Regulatory Small RNA Qrr2 Is Expressed Independently of Sigma Factor-54 and Can Function as the Sole Qrr Small RNA To Control Quorum Sensing in Vibrio parahaemolyticus

Abstract

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.

Transcriptomic Analysis of Vibrio parahaemolyticus Underlying the Wrinkly and Smooth Phenotypes

“…In V. parahaemolyticus , only a few sRNAs have been experimentally studied. For example, the Qrr sRNAs are trans -acting sRNAs that affect quorum sensing, CPS production, motility, and metabolism ( 20 , 21 , 74 ). The sRNA Spot42 binds to the ribosomal binding site and initiation codon of VP1682 mRNA, encoding a chaperone protein of T3SS1 to posttranscriptionally repress the translation of this mRNA ( 75 ).…”

A class IV adenylate cyclase CyaB is required for capsule polysaccharide production and biofilm formation inVibrio parahaemolyticus

“…Biofilm assays were performed as previously described (39, 40, 57). Briefly, overnight cultures were used to inoculate strip wells in a 1:40 dilution in LBS.…”

“…Capsule polysaccharide (CPS) production is a key component of biofilm and is positively controlled by the quorum sensing master regulator OpaR in V. parahaemolyticus (34,36,(38)(39)(40)44). To determine whether CRP plays a role in CPS formation, we examined the Δcrp and cya mutants on HI medium supplemented with congo red and CaCl2, incubated for 48 hrs at 30˚C.…”

“…Consumption of raw or undercooked shellfish infected with V. parahaemolyticus causes gastroenteritis in human and is the leading cause of bacterial seafood borne gastroenteritis worldwide (32,33). In this species, the QS master regulator OpaR (LuxR homolog) positively regulates biofilm by directly, positively regulating cpsQ and the cspA-K loci required for CPS production, a major component of biofilm (34)(35)(36)(37)(38)(39)(40). The role of cAMP-CRP in V. parahaemolyticus physiology has not been previously examined.…”

The Regulatory Functions of σ54 Factor in Phytopathogenic Bacteria

“…In P. putida , it also affects the utilization of nitrate, urea, and uncharged amino acids as nitrogen sources, as well as of lysine, C 4 -dicarboxylates, and alpha-ketoglutarate as carbon sources [ 15 , 16 ]. In addition, σ 54 factor controls bacterial growth [ 17 , 18 , 19 ], nitrogen and carbohydrate assimilation [ 20 , 21 ], swimming and twitching motility [ 22 ], biofilm formation [ 23 , 24 ], exopolysaccharide (EPS) production [ 23 ], quorum sensing [ 25 , 26 ], type VI secretion systems (T6SS) [ 27 , 28 ], virulence [ 29 , 30 ], environmental adaptation [ 31 , 32 ], and antibiotic resistance [ 33 , 34 ] in other bacteria.…”