Gene Regulation - Signalling

Efficiency Poor RBS

SequesteringRiboswitches

Stringent Response

Transcription

Replication

Translation

DNA

mRNA

Protein

ProcessingStabilityStatus

StabilityAmount

Adenylation

Coupling

InitiationElongation

TerminationTemporalTandem Promoters

Signals: intra- and extracellular

MethylationSuperhelicity

Rearrangement

Gene Regulation

Two component response- regulator system

Covalent modification of the effector by the modulator

Involves phospho-relay

Three component response-regulator system: Quorum sensing

Modification?

Involved diffusible molecules, the acyl-homoserine lactones

Regulation via Signaling

Sense minute fluctuations in chemical and physical conditions

The process:

Stimulus detection

Signal processing including amplification

Integration of sensory inputs

Production of appropriate output responses

Two components: Sensor and Response Regulator

They have “transmitters” and “receivers”

Input domain

Transmitter

Sensor

Receiver Output domain

Response Regulator

Input

Signal

Output

Signal

Two-Component Signal Transduction

CytoplasmIM

Input domain Transmitter

Sensor

Receiver Output domain

Response Regulator

Input

Signal

Output

Signal

Transmitters and receivers communication involves phosphorylation activities

Transmitters:

Autokinase activity

Attach phosphate from ATP to histidine --> Histidine kinases

High-energy intermediate that transfers the Pi to and Aspartate residue on the “Receiver”

Localization

Transmitter: Cytoplasmic membrane

Receiver: Cytoplasm

+ P

Periplasm

Two-Component Signal Transducer Components

Three reactions:

(I) ATP + His <--> ADP + His ~ P

(II) His ~ P + Asp <--> His + Asp ~ P

(III) Asp ~ P + H2O <--> Asp + Pi

These reactions transduce information - also called the “phospho-relay’

(I) The phosphoryl group in ATP is first transferred to a histidine side-chain

in response to a stimulus

(II) The phosphoryl group is then transferred from the phosphohistidine residue to an aspartate side-chain --> phosphorylation induced conformational change --> eliciting response

stimulate or repress the transcription of specific genes

(III) Finally the phosphoryl group from a phosphoaspartae residue is transferred to water

Always have to function in pairs

Widespread - prokaryotic and eukaryotic

Two-Component Signal Transducer Components

The genes for the two components are linked.

Part of an operon

Sensor Response Regulator

SensorResponse Regulator

Two-Component Signal Transduction System

Localized in the cytoplasm

Stimulate or repress the transcription of specific genes - DNA-binding Domains

Conserved regions

N-terminal Phosphorylation Domain

C-terminal DNA-binding Domain

Example:

OmpR circuitry

Activates transcription of ompF and ompC

Represses transcription of ompF

Histidine kinases Reference Pageshttp://www.uni-kl.de/FB-Biologie/AG-Hakenbeck/TGrebe/HPK/Classification.htm

Receiver

Phosphorylation Domain

Output domain

DNA-binding

Domain

Response Regulators

http://info.bio.cmu.edu/Courses/03441/TermPapers/99TermPapers/TwoCom/osmoregmech.html

The proteins involved are

EnvZ Sensor Kinase

OmpR Response Regulator

Three reactions:

(I) Mg-ATP + EnvZ-His <--> ADP + EnvZ-His ~ P

(II) EnvZ- His ~ P + OmpR-Asp <--> EnvZ-His + OmpR-Asp ~ P

(III) OmpR-Asp ~ P + H2O <--> OmpR-Asp + Pi

The phosphorylated residues:

His 243

Asp 55

Osmoregulation

Cytoplasmic membrane protein with at least two membrane spanning domain

Unconserved regions provide the specificity (80 %)

Highly conserved regions

Sensory Domain ~115 residues

Catalytic Domain ~270 residues

Signal Transduction

Domain

IM

H Box (His-243)

N Box - Kinase domain

G Box - ATP Binding

EnvZ

Histidine phosphorylation is NOT intra-molecular, requires a second monomer

Can also act as phosphatase - dephosphorylate the response regulators

Sensor Kinases or Histidine Protein Kinases

http://www.science.siu.edu/microbiology/micr425/425Notes/03-Osmoreg.html

OmpR bindig Sites: High and Low affinity

Low OP

OmpR-P form binds to the high affinity site --> activate ompF transcription

High OP

Increase in OmpR-P,

• Bind to both high & LOW affinity sites --> inhibit ompF transcription

• Bind to ompC promoter --> activate transcription

• Activate micF expression --> inhibit ompF translation

OmpR Regulation of OmpF and OmpC

Gene Regulation: Regulation of Luminesence in

Marine Bacteria

Light Production Costs Energy and Is Regulated…..

Bacterial Bioluminescence

Chemiluminescence from an Enzyme

Lets Light Up Some Terms:

Fluorescence

Phosphorescence

Luminescence

Energy Source? Excited States?

Excited State Life Times?

Bioluminescence

Energy Change During a Chemical Reaction –

(regardless of the energy source: chemical or light)

Substrate(s) Product(s) + Energy

or

Substrate(s) + Energy Product(s)

Thermodynamics

In honor of Lucifer !

In Greek Mythology Lucifer is the Bearer of Light

In Christian Mythology….

Luciferase = Enzyme Catalyzing Bioluminescent Reaction

Luciferin = small molecule whose excited state produces light

Bioluminescent Enzymes Luciferases

FMNH2 + O2 + Fatty Aldehyde FMN + Fatty Acid + H2O + hv

(Light)

Fatty-CHO Fatty-COOH

Reduced (energy rich) oxidized (energy poor)

FMNH2 FMN

both oxidized by O2

Luciferase is a Mixed Function Oxidase

Bacterial Luciferase Reaction

E = hν

Energy = Planck’s Constant x vibrational frequency of light

The smaller the Wavelength (λ), the larger the v.

Planck-Einstein Equation

Proton Motive Force

1. ATP

2. Rotate Flagella

3. Active Transport

4. Reversed electron transport

It costs energy to produce light !

Bacterial BioluminescenceA Shunt from Electron Transport

Luciferase Assay

Fast and Slow Luciferases The Enzyme Assay

Nealson, Platt and Hastings, 1970. Their Photobacterium fischeri MAV was reclassified Beneckea harveyi and now Vibrio harveyi MAV.

Is Luciferases Constitutive or Inducible?

Shewanella woodyi Photobacterium phosphoreum WSU

Is Luciferases Constitutive or Inducible?

is called AUTOINDUCTION

bacterial growth results in production of “autoinducer”…. a small diffusible molecule that when it accumulates in the medium to sufficient concentration induces the synthesis of luciferase.

Nealson, Platt and Hastings, 1970

Also called Quorum Sensing (since 90’s) … the molecules are called AutoInducers. AI-1, AI-2, etc.

Phenomenon of Luciferase Induction

The number of members of a group or an organization required to be present to transact business legally, usually a majority. - Webster

Quorum

Quorum Sensing - 1995

Widespread phenomena but have to go through the drill:

“Every novel idea in science passes through three stages:

1. First people say it isn’t true, 2.Then they say it’s true but not important, and3. Finally they say it’s true and important, but not new”

Peter Greenberg

What was unique?

Covalent Modification is not NEW – but new for bacteria

Diffusible chemicals to mediate regulation is NEW for bacteria

The Inevitable Science Drill

0.01

0.1

1

10

A 6

60 n

m

1

10

100

1000

LUC

IFE

RA

SE

AC

TIV

ITY

(qp

s X

107 )

0 2 4 6 8 10

TIME (hours)

Population density-dependent OR Growth-phase-dependent

Cell-Density Dependent Expression

Luminescent bacteria in marine environment

Why do they produce light??

The genes that regulate luminescence are lux genes

Intracellular communication

Quorum sensing

60s:The first report on cell-density dependent phenomenon in the marine bacterium Vibrio fisheri and V. harveyi

by Nealson, Hastings, Eberhard, Makemson.

70s: V. fisheri lives in specific light organs (Euprymma ecolopsis) as pure culture (107/ml). Bioluminescence phenomenon - at stationary phase called autoinduction

70’s-80s: The details of the regulation and mechanisms of luminescence were worked out. The genes involved in autoinduction, luxR and luxI, were cloned. Other effectors: Arginine, glucose (c-AMP), iron, oxygen, salt.

90s: The term quorum sensing was coined. Discovery of LuxR-LuxI systems in many gram negative bacteria.Homology of LasR in Pseudomonas aeruginosa to LuxR.Discovery of anti-quorum sensing molecules - Furanone

History

Euprymna scolopes

A network of cellular transduction mechanisms.

Integrate signals from the bacterial environment to control gene expression,

and thereby the bacterial phenotype.

Relies on the accumulation of small diffusible, extracellular signaling molecules to modulate the transcription of target operons.

Autoinducers/Communication Molecules/Bacterial Pheromones.

Interaction between autoinducers and a positive transcriptional activator.

Three-component system

Cell-to-cell Communication

Quorum Sensing Molecules

Signaling molecules

(Acyl homoserine lactone/ AHL)

and many others

Synthetase

(LuxI homologue)Quorum-sensing Signal Generators - “I”

Regulator

(LuxR homologue)

Signal Receptors - “R”

Three Component System

luxI C D A B E GluxR(not drawn to scale)

Subunits of luciferase

Fatty acid reductasecomplex

Lux system - Vibrio fischeri

Genes involved in the luminescence

Found in diverse microorganisms

Affects various physiologic functions

Genes are linked but NOT co-transcribed

Divergently or independently transcribed

LuxI and LuxR Family

Synthetase for autoinducers forms amide bond between acyl-acyl carrier protein and S-adenosyl methionine

N-acyl-homoserine lactones (AHLs)

Differ in the structure of their N-acyl side chains (sat/unsat)Vary between 4 to 14 carbons

O

N

H

homoserinelactone

Species Specificity

O

Examples:N-butanoyl-L homoserine lactone (BHL) N-hexanoyl-L-homoserine lactone (HHL) N-octanoyl-L-homoserine lactone (OHL)

LuxI Family

Transcriptional regulators

lux Box Centered around -40

R N S T G Y A X G A T N X T R C A S R TG G G T G T A G G A T G G T G C A G G TA A C C A A A A C A

T T T T C C C C - -

N C250 aaAI - binding

(79-127)DNA- binding

(160-250)HTH

Regulator Domain Activator Domain

Multimerization(120-60)

TranscriptionalActivation(230-50)

LuxR Family

luxIluxR

LuxI

OOHNH

O

LuxR

+

PassiveDiffusion

LuxI-LuxR System

Vibrio anguillarum(Milton et al 1997)

vanI/vanR N-(3-oxodecanoyl)-L-homoserine lactone (ODHL)

ainS/ainRVibrio fischeri(Kuo et al 1994;Gilson et al 1995)

N-octanoyl-L-homoserine lactone (AI-2; OHL)

luxI/luxR N-3-oxohexanoyl-L-homoserine lactone (AI-1; OHHL)N-hexanoyl-L-homoserine lactone (HHL)

Vibrio fischeriPhotobacterium fischeri(Engebrecht & Silverman , 1984;Kuo et al 1994)

rhiI/rhiRRhizobium leguminosarum(Cubo et al 1992; Gray et al 1996;Schripsema et al 1996)

N-(3R-hydroxy-7-cis-tetradecanoyl)-L-homoserine lactone(HTHL; Small bacteriocin )

solI/solRRalstonia solanacearum(Flavier et al 1997)

N-hexanoyl-L-homoserine lactone (HHL) &N-octanoyl-homoserine lactone (OHL)

carl/carRErwinia carotovora(McGowan et al 1995)

N-(3-oxohexanoyl)-L-homoserine lactone (OHHL)

Erwinia stewartii(Beck von Bodman &Farrand 1995)

esaI/esaR N-(3-oxohexanoyl)-L-homoserine lactone (OHHL)

Enterobacter agglomerans(Swift et al 1993)

eagl/eagR N-(3-oxohexanoyl)-L-homoserine lactone (OHHL)

traI/traRAgrobacterium tumefaciensTi plasmid(Zhang et al 1993;Hwang et al 1994)

N-(beta oxo-octan-1-oyl)-L-homoserine lactone ( BOHL) & N-(hexan-1-oyl)-L-homoserine lactone (HHL);Diffusible signal molecule, conjugation factor (CF)

BACTERIUM GENES AUTOINDUCER MOLECULES

Quorum sensing systems - a sample

Bioluminescence Conjugal transferVibrio anguillarum, V. fischeri, V. harveyi Agrobacterium tumefaciens Ti plasmid

Plant-microbe interactions Swarming motilityRhizobium leguminosarum Serratia liquefaciensR. meliloti - rhizosphere-specific genes

Cell divisionVirulence determinants Escherichia coli Pseudomonas aeruginosa - elastase, pyocyanin etcRalstonia solanacearum - a phytopathogen Extracellular lipase synthesisAeromonas hydrophila - serine proteases Streptomyces lividans 66Vibrio cholerae - HA/proteaseBordetella pertussis - pertussis toxin Pigment productionErwinia carotovora - a phytopathogen Chromobacterium violaceum - violaceinStaphylococcus aureus

Antibiotic production Polysaccharide productionErwinia carotovora - carbapenem, -lactam antibiotic Klebsiella pneumoniaeRhodobacter sphaeroides Escherichia coli - colanic acid Pseudomonas aureofaciens- 3 phenazine antibioticsErwinia stewartii - Stewart's wilt of sweet corn Genetic competence Gram +ve lactic acid bacteria - antimicrobial peptide Streptococcus pneumoniaePseudomonas solanacearum - plant pathogen Bacillus subtilis

AHL molecules and their functions

• A cell-density dependent phenomenon.• A global mode of gene regulation, including virulence gene expression.• Alters bacterial behavior/phenotypes.• Involves intra-, inter-species and inter-kingdom communication.

Bacteria act as a community

Quorum Sensing

Classical Paradigm Current Perspectives

Bacteria exist as individuals. Bacteria can act in groups.

Consistent phenotypes. Cell-density dependent phenotypes.

One species, one disease. (Koch postulate)

Multiple species, one disease. (Cross-talk)

Non-interaction between host and pathogens

Close intimacy between host and pathogens.

Courtesy: KKF

Changes in Perspectives

rhlIrhlR

RhlIRhlR

+

TesterP.

aeruginosa

PassiveDiffusion

O

OH

NH

O

+

MonitorE. coli

PluxI-gfpluxR

LuxR

gfpluxR

GFPLuxR

GFP-based detection

(ii)

(i)(a)

(b) (c)(d)

Cell-density dependent expression with GFP monitor