphorylated form of Spo0A acts as a

transcription factor, being the key activator of

sporulation genes. The connectors Sda and KipI

block activation of the phosphorelay by inhibiting

KinA autophosphorylation. The connectors RapA,

RapB, RapE, and RapH promote

dephosphorylation of the response regulator

Spo0F-P; the connectors Spo0E, YisI, and YnzD act

in a similar way on Spo0A-P.

The expression of connectors is controlled by

factors such as growth conditions, status of the

DNA replication machinery, and development of

competence (through the action of ComA and

ComK the key regulators of competence genes).



Inhibiting response regulator

dephosphorylation

TCS connectors can promote activation of response regulators and sensor kinases.

The connector-mediated pathway from S. enterica. The low Mg2+ signal activates the

PhoP/PhoQ TCS, which triggers the expression of the connector PmrD. PmrD binds to

the phosphorylated form of the response regulator PmrA, thereby protecting it from

dephosphorylation.


PmrA-P binds to DNA and regulates its target promoters. PmrA-P represses

transcription of the pmrD gene, thus establishing a negative feedback loop controlling

PmrD expression. The PmrA/PmrB TCS can be activated directly by the Fe3+ signal.


Inhibiting recruitment of RNA polymerase

to promoters

TCS connectors can inhibit binding of activated response regulators to DNA or prevent RNA polymerase from interacting

with a response regulator.

The ComA/ComP TCS from B. subtilis responds to the extracellular quorum-sensing signal, the peptide ComX. Upon

activation, ComA promotes transcription of the srf operon, which leads to development of the competent state.

Binding of ComA to DNA is inhibited by the connectors RapC, RapF, and RapH.

These connectors, in turn, are deactivated upon binding to the corresponding Phr

peptides. The action of ComA is also inhibited by the connector Spx, which disrupts

the interaction between ComA and RNA polymerase.




POTENTIAL TARGETS FOR ANTIMICROBIAL

THERAPY

The search for structurally unique antibiotics that inhibit newmolecular targets has led researchers to

prokaryotic two-component systems.

Two-component systems are attractive for several reasons. First of all, they are widespread in bacteria and,

sofar, absent in mammals. Therefore, general HK or RR inhibitors could potentially be broad-spectrum

antibiotics.


Perhaps the most attractive reason for targeting two-component systems is that they are used

by pathogenic bacteria to control the expression of virulence factors required for infectivity.

Several well-characterized virulence systems are the A. tumefaciens VirA/VirG system, the B.

pertussis BvgA/BvgS system, and the Salmonella typhimurium PhoP/PhoQ system. Interestingly, some bacteria have developed two-component systems that regulate

resistance to certain chemotherapeutics.

These include the vancomycin resistance systems in Enterococcus faecalis (VanR/VanS)

and Streptococcus pneumoniae (VncS/VncR), as well as the system associated with

tetracycline resistance in Bacteroides fragilis (RprX/RprY).




Hexose phosphate is an important carbon source within the cytoplasm of host cells. Bacterial pathogens that invade, survive, and multiply within various host epithelial cells exploit hexose phosphates from the host cytoplasm through the hexose phosphate transport (HPT) system to gain energy and synthesize cellular components.

In Escherichia coli, the HPT system consists of a two-component regulatory system (UhpAB) and a phosphate sensor protein (UhpC) that tightly regulate expression of a hexose phosphate transporter (UhpT).

Staphylococcus aureus also can invade, survive, and multiply within various host epithelial cells. the HPT system in S. aureus that includes the hptRS (a novel two-component regulatory system), the hptA (a putative phosphate sensor), and the uhpT (a hexose phosphate transporter) genes.

We demonstrated that both hptA and hptRS are required to positively regulate transcription of uhpT in response to extracellular phosphates, such as glycerol-3-phosphate (G3P), glucose-6-phosphate (G6P), and fosfomycin.

disruption of the hptA, hptRS, or uhpT gene impaired the growth of bacteria when the available carbon source was

limited to G6P, impaired survival/multiplication within various types of host cells, and increased resistance to

fosfomycin.

the HPT system plays an important role in adaptation of S. aureus within the host cells and could be an important target

for developing novel antistaphylococcal therapies.



Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life-threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or artificial implants.

During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca2) are elevated. P. aeruginosa responds to externally added Ca2 through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2 level, followed by its removal to the basal submicromolar level.


we used microarray analysis to characterize the global transcriptional response of P. aeruginosa to elevated external Ca2 levels. we identified the TCS PA2656-PA2657 (here referred to as carSR, for calcium regulator sensor and regulator), whose transcription is highly induced by elevated Ca2 in planktonic cultures of P. aeruginosa PAO1.

Using deletion mutations and microarray analysis, we identified the regulatory targets of carSR, which include the hypothetical proteins PA0320 and PA0327. Further characterization of PA0320 and PA0327 indicate that they play roles in maintaining Ca2 homeostasis. PA0327 also influences the production of the virulence factor pyocyanin and swarming motility in a Ca2-dependent manner.



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Qazvin university of medical science Journal Club & MSc ...file.qums.ac.ir/repository/sm/گروه آموزشی میکروب شناسی...The HPK has two domains. The input domain is