Presentation on a scientific publication describing a method for modulating the symbiotic colonization in the Hawaiian bobtail squid.

1. H-NOX-MEDIATED NITRIC OXIDE
SENSING MODULATES SYMBIOTIC
COLONIZATION BY VIBRIO
FISCHERI
Yanling Wang et. Al.
Presented by Lucas Man

2. Vibrio fischeri and Euprymna scolopes
• Hawaiian bobtail squid and its bioluminescent
symbiont
• V. fischeri colonizes the light organ
• Bioluminescence provides defense through
counterillumination
Pic source: Discover Magazine; V. fischeri genome project

3. Vibrio fischeri and Euprymna scolopes
• Colonization of the light organ involves:
• Secretion of mucus into seawater by E. scolopes
• V. fischeri migrating and aggregating in mucus before
traveling into pores and through ducts to reach crypts in
light organ
• Mucus shed by E. scolopes contains high levels
of nitric oxide
Pic source: Cell Microbiol

4. Bonus paper: “NO means „yes‟ in the squid-vibrio symbiosis:
nitric oxide (NO) during the initial stages of a
beneficial association” – SK. Davidson et. al. Cell Microbiol.
• Found active NO
synthase and NO-filled
vesicles in secreted
mucus
• Also discovered that NO
synthase activity drops
once colonization of the
light organ is complete Pic source: Cell Microbiol

5. Nitric Oxide (NO)
• In high concentrations, it can serve as a
antimicrobial
• It can also serve as a signaling molecule
• Key biological messenger in vertebrates
• H-NOX: heme NO/oxygen-binding protein
• High sequence identity with soluble guanylate cyclase
(sGC), a eukaryote NO receptor
• Found in V. fischeri

6. Hypothesis
• NO is a symbiotic signal
• H-NOX can sense host-derived NO
• H-NOX can regulate V. fischeri genes
H-NOX protein
Pic source: PDB

7. Experiment
• Step 1: Determine if H-NOXvf binds NO with high affinity
• Method: Spectroscopic characterization of H-NOXvf complexes with
NO, CO and O2
• Results:
• Stable complexes with NO and CO
but not O2
• UV peak positions closely match
those of sGC
Fig. 1

8. Experiment
• Step 2: Determine genes that might be regulated by NO in
the presence of H-NOXvf
• Method:
• Compare transcriptional profile of wild-type cells to hnoX-insertion
mutant cells (YLW1 cells)
• Cells were cultured in 4 experimental groups: wild-type, wild in the
presence of NO (released from DEA-NONOate), mutant, mutant in the
presence of NO; 3 replicates in each experimental group
• Extract total RNA from cultures
Wild-type Wild-type Mutant Mutant
+NO +NO

9. Experiment
Fig. 2
• Results:
• Bacterial defensive responses to
NO not dependent on H-NOX
• In wild-type cells, NO down-
regulated hemin-use genes, in
mutant cells these genes were
unaffected by NO exposure
• Many of the genes that were
down-regulated contained a motif
similar to the FUR regulon

10. Experiment
• Step 3: Test growth rates of wild-type and mutant
cells with hemin as primary iron source
• Hypothesis: mutant cells will grow more rapidly than wild-type
cells in a medium in which hemin is the primary iron source
when exposed to NO
• Method:
• Cultured wild-type and mutant cells in medium with only hemin
as iron source
• Treated one-half of each culture with NO twice
• Also cultured mutant and wild-type cells either carrying a
plasmid vector or the plasmid vector containing the H-NOX gene

11. Experiment
• Results:
• Mutant cells grew faster on
hemin when NO is present
• When H-NOX gene is added
with the plasmid vector to
mutant cells, growth with NO
matches wild-type cells
• Supports hypothesis that H-
NOX mediates hemin-use in
presence of NO
Fig. 3

12. Experiment
• Step 4: compare colonization competence of wild-
type and mutant cells
• Method: grew juvenile squids in seawater either
containing mutant or wild-type V. fischeri
• Also grew juvenile squids in a mixed culture containing
1:1 ratio of mutant and wild-type cells to compare
competition
• Used onset of luminescence as marker for colonization

13. Experiment Fig. 4
• Results:
• H-NOX mutant actually more
proficient in initiation of
colonization
• H-NOX mutant had 10-fold
higher colonization efficiency
(density of cells required to
colonize ½ of a juvenile squid
cohort)

14. Experiment
• Results:
• Mutant cells outcompeted the wildtype by 16-fold after
24 hours, advantage dropped to 3-fold by 48 hours
• Wild-type cells had an advantage over mutant in the
Fig. 4
presence of added iron
• NO synthase inhibitor
reduces mutant advantage
• Vector insertion of HNOX
gene removes mutant
advantage

15. Conclusions
• H-NOX senses NO, represses V. fisheri’s ability to use
hemin, suppresses rapid growth
• Why? What could be the advantage?
• Possibility: high intracellular concentration of iron can generate
toxic levels of hydroxyl radicals
• Host already generates a high level of oxidants, hemin accumulation
could lead to toxic levels
• H-NOXvf-NO could prime V. fischeri for oxidative stress and protect the
symbiont until oxidants have been reduced
• Oxidative stress could cause harmful mutations
• Could explain why competitive advantage of the mutant decreased after
the first 24 hours (Figure 4)

16. Conclusions
• The take-home message:
• H-NOX in V. fischeri is an NO sensor that influences the
expression of genes associated with hemin acquisition
(and possibly others as well)
• Pathway is still unknown, it is possible that H-NOXvf-NO targets
the FUR regulon
• H-NOX and NO sensing plays a role in symbiotic
colonization by V. fischeri
• Host-symbiont biomolecular cross-talk

17. Questions?