1.5 regulation of bacterial gene expression

How do anti-virulence strategies rely on gene regulation

Many virulence factors are regulated by environmental factors, so if we can inhibit this virulence expression then we can help prevent and treat disease

Operon

A cluster of genes under the control of a single promoter, allowing coordinated regulation of gene expression in bacteria.

Where does polymerase bind in the operon

Polymerase binds to the promoter region of the operon, initiating transcription of the downstream genes.

Where do activators/repressors bind in the operon

To the operator sequence which is downstream to the promoter

What is the structure of an operon

Promoter, operon, then genes

What is positive regulation of an operon

Polymerase cannot transcribe until activator binds to operator

What is negative regulation of an operon

When the repressor is bound to the operator, polymerase activity is inhibited

The lac operon is an example of what

Negative regulation

What is the Lac operon repressor

LacI

What happens to the Lac operon in the absence of lactose

The repressor, LacI prevents transcription by binding to the operator, blocking RNA polymerase from accessing the promoter.

What is the role of lactose in the Lac operon

It is the inducer, which removes the repressor and allow transcription

What happens to the Lac operon in the presence of lactose

Lactose binds to the repressor and removes it to allow transcription of Lac operon

Inducer

Binds to the repressor to inactivate it

Corepressor

Binds to the repressor to activate it

Corynebacterium diphtheriae diphtheria toxin is only produced in what type of conditions

Low iron

How is Corynebacterium diphtheriae diphtheria toxin production regulated

protein DtxR

C. diphtheriae protein DtxR regulation process in high iron conditions

DtxR protein binds to iron and represses the transcription of the diphtheria toxin-encoding gene

C. diphtheriae protein DtxR regulation process in low iron conditions

DtxR protein cannot bind to iron, so it does not repress the transcription of the diphtheria toxin-encoding gene, allowing toxin production

Within the diphtheria toxin regulation, which are the corepressors

Iron and DtxR

An E. coli strain was isolated that has
a mutation in lacI. This mutation
results in the encoded protein being
unable to bind to lactose but is
functional otherwise. What would be
the consequences of this mutation?

unable to express the lac operon
genes, regardless of whether lactose was
present or not

two-component system (TCS)

A regulatory mechanism in bacteria that uses a membrane spanning sensor kinase and a cytoplasmic response regulator to control gene expression in response to environmental stimuli.

Sensor kinase structure

Input——-Transmitter

Response regulator structure

Receiver———Output

Upon sensing an extracellular signal, sensor kinases ______

Auto phosphorylate

On what residue of the sensor kinase does autophosphorylation occur

histidine (H) residue in the transmitter domain

What happens after the kinase autophosphorylates

transfers the phosphate group to the response regulator.

What residue is phosphorylated on the response regulator

aspartate (D) residue located within the receiver domain of the response regulator

What happens after the response regulator is phosphorylated

It promotes DNA-binding, usually by promoting dimerization

What types of sequences does the response regulator typically bind to

direct-repeat or inverted-repeat DNA sequences.

Quorum sensing

the production and release by individual bacteria of molecules called autoinducers that modulate gene expression in response to bacterial population density

Quorum sensing facilitates

coordinated responses by bacterial populations, e.g.:
 biofilm formation
 virulence factor secretion
 competence
 bioluminescence

Agr system of S. aureus

a quorum sensing system that regulates virulence gene expression in response to population density.

AgrB

a membrane protein involved in the processing and export of autoinducer peptides (AgrD) for the Agr system.

AgrD

Peptide which is processed to an autoinducer that activates the Agr system.

AgrC

A receptor protein that detects the autoinducer and initiates the signaling cascade in the Agr system.

AgrA

Response regulator that binds to the P2 and P3 promoters driving production of RNAII and RNAIII when phosphorylated

RNAIII

a small regulatory RNA (sRNA; see later) that targets multiple virulence factor-encoding mRNAs

Within the S. aureus AGR system, which is the membrane spanning receptor for the autoinducer

AgrC

Within the S. aureus AGR system, which is the response regulator

AgrA

Within the S. aureus AGR system, which is the autoinducer

Autoinducing peptide (AIP) processed from AgrD

What is the second quorum sensing system that interacts with Agr

RNAIII system

Role of RNAIII in the secondary quorum sensing system

Becomes an autoinducer called RNAIII activating peptide (RAP)

Within the secondary quorum sensing system, what happens when RAP is detected

Autophosphorylation of target of RAP (TRAP)

What happens when TRAP is activated

enhances Agr function, so increased virulence

other staphylococcal species produce a peptide that inhibits RAP called _____

RNAIII inactivating peptide (RIP)

What does RIP do

RIP inhibits the activity of RAP, thereby reducing the expression of virulence factors

Why would other staphylococci produce RIP

To combat the competition for resources and reduce virulence in neighboring strains.

How might our knowledge of Agr, RAP, and RIP
be exploited, via anti-virulence approaches, to
create a novel therapy

We could use RIP to target and inhibit RAP, thereby reducing virulence in pathogenic staphylococci.

How might our knowledge of Agr, RAP, and RIP
be exploited, via anti-virulence approaches, to
create a novel vaccine

vaccine targeting RAP could induce antibodies that neutralize the autoinducer, thereby preventing the activation of virulence factors regulated by the Agr system and protecting against staphylococcal infections

sRNAs

are small non-coding RNAs that regulate gene expression

Cis acting sRNA

act on the same molecule of RNA they are transcribed from, typically affecting gene expression by binding to complementary sequences within the same transcript

Transacting sRNA

act on different RNA molecules, often silencing gene expression by binding to target mRNAs from different genes

Activating sRNA

are small non-coding RNAs that enhance gene expression by promoting the stability or translation of target mRNAs.

Repressing sRNA

are small non-coding RNAs that inhibit gene expression by destabilizing target mRNAs or preventing their translation.

Base-paring sRNA

regulate gene expression by directly binding to complementary sequences on target mRNAs

Non base-pairing sRNA

influence gene expression without binding directly to mRNA. They might interact with proteins that regulate transcription or translation

SodB

Converts superoxide into hydrogen peroxide and oxygen

RyhB sRNA of E. coli

sRNA which represses sodB mRNA translation by binding to its 5’-untranslated region (5’-UTR) and
occluding the ribosome binding site (RBS)

How can sRNA modify expression of target mRNA

Usually by reducing stability but sometime enhancing

Which of the following is true about
autoinducers?
(1) they usually bind to small regulatory RNA
molecules to bring about regulatory activity
(2) homoserine lactones are the class most used by
Gram-positive organisms
(3) they bring about changes in gene expression
only when present at high concentration
(4) they bind to the promoter sequence of select
genes and inhibit transcription
(5) they do not require any other regulatory
element (e.g., proteins or RNAs) to bring about
regulatory activity

they bring about changes in gene expression
only when present at high concentration

Phase variation

the reversible switching of protein expression in bacteria between ON and OFF states as a way of responding to rapidly changing environments

How does phase variation occur

via DNA alterations and can occur at frequencies that are much higher than classical mutation rates

Since phase variation results in extremely high levels of DNA alterations, how does this affect the population of

leading to heterogeneity within a population of bacteria

Mechanisms of phase variation

Inversion
gene conversion
Slipped strand mispairing

How is the expression of E. coli type I fimbriae regulated

Phase variation using site specific inversion of a segment which includes the promoter

FimA

encodes the major subunit of type I fimbriae

Type I fimbriae

cell surface proteins that are essential to colonize a
host, is regulated by a phase variation

FimB and FimE

Recombinases which control site specific inversion of FimA promoter

Why would e coli want phase variation in the FimA gene

FimA is targeted by the immune system, so varying the expression of it helps them survive

Neisseria gonorrhoeae can cause repeated infections in an individual by ____

the alteration of Type IV pili through gene conversion

PilE

the major component of Type IV pili in Neisseria gonorrhoeae, involved in adhesion and immune evasion.

pilS

multiple homologous but non-transcribed
genes involving the type IV pili

How does the Neisseria gonorrhoeae gene conversion occur

Recombination between pilE and one or more pilS genes creates new pilE genes that produce antigenically distinct proteins

Controversy surrounding Neisseria gonorrhoeae gene conversion in the type IV pili

Most consider it antigenetic variation instead of true phase variation

Slipped-strand mispairing (SSM)

When there are multiple
repeats (e.g., ATCGATCGATCGATCG or AAAAAAAAAA) the DNA polymerase can “slip”,
resulting in an increase or decrease in repeat number

How does the change in repeats affect expression (SSM) if the repeat is in the promoter

then SSM can turn
transcription ON/OFF (e.g., by altering the spacing between the -10 and -35 promoter elements

How does the change in repeats affect expression (SSM) if the repeat is in the gene

then SSM can lead to the formation of a premature stop
codon or insert/delete codons. This can be reversed

Whole-genome sequencing of group A Streptococcus (GAS) isolates identified that
invasive disease isolates often harbor mutations in the genes encoding the CovR/S TCS.
Why is this not phase variation

because phase variation typically involves reversible changes in gene expression, while mutations in CovR/S TCS lead to stable, non-reversible alterations in gene function.
Also isolates have different mutations