Bacterial cells alter gene expression in response to changes in population density in a process called quorum sensing (QS). In
Vibrio harveyi
, LuxO, a low cell density activator of sigma factor-54 (RpoN), is required for transcription of five non-coding regulatory sRNAs, Qrr1-Qrr5, which each repress translation of the master QS regulator LuxR.
Vibrio parahaemolyticus
, the leading cause of bacterial seafood-borne gastroenteritis, also contains five Qrr sRNAs that control OpaR (the LuxR homolog), controlling capsule polysaccharide (CPS), motility, and metabolism. We show that in a Δ
luxO
deletion mutant,
opaR
was de-repressed and CPS and biofilm were produced. However, in a Δ
rpoN
mutant,
opaR
was repressed, no CPS was produced, and less biofilm production was observed compared to wild type. To determine why
opaR
was repressed, expression analysis in Δ
luxO
showed all five
qrr
genes were repressed, while in Δ
rpoN
the
qrr2
gene was significantly de-repressed. Reporter assays and mutant analysis showed Qrr2 sRNA can act alone to control OpaR. Bioinformatics analysis identified a sigma-70 (RpoD) -35 -10 promoter overlapping the canonical sigma-54 (RpoN) -24 -12 promoter in the
qrr2
regulatory region. The
qrr2
sigma-70 promoter element was also present in additional
Vibrio
species indicating it is widespread. Mutagenesis of the sigma-70 -10 promoter site in the Δ
rpoN
mutant background, resulted in repression of
qrr2.
Analysis of
qrr
quadruple deletion mutants, in which only a single
qrr
gene is present, showed that only Qrr2 sRNA can act independently to regulate
opaR
. Mutant and expression data also demonstrated that RpoN and the global regulator, Fis, act additively to repress
qrr2
. Our data has uncovered a new mechanism of
qrr
expression and shows that Qrr2 sRNA is sufficient for OpaR regulation.
Importance
The quorum sensing non-coding sRNAs are present in all
Vibrio
species but vary in number and regulatory roles among species. In the Harveyi clade, all species contain five
qrr
genes, and in
V. harveyi
these are transcribed by sigma-54 and are additive in function. In the Cholerae clade, four
qrr
genes are present, and in
V. cholerae
the
qrr
genes are redundant in function. In
V. parahaemolyticus
,
qrr2
is controlled by two overlapping promoters. In an
rpoN
mutant,
qrr2
is transcribed from a sigma-70 promoter that is present in all
V. parahaemolyticus
strains and in other species of the Harveyi clade suggesting a conserved mechanism of regulation. Qrr2 sRNA can function as the sole Qrr sRNA to control OpaR.