Hardik Shah - Lecture on Quorum Sensing

QUORUM SENSING

Hardik Shah - Lecture on Quorum Sensing

The ability of bacteria to monitor their own population density

and modulate gene expression.

Number of different mechanisms best characterized one is

acyl-HSL based system.

First described in marine bacterium Vibrio fischeri. (host -

Hawaiian bobtail squid, Euprymna scolopes).

VF produce autoinducer regulate the production of light

producing enzyme Luciferase.

Other examples Plant pathogens - Erwinia sp., and Agrobacterium tumefaciens. Opp. Human pathogen Pseudomonas aeruginosa.

Hardik Shah - Lecture on Quorum Sensing

Hardik Shah - Lecture on Quorum Sensing

Hardik Shah - Lecture on Quorum Sensing

Hardik Shah - Lecture on Quorum Sensing

Hardik Shah - Lecture on Quorum Sensing

Hardik Shah - Lecture on Quorum Sensing

luxI/luxR system in Vibrio fischeri

In these systems, the canonical I gene encodes the acyl-HSL synthase, R gene encodes the regulatory protein, usually an activator. AinS N-octanoyl HSL (C8-HSL) and the LuxS autoinducer

2 (AI-2) signal synthases and Their cognate sensory histidine kinases LuxN and LuxP/Q, respectively synergistically regulates the regulatory circuit.

Hardik Shah - Lecture on Quorum Sensing

Acyl HSL
VF : - 3 - oxo haxanoyl HSL

Techniques used for study: - HPLC & NMR combined.

Dual QS Systems Ps. Aeruginosa - With two distinct acyl HSL
Butyryl HSL

3 - oxo dodecanoyl HSL

HSL with longest acyl chain tetradecanoyl HSL by R. leguminosarum

and with shortest acyl chain butyryl HSL.

Hardik Shah - Lecture on Quorum Sensing

Synthesis of HSL
Study of different systems indicated that VF luxI gene only

gene required to direct synthesis of particular acyl HSL.

e.g. A. tumefaciens (traI) and P. aeruginosa (lasI)

Substrates for synthesis

SAM (s adenosyl metheonine)
methionine is converted to SAM via SAM synthetase (metK). Required for Different NA-methylation reactions, Synthesis of polyamines like spermidine and Variety of step in single carbon metabolism

Acyl ACP
Intermediate of FA biosynthesis Can act as substrate in lipid biosynthesis

Hardik Shah - Lecture on Quorum Sensing

Enzymatic Mechanism and Inhibitors

Ping pong reaction mechanism
Enzymes

for fatty acid synthesis and LuxI both has common sub.

Acyl group from acyl-ACP is

transferred to active site residue on acyl-HSL synthase.

Enz-acyl intermediate binds to

SAM forming Amide bond (betn acyl & amino grp of SAM) with lactonization of SAM yielding HSL ring.

Hardik Shah - Lecture on Quorum Sensing

INHIBITORS OF ACYL-HSL SYNTHESIS

Inhibitor includes (for RhlI) Reaction products 5- methylthioadenosine and holo-ACP. S-adenosylhomocysteine and sinefungin.

Hardik Shah - Lecture on Quorum Sensing

LuxI type proteins

20 LuxI family members.

Vary in length of 194-226 aas.

10 completely conserved residues, most in N-half. 7/10 are charged residues.

N-terminal portion highest homology

C-terminal little homology All above leads to finding that carboxy terminus is

involved in acyl side chain specificity.

Hardik Shah - Lecture on Quorum Sensing

Mutagenesis studies indicated region between 25-70 aa

residues may constitute active site of protein involved in amide bond formation.
Region between 104-164 aas critical in binding of acyl

chain.
For RhlI similar trends as in LuxI. 8 critical residues reqd. for acyl-HSL synthesis (7 as in LuxI, 1 unique) no residue in c-termini region critical for its activity.

Hardik Shah - Lecture on Quorum Sensing

AinS family of Acyl-HSL synthase

Two genes not homologous to luxI family (ainS and luxM)

with 34% identity between both

ainS Found in V. fischeri 46-kD protein with 218 aas Responsible for production of octanoyl-HSL Contains lux boxes upstream of its promoter Compete with 3-oxohexanoyl-HSL for binding to LuxR. luxM Found in V. harveyi 25-kD protein with 216 aas Responsible for production of 3-OH-butanoyl-HSL Positively regulates bioluminescence in V. harveyi.

Hardik Shah - Lecture on Quorum Sensing

HSL ring is obtained from SAM.

Acyl side chain in octanoyl-HSL may be from either

octanoyl-CoA or octanoyl-ACP. Using both substrates may represent a strategy, the cell uses to ensure that it has substrates for acyl-HSL synthesis during different physiological conditions.
Inhibitors includes S-adenosylhomocysteine and holo-

ACP, indicating that

Hardik Shah - Lecture on Quorum Sensing

Acyl-HSL release and accumulation

Freely diffuse across bacterial envelope (passive diffusion). In VF, distribution of 3-O-6-HSL is dictated by its conc gradient across

bac. Envelope.
In PA, MexAB-OprD efflux system is general transporter of

hydrophobic residues from p-lipid bilayers and play role in release of 3-O-12-HSL. 3-O-12-HSL is a substrate for this efflux system.
Acyl-HSL accumulation --- primary mech dictating interaction with

LuxR type receptor. In light organs of symbionts --- provide environment for VF.
In other bacteria, mechanism probably involves formation of

aggregates or surface associated biofilms. Multiple pathways for induction. Incl. flow characteristics, dimension and diffusion characteristics and chemical nature of environment.

Hardik Shah - Lecture on Quorum Sensing

LUXR TYPE PROTEINS

Diff proteins with 18-23% sequence identity. 2 clusters strongly conserved (66-138 and 183-229 aas),

HSL interaction and DNA binding motif.

C-terminal regions of LuxR type proteins --- homology

with prok transcription factor (two component)

Steps in role of LuxR proteins Recognition and binding of acyl-HSL Conf. change and alteration in multimerization on a-HSL binding Binding or release of sp. Sites proximal to target genes Transcriptional activation.