Yip et al. 2005.
A novel, conserved cluster of genes
promotes symbiotic colonization
and _54-dependent biofilm
formation by Vibrio fischeri
Molecular Microbiology 57 (5), 1485-1498.
Vibrio fischeri – Exclusive symbiont in
the light organ of the squid
•
Gram-, motile heterotroph, ~0.6 _m in diameter.
•
Produces bioluminescence
Tetradecanal + FMNH2 + O2
•
Tetradecanoic acid + FMN + H2O + light(490nm)
Discovery of quorum sensing
– acyl-homoserine lactone
http://ergo.integratedgenomics.com/Geno
mes/VFI/vibrio_fischeri.html
http://microbiology.unh.edu/faculty/Whistler/
Hawaiian bobtail squid
- Euprymna scolopes
• 3-4 cm cephalopod
• 10 legs
• Lives in a shallow water
Habitats around Hawaii
• Nocturnal animal: burrowing
into the sand on daytime,
and cruise around at night.
• Has a light organ in the
center of its mantle cavity
Symbiont growth
The host behavior and the associated
symbiont density in the light organ
Time (Day/night cycle)
Nyholm, S.V., and McFall-Ngai, M. (2004). Nat Rev Microbiol 2: 632–642.
How do the cells enter into the light
organ?
Nyholm, Spencer V. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 10231-10235
The internal components of the squid
light organ at hatching
Nyholm, S.V., and McFall-Ngai, M. (2004). Nat Rev Microbiol 2: 632–642.
The progression of light-organ
colonization
Nyholm, S.V., and McFall-Ngai, M. (2004). Nat Rev Microbiol 2: 632–642.
Gradual, symbiont-induced regression of
the ciliated epithelium of the juvenile
light organ
0h
24h
48h
96-120h
Nyholm, S.V., and McFall-Ngai, M. (2004). Nat Rev Microbiol 2: 632–642.
Early events and signals during
onset of symbiosis
0
Mucus shedding
1
Peptidoglycan signal
Onset of aggregation
2
• Nitric oxide attenuation
• Duct constriction
Apoptosis
Increase of microvillar
density, mucus secretion
and cell swelling
Epithelium regression
4-5
6
~9-10
12
48
96
hr
• V. fischeri dominance
• Migration into ducts and crypts
LPS signal
Induction of bioluminescence
• Colonization complete
• Growth ceases
LPS and peptidoglycan signals
Primary bacterial factors for
symbiosis
• OmpU (Outer membrane protein) – Initiation
• Pgm (Metabolic enzyme) – Growth to high density
• GlnD (Nitrogen and siderophore reg.) – Persistence
• AinS, LuxS and LuxO – Quorum sensing
• RscS, GacA and FlrA – Two-component regulators
• _54 – Controls flagella and bioluminescence(works
with FlrA and LuxO respectively)
_ 54
• Encoded by rpoN
• Consensus sequence for binding is
mrNrYTGGCACG-N4-TTGCWNNw
• Recognition positions: -24/-12
• _54-dependent expression absolutely
requires an activator
• rpoN mutant fail to initiate symbiotic
colonization, due to loss of motility or other
_54-dependent phenotypes?
Questions to be answered?
• What’s the new genes required for
symbiotic colonization?
• How are the new genes regulated for
transcription?
• What’s function of these new genes?
• Are there homologous genes in other
related species?
Questions to be answered?
• What’s the new genes required for
symbiotic colonization?
• How are the new genes regulated for
transcription?
• What’s function of these new genes?
• Are there homologous genes in other
related species?
What’s the new genes required for
symbiotic colonization?
• Experiments
– Construction of a Tn-mutagenized library
– Isolation of symbiosis-defective mutants
– Investigation of the isolated mutants with
known symbiosis factors
– Southern blot analysis, cloning,
sequencing and BLAST analysis on the
target genes.
Isolation of symbiosis-defective
mutants
Four mutants were defective in symbiotic bioluminescence due to either a delay in or an absence of
colonization.
Investigation of the isolated mutants
with known symbiosis factors
• No apparent difference in motility among
mutants and the wild type strain
• All four mutants contained OmpU similar to
the wild type strain
• The isolated mutants were not defective for
known symbiotic factors.
Southern blot analysis, cloning,
sequencing and BLAST analysis
• All mutants carried a single Tn insertion
• All insertions were mapped within a cluster
of 21 genes.
• All genes are in the same orientation
• Most genes were predicted to be involved in
synthesis or export of LPS, capsule or PS.
Additional genes were tested for
symbiotic colonization in the cluster
• Each of the 5 genes
were also required for
symbiosis
• The cluster functions in
symbiotic colonization
I J K L M N O P QR
A
VF 103
A 8
VF 103
A1 9
04
0
ABC DE F G H
VF
VF
A1
01
9
• The syp does not extend
to VFA1038 or beyond
The cluster was termed syp for
symbiosis polysaccharide
Questions to be answered?
• What’s the new genes required for
symbiotic colonization?
• How are the new genes regulated for
transcription?
• What’s function of these new genes?
• Are there homologous genes in other
related species?
How is the syp regulated for
transcription?
• Experiments
– Predicted target sequences were searched by
bioinformatics tools
• Sequence analysis revealed a putative _54dependent regulator, SypG in the syp.
• Use of PromScan program to search for _54dependent promoter sequences
• Use of MEME (Multiple EM for Motif Elicitation)
system to search for a potential enhancer
consensus sequence for an activator.
How is the syp regulated for
transcription?
• Experiments
– Transcription control experiments were
performed by using lacZ reporter fusions and
primer extension.
SypG - a putative _54-dependent
regulator in the syp
Putative _54 binding sites and
potential enhancer binding sequences
Putative _54 binding sites
sypA
sypI
sypM
sypP
TTGGCACNNNNNNTGCN
Putative enhancer binding
sequences
sypA
sypI
sypM
sypP
AAATCNATTCTCAATNTGAGAA
Primer extension-based mapping
sypA
sypI
sypM
sypP
Transcription of the syp genes
• Use of two reporter strains
of Tn mutants, sypD and
sypN, each was fused with
a promoterless lacZ
oriented correctly for sypdependent transcription.
• Two groups, vector
control or the sypGexpressing multicopy
plasmid.
• Construction of double
mutants (in rpoN and
either sypD or sypN)
Transcription of the syp genes
• No syp expression in
laboratory conditions
• Multicopy expression of
sypG induced
transcription of the two
lacZ reporters by 37 to 70
fold.
• Double mutants in either
rpoN and sypD or sypN
revealed that the induction
of syp transcription by
sypG depends on _54.
Questions to be answered?
• What’s the new genes required for
symbiotic colonization?
• How are the new genes regulated for
transcription?
• What’s function of these new genes?
• Are there homologous genes in other
related species?
What’s function of the syp cluster?
• No study in vivo
• Biofilm formation study was performed
in laboratory conditions
Biofilm formation by syp mutants
and their parent
Questions to be answered?
• What’s the new genes required for
symbiotic colonization?
• How are the new genes regulated for
transcription?
• What’s function of these new genes?
• Are there homologous genes in other
related species?
Are there homologous genes in
other related species?
• Methods
– BLAST analysis using the TIGR database.
• Results
I J K L M N O P QR
Syp cluster
A
VF 103
A 8
VF 10
A1 39
04
0
ABC DE F G H
VF
VF
A1
01
9
– Homologue of almost all of the syp genes were
present and similarly clustered in V.
parahaemolyticus and V. vulnificus.
– Genes of VFA1038-1040 are conserved, but not
closed to syp in other organisms.
Significance of the study
• A new, conserved cluster (syp) was
found to be required for the SquidVibrio symbiosis.
• Transcription of the syp cluster is
controlled by _54-containing RNAP.
• Homologous clusters of the syp genes
may play a role in promoting pathogenhost interactions.