Transcript The Small RNA Chaperone Hfq and Multiple Small RNAs Control

The Small RNA Chaperone Hfq and
Multiple Small RNAs Control Quorum
Sensing in Vibrio harveyi and Vibrio
cholerae
Derrick H. Lenz,1,3 Kenny C. Mok,
Brendan N. Lilley,1,4 Rahul V. Kulkarni,
Ned S. Wingreen,1,2 and Bonnie
1Department
of Molecular Biology Princeton University Princeton,
New Jersey 08544 2NEC Laboratories America, Inc. 4 Independence
Way Princeton, New Jersey 08540
Introduction

Quorum Sensing
 Regulation
system that recognizes a signal
produced by the bacterium
 Signals
 Senses
are autoinducers
the concentration of a particular
species
 Can control either activators or repressors
Introduction

Autoinducers
 Can
promote intraspecies or interspecies
communication
 Controls
 Bioluminescence
 Siderophore
production
 Colony morphology
 Metalloprotease production
 Type III secretion
Quorum Sensing Circuits






Autoinducers Al-1 & Al-2
produced by synthases LuxM
& LuxS
LuxN & LuxPQ detection of
autoinducers
Systems converge to LuxU
Transmission of signal to
LuxO
LuxO requires σ54 to function
LuxR required for expression
Figure 1. (A) Two quorum-sensing
systems function in parallel to regulate
gene expression in V. harveyi.
Pentagons and triangles represent Al-1
and Al-2, respectively.
Quorum Sensing Circuit






Autoinducers CAl-1 & Al-2
produced by synthases CqsA
& LuxS
CqsS & LuxPQ detection of
autoinducers
Third system not yet
identified
Systems converge to LuxO
LuxO requires σ54 to function Figure 1. (B) Three quorum-sensing systems
function in parallel to regulate gene expression in V.
HapR required for
cholerae. The functions making up the third circuit
(denoted System 3) remain to be identified.
Diamonds and triangles represent Cal-1 and Al-2,
expression
respectively. In both circuits, phosphate flows in
the direction indicated by the arrows at low cell
density and in the opposite direction at high cell
density.
Circuit Operation

Low Cell Density
 Low
concentration of autoinducer
 Sensors act as kinases
 Transfer of phosphate via LuxU to LuxO
 LuxO-P is active and negatively regulates lux
Circuit Operation

High Cell Density
 High
Concentration of autoinducers
 Sensors act as phosphatases
 Phosphate flow is reversed
 Dephosphorylation and inactivation of LuxO
 LuxR/HapR bind to lux promoter and activate
transcription
Revealing Quorum Sensing
Repressor Hfq
LuxO D47E used to identify quorum sensing repressor
 Site of phosphorylation is altered
 LuxO D47E protein locked and mimics LuxO-P
 40,000 transposon insertion mutants generated, 85
were bright
 82 contained transposon insertions in either luxO or
rpoN, genes encoding σ54
 Three did not have mutations of these genes
 A V. harveyi genomic cosmid library was introduced
into one mutant w/o luxO or rpoN mutations (BNL211)

Revealing Quorum Sensing
Repressor Hfq
All cosmids w/ dark phenotype contained overlapping
regions of DNA
 Cosmid pBNL2014 mutated w/ Tn5lacZ to find lux
repression region
 Region cloned and sequenced
 Found to contain gene hfq

Figure 2A
(A) The hfq locus in V. harveyi, miaA and hflC were not fully
sequenced (unsequenced regions are denoted by light-colored
shading).
Requirement for Quorum Sensing
Repression
Question: Is hfq required for quorum
sensing repression?
 Strains/mutants

 Wild-type
(WT)
 luxO
 luxO
D47E
 hfq
 luxO
D47E, hfq
Figure 2. (B) Bioluminescence assays for
V. harveyi strains are: BB120 (WT,
squares), JAF78 (luxO::cmr, diamonds),
JAF548 (luxO D47E, open triangles),
BNL258 (hfq::Tn5lacZ, circles), and
BNL211 (luxO D47E, hfq::Mini-MulacZ,
closed triangles). Relative light units for
V. harveyi are defined as counts min-1 ml-1
x 103/cfu ml-1.
Figure 2. (C) Bioluminescence assays
for V. cholerae strains are: MM227
(WT, squares), MM349 (luxO,
diamonds), BH48 (luxO D47E, open
triangles), DL2078 (hfq, circles), and
DL2378 (luxO D47E, hfq, closed
triangles). Relative light units for V.
cholerae are defined as counts min-1
ml-1/OD600nm.
In (B) and (C), the dotted lines represent the limit of detection for light.
Regulation of Virulence Genes

Western blotting of TcpA
production was measured
to show that Hfq is not
restricted to nonnative
lux target in V. cholerae






Wild type: TcpA present
luxO: TcpA absent
luxO D47E: TcpA present in
high levels
hapR: TcpA present in high
levels
Hfq: low TcpA production
luxO D47E, hfq & hapR, hfq:
Hfq acts downstream of LuxO
and upstream of HapR
Figure 2. (D) V. cholerae strains analyzed for TcpA production by Western blot
are: C6706str2 (WT), MM307 (luxO), BH38 (luxO D47E), MM194 (hapR),
DL2066(hfq), DL2146 (luxO D47E, hfq), and DL2607 (hapR, hfq)
Hfq IS Required for Quorum Sensing
Repression

Predictions
 Quorum
sensing repression occurs
posttranscriptionally
 There must be one or more sRNA involved
 At low cell density, the LuxO-P- σ54 complex
activates the transcription of the gene(s)
encoding the sRNA(s)
Prediction 1. Quorum sensing repression
occurs posttranscriptionally


Northern blots used to determine the effect of hfq
mutations on luxR and hapR mRNA stability.
Rifampicin added to terminate transcription
Figure 3. Hfq Regulates the Expression of luxR and hapR Posttranscriptionally
(A) Non-steady-state Northern blots were used to analyze luxR/hapR transcript
stability in the following: V. harveyi JAF548 (luxO D47E) and BNL211 (luxO
D47E, hfq::Mini-MulacZ); and V. cholerae BH38 (luxO D47E) and DL2146 (luxO
D47E, Δhfq)
Prediction 1. Quorum sensing repression
occurs posttranscriptionally


Western blots show that the increased stability of the
luxR and hapR mRNAs in the hfq mutants lead to
increased levels of the LuxR and HapR proteins
Low cell density, Hfq destabilizes the luxR and hapR
mRNA
(B) Western blots on lysates of V. harveyi BB120 (WT), JAF548 (luxO D47E), BNL258
(hfq::Tn5lacZ), BNL211 (luxO D47E, hfq::Mini-MulacZ), and V. cholerae C6706str2
(WT), BH38 (luxO D47E), DL2066 (Δhfq), DL2146 (luxO D47E, Δhfq) measured LuxR
and HapR protein, respectively.
LuxO-P Regulation of hapR is
Posttranscriptional and Requires Hfq
Constructed chromosomal hapR-lacZ
transcriptional, translational, and promoter
fusions
 Measured their activities in V. cholerae
strains

 Wild-type
 luxO
D47E
 hfq
 luxO
D47E
• The transcriptional and
translational are repressed
in the luxO D47E strain, and
repression requires Hfq.
• LuxO D47E does not
repress the hapR-lacZ
promoter fusion.
• Results suggest that LuxOP regulation of hapR is
posttranscriptional
Identification of sRNAs using
Bioinformatics

Parameters
 Upstream
region of the sRNA locus must
contain a σ54 binding site
 Assumed sRNAs have Rho-independent
terminators
 Restricted search to regions between
annotated genes
 sRNAs must be conserved in V. cholerae, V.
parahaemolyticus, and V. vulnificus
Identification of sRNAs using
Bioinformatics

Two techniques used to find potential σ54 binding
sites

PATSER

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
Weight matrix constructed w/ compiled set of approx. 180
σ54 binding sites from multiple bacterial species
Includes all binding sites upstream of genes in V. cholerae
known to be regulated by σ54
Upstream regions of known V. cholerae σ54 genes
were extracted


Using CONSENSUS searched for 16 bp motif
Aligned set of binding sites used to construct σ54 weight
matrix
Identification of sRNAs using
Bioinformatics
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Four intergenic regions
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Conservation across the specified vibrio genomes
Contained Rho-independent terminators
Figure 5
(A) Multiple sequence alignment of the qrr genes encoding the sRNAs identified in V.
cholerae, V. parahaemolyticus, V. vulnificus, and V. harveyi.
Identification of sRNAs using
Bioinformatics

V. parahaemolyticus & V. vulnificus
 Five
intergenic regions
 Conservation
across the specified vibrio genomes
 Contained Rho-independent terminators

V. harveyi is most closely related to V.
parahaemolyticus
 Assumed
that V. harveyi has five sRNAs
Identification of sRNAs using
Bioinformatics

RNAFOLD

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Prediction of secondary structures
Qrr 2, Qrr3, & Qrr4 very similar
Loop composition variable, stem conserved
(B) Lowest-energy secondary-structural predictions for the Qrr sRNAs identified
in V. cholerae. Bold typeface indicates regions conserved across all sRNAs in V.
cholerae, V. parahaemolyticus, and V. vulnificus.
Identification of sRNAs using
Bioinformatics

Using LALIGN


Aligned complement of hapR untranslated upstream
region with Qrr 1-4
Aligned complement of luxR untranslated upstream
region with Qrr 1
(C) Alignment of the complement of the hapR UTR with a portion of the Qrr sRNAs
identified in V. cholerae. (D) Alignment of the complement of the luxR UTR with a
portion of the Qrr1 identified in V. harveyi.
LuxO-P- σ54 Controls the Expression
of the sRNA loci

Question: Are sRNAs
regulated by LuxO-Pσ54?





Northern blot used to
quantify transcript
levels
hapR+: qrr4 is
regulated
hapR-: qrr2 & qrr3 are
regulated
Unable to detect qrr1
Detection of Qrr4
from V. harveyi

Expression induced by
LuxO D47E
Figure 6. (A) V. cholerae C6706str2 (WT), MM307
(ΔluxO), BH38 (luxO D47E), BH76 (ΔrpoN) was probed fo
sRNAs Qrr1, Qrr2, Qrr3, and Qrr4, and V. cholerae rpsL is
shown as the loading control. (B) RNA isolated from V.
harveyi qrr1 and for sRNA Qrr4 with a probe made agains
V. cholerae qrr4. V. harveyi rspL is shown as the loading
control.
LuxO-P- σ54 Controls the Expression
of the sRNA loci

qrr1 transcriptional
reporter


Fusion of upstream
region of V. cholerae
qrr1 to luciferase
operon
Results indicate qrr1
is regulated by LuxOP- σ54
(C) Single time point RLU for V. cholerae
strains DL3212 (luxO) and DL3213 (luxO
D47E) containing the qrr1-lux
transcriptional fusion in trans.
sRNAs Involved in Quorum Sensing
Repression

Individual roles of
sRNAs


Presence of any one
sRNA expresses
density-dependent
bioluminescence
similar to WT
Deletion of all sRNAs
eliminates quorum
sensing repression
Figure 7. (A) Bioluminescence assays
were performed on V. cholerae.
sRNAs Involved in Quorum Sensing
Repression
Overexpression of
one sRNA results in
quorum sensing
repression
 Epistasis test


The four sRNAs in V.
cholerae are epistatic
to LuxO-P in regulation
of tcpA
(B) Single
time point
RLU for V.
cholerae
strains.
Western
blots probed
for hapR and
TcpA from V.
cholerae.
Accumulation Rate


If rate of synthesis
of sRNA exceeds
that of its target,
sRNA can
accumulate in the
cell
If rate of synthesis
of a target exceeds
that of its regulatory
sRNA, the message
can accumulate in
the cell
Conclusion

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Hfq is an RNA chaperone for a large number of sRNAs
Presence of multiple sRNAs is important in fine tuning
the transition between low to high cell density by
allowing the influence of additional regulatory inputs.
Simultaneous inactivation of all four sRNAs is necessary
to eliminate Hfq-mediated quorum sensing repression
Overexpression of only one sRNA is sufficient for
repression
Simultaneous presence of multiple autoinducers is
required to reverse the direction of phosphoflow through
the system and initiate the transition between low to
high cell density.
References
Lenz, D. H., K. C. Mok, B. N. Lilley, R. V.
Kulkarni, N. S. Wingreen, and B. L. Bassler.
2004. The small RNA chaperone hfq and
multiple small RNAs control quorum sensing in V.
harveyi and V. cholerae. Cell. 118:69-82.
 Salyers, A. A., and D. D. Whitt. 2001.

Microbiology: Diversity, Disease, and the
Environment, pp. 107. Fitzgerald Science Press,
Inc., Bethesda.