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Multiple Small RNAs Act Additively Multiple Small RNAs Act Additively to Integrate Sensory Information and to Integrate Sensory Information and

Control Quorum Sensing Control Quorum Sensing inin Vibrio harveyiVibrio harveyi

Speaker: Yu-Chen HsuAdvisor: Dr. Li-Kwan ChangDate: 05.27.2008Place: The 6th Classroom

Kimberly C. Tu and Bonnie L. Bassler

GENES & DEVELOPMENT 21:221-233, 2007

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What will we learn?What will we learn?

Vibrio harveyi

Quorum Sensing(QS)

“Qrrs” (QS regulatory RNAs) additively control light production phenomenon called bioluminescence

http://www.molbio1.princeton.edu/labs/bassler/links.htm http://genome.wustl.edu/genome.cgi?GENOME=Vibrio%20harveyi

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Bioluminescent marine bacterium

Exist free swimming in seawater, adhered to surfaces of marine animals as biofilm, and in parthogenic associations with marine hosts

Use Quorum Sensing to control bioluminescence and other functions

V. harveyiV. harveyi

http://www.nyas.org/ebrief/miniEB.asp?ebriefID=544

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Quorum SensingQuorum SensingBacterial cell-cell communication that allows bacteria

to monitor their population density

Induce gene expressions that are effective when a large number of bacteria act together but insignificant when the population is small (Bioluminescence)

Via production, secretion, and detection of extracellular autoinducers (AIs): CAI-1, HAI-1, AI-2

Genes & Development 21:221-223

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Quorum Quorum SensingSensing (cont.)(cont.)

Secret AIs out to the environment

Low cell density (LCD): extracellular AIs can’t be detected

High cell density (HCD): AIs diffuse back into cells, detected by

sensors on the membrane

Low cell density (LCD): extracellular AIs can’t be detected

High cell density (HCD): AIs diffuse back into cells, detected by

sensors on the membrane

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lux

AI-sensor

CAI-1

HAI-1

AI-2

V. harveyi secrete AIs out of cell

PPPP

LuxULuxU

PP

LuxOLuxO

lux genelux gene

PPPP

PP

activatedactivatedLuxOLuxO PP

PPPP

σσ5454

5 Qrr sRNAs

luxR geneluxR gene

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When cell density is lowWhen cell density is low……HIGHHIGH

AIs flow AIs flow backbackinto cells !into cells !

LuxULuxU

LuxOLuxO

luxRluxR

luxlux

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When cell density is highWhen cell density is high……

PP

PPactivatedactivatedLuxOLuxO

phosphatase

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V. harveyi &V. cholerae have V. harveyi &V. cholerae have similar QS circuitssimilar QS circuits

Cell 118: 69–82

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How Qrrs interfere with How Qrrs interfere with luxRluxR not not fully resolvedfully resolved

In the closely related species V. cholerae, previous studies found out that …

1.V. cholerae has 4 Qrrs.2. These Qrrs interfere luxR redundantly.

Does Qrrs of V. harveyi act the same way?

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V. cholerae V. harveyi

4 Qrrs 5 Qrrs

Repress luxR redundantly

Repress luxR additively

NO!NO!NO!

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V. harveyiV. harveyiQuorumQuorum--Sensing SystemSensing System

repress luxR additively

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V. cholerae has 4 Qrrs and they act additively. How about V. harveyi?

How many Qrrs?

How do they act?

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5 Qrr sRNAs were identified5 Qrr sRNAs were identified4 Qrrs of V. cholerae were identified using a bioinformatics

approach. However, the genome of V. harveyi has not yet been fully sequenced.

The DNA sequence of qrr1 was available.qrr2-qrr4 were identified using genetic screen based on

differential fluorescence induction and fluorescence-activated cell sorting.

qrr5 was identified by scanning the partially sequenced genomic DNA sequence.

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Qrrs Complementary to Qrrs Complementary to luxRluxR mRNA 5mRNA 5’’--UTRUTR

Prove that these Qrrs can bind to luxR mRNA to degrade it.

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qrrqrr regulated by QS confirmedregulated by QS confirmed

HCD

LCD

HCD: Qrrs luxRLCD: Qrrs luxR

Qrr5 levels differ marginally

Qrr5 levels differ marginally

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V. cholerae has 4 Qrrs and they act additively. How about V. harveyi?

How many Qrrs?

How do they act?

5 Qrrs

How do qrr expression transit from LCD to HCD?

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Qrrs expression transitioning Qrrs expression transitioning from LCD to HCDfrom LCD to HCD

dilution

AI reaches threshold

Overnight growth

Overnight growth Light

output decreases

Light output

increasesqrr expressedbut light is

minimalat LCD

qrr expressedbut light is

minimalat LCD

qrr expressiondecreases

but light increasesat HCD

qrr expressiondecreases

but light increasesat HCD

Qrr4>Qrr2>Qrr3>Qrr1>Qrr5Qrrs can be expressed

differently?

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V. cholerae has 4 Qrrs and they act additively. How about V. harveyi?

How many Qrrs?

How do they act?

5 Qrrs

How do qrr expression transit from LCD to HCD?qrr and lux expression are inverse

Qrrs can be expressed differently?

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Qrrs can be expressed differently?Qrrs can be expressed differently?

Engineered quadruple deletion mutants that lacked all but one qrr gene.

QS-activated gene: lux (Bioluminescence)

QS-repressed gene: qrgA (qrgA-gfp reproter fusion, fluorescence measured)

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Qrr represses Qrr represses luxlux: : Qrr4>Qrr2>Qrr3>Qrr1>Qrr5Qrr4>Qrr2>Qrr3>Qrr1>Qrr5

Each Qrr is capable of repressing luxR: Qrr4 > Qrr2 > Qrr3 > Qrr1 > Qrr5

Parellels the strength of qrr expression

qrr4 is the most highly expressed, so Qrr4 can repress luxR the most.

qrr4+ mutant most closely resembles WT

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Qrrs repress Qrrs repress luxRluxR additively,additively, only only 4 are required4 are required

Single, double and triple qrr mutants were also assayed for light production. Single mutants showed the greatest luxrepression. When additional qrrs were inactivated, luxrepression decreases.

Qrrs repress luxR additively

qrr5 behaves no differently than the all-qrr-null mutant, indicating that qrr5 is not expressed and does not regulate QS in V. harveyi.

Only 4 of the 5 Qrrs are required

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Qrr activates Qrr activates qrgAqrgA: : Qrr4>Qrr2>Qrr1>Qrr3>Qrr5 Qrr4>Qrr2>Qrr1>Qrr3>Qrr5

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The regulatory heirarchy extended The regulatory heirarchy extended to the entire QS regulonto the entire QS regulon

Qrr4>Qrr2>Qrr1,3>Qrr5

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V. cholerae has 4 Qrrs and they act additively. How about V. harveyi?

How many Qrrs?

How do they act?

5 Qrrs

How do qrr expression transit from LCD to HCD?qrr and lux expression are inverse

Qrrs can be expressed differently?Qrrs repress luxR additively Only 4 of the 5 Qrrs are required

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Titration model of Qrrs and Titration model of Qrrs and luxRluxR mRNAmRNA

Qrr4 LuxluxR mRNA Lux

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V. cholerae has 4 Qrrs and they act additively. How about V. harveyi?

How many Qrrs?

How do they act?

5 Qrrs

How do qrr expression transit from LCD to HCD?

Qrrs can be expressed differently?

Qrrs repress luxR additively

Only 4 of the 5 Qrrs are requiredAIs alter the balance betwn Qrrs and luxR mRNA to control QS.

Qrr5 can not function at all?

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Qrr5 expressed in Qrr5 expressed in E. coliE. coli but but not not V. harveyiV. harveyi

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Qrr5 functions in Qrr5 functions in V. harveyiV. harveyi when overexpressedwhen overexpressed

LuxR protein levelsdecrease when Qrr5 is overexpressed !

LuxR protein levelsdecrease when Qrr5 is overexpressed !

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Qrr5 expressed in E. coli but not V. harveyi

Qrr5 functions in V. harveyi if overexpressed

A repressor function that is not in E. coli exists in V. harveyi and keeps qrr5 expression off.

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V. cholerae has 4 Qrrs and they act additively. How about V. harveyi?

How many Qrrs?

How do they act?

5 Qrrs

Qrrs repress luxR additively

Only 4 of the 5 Qrrs are requiredAIs alter the balance betwn Qrrs and luxR mRNA to control QS.

How rapidly the Qrrs respond to changes in AI concentration?

Qrr5 can not function at all?Yes, when overexpressed.

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How rapidly the Qrrs respond to How rapidly the Qrrs respond to changes in AI concentration?changes in AI concentration?

Add exogenousAIs to double AIsynthase mutant.

Add exogenousAIs to double AIsynthase mutant.

Half washed w/ fresh medium to eliminate AI conc. mimicking

HCD to LCD

Half washed w/ fresh medium to eliminate AI conc. mimicking

HCD to LCD

Half washed w/ cell-free spent

culture to maintain AI conc.

Half washed w/ cell-free spent

culture to maintain AI conc.

AIlux expression

AIlux expression

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LuxR protein levels show similar LuxR protein levels show similar pattern with bioluminescencepattern with bioluminescence

AILuxR expression

AILuxR expression

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Qrrs levels show a reverse patternQrrs levels show a reverse pattern

AIQrrs

AIQrrs

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sRNAs can response to AI conc. rapidly, mediating a rapid response by altering luxR levels, therefore downstream lux gene expression is regulated.

How rapidly the Qrrs respond to changes in AI concentration?

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V. cholerae has 4 Qrrs and they act additively. How about V. harveyi?

How many Qrrs?

How do they act?

5 Qrrs

Qrrs repress luxR additively

Only 4 of the 5 Qrrs are requiredHow rapidly the Qrrs respond to

changes in AI concentration?

Qrr5 can not function at all?

How do qrr expression transit from LCD to HCD?Qrrs act differently: 4 > 2 > 1, 3 > 5

Yes, when overexpressed.sRNAs can response to AI conc. rapidly, mediating a rapid response by altering luxR levels, therefore downstream lux gene expression is regulated.

V. harveyi QS model

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V. V. harveyiharveyi QS modelQS model

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Thank you for your Thank you for your attention!attention!

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Only Qrr1 was identified by analogous analysis as its DNA sequence was available.

Introduce a V. harveyi random promoter-gfp library into an E. coli strain carrying V. harveyi luxO D47E (mimics low cell density)

sort out high levels of GFPluxO-null (mimics high cell density)sort out low levels of GFPluxO D47E (mimics low cell density)sort out high levels of GFPqrr2, qrr3, qrr4 were found

Qrr5 was identified by scanning the partially sequenced V. harveyi genomic DNA sequence.

Identify 5 Qrrs

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AnsAns: : VibrioVibrio harveyiharveyi bioluminescence bioluminescence plays a role in plays a role in

stimulation of DNA repairstimulation of DNA repair

Why bioluminescent?Why bioluminescent?

AgataAgata CzyzCzyz11,, BorysBorys WrWróóbelbel22 andand GrzegorzGrzegorz WegrzynWegrzyn11

MicrobiologyMicrobiology (2000),(2000), 146146, 283, 283--288.288.

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Why bioluminescent? (cont.)Why bioluminescent? (cont.)

…luxA, luxB and luxD mutants (unable to emit light) of V. harveyi are significantly more sensitive to UV irradiation when cultivated in the dark after irradiation than when cultivated under a white fluorescent lamp…

… It is proposed that luminescent bacteria have an internal source of light which could be used in DNA repair by a photoreactivation process. Therefore, production of internal light ensuring effective DNA repair seems to be at least one of the biological functions of bacterial luminescence.

Microbiology (2000), 146, 283-288