Table 1.
Strains and plasmids used in this study.
Fig 1.
Structure of the hfq locus (A) and neighboring gene expression in Vibrio alginolyticus ZJ-T and derivative strains (B). A. The hfq gene is downstream of the miaA gene (locus ID = BAU10_13965) and upstream of 3 genes (hflX, K, C) likely to form an operon. A putative Sigma70 promoter identified upstream of hfq is indicated. 246 bps of the hfq ORF, from the 6th codon to the stop codon not included (marked by a star) were deleted as described in Materials and Methods, giving rise to the Δhfq-T strain. This mutation does not affect the potential promoter of the operon, neither the ribosome binding site of the downstream gene. A wild type hfq gene was reinserted at the Δhfq locus of the Δhfq-T strain by insertion of pNQ705-1-hfq by homologous recombination (see Materials and Methods for details) generating a duplication of the locus, corresponding to the fragment inserted in pNQ705-1, from primer hfq-A to primer hfq-D. The depicted situation corresponds to an insertion of the plasmid downstream of the hfq deletion. B. Relative expression of hfq, the upstream gene (miaA) and the downstream gene (hflX) in derivative strains compared to WT. Relative expression (normalized to the WT level for each gene) was determined by qPCR as described in Materials and Methods. Error bars correspond to standard deviations from three biological replicates. Statistically significant differences are indicated (***p < 0.001).
Fig 2.
hfq deletion affects colony morphology and extracellular polysaccharide production.
A. Colony morphology was observed with a BIO-RAD Gel DocTM XR+ imager. hfq deletion led to a colony morphology change from translucent and smooth to opaque and rugose. B. Amounts of extracellular polysaccharides of ZJ-T, Δhfq-T and hfq+-T were assayed in triplicates with alcian blue (top) and quantified by spectroscopy (bottom). Error bars correspond to standard deviations. Statistically significant differences are indicated (* p < 0.05)
Table 2.
Effect of hfq deletion on growth rate and total growth in rich medium and minimal medium M63 supplemented with various carbon sources.
Values correspond to the mean of three independent cultures. Significant statistical differences between strains and WT are indicated (*p < 0.05, **p < 0.01, ***p <0.001). Growth rates are expressed as generation times.
Table 3.
Effect of hfq deletion on growth rates and total growth in minimal medium M63 supplemented with various amino acids as both carbon and nitrogen sources.
Values correspond to the mean of three independent cultures. Significant statistical differences between strains and WT are indicated (*p < 0.05, **p < 0.01, ***p <0.001).
Fig 3.
Δhfq-T showed increased sensitivity to CuSO4 and H2O2.
Serial dilution of cultures of ZJ-T, Δhfq-T and hfq+-T were spotted on TSB agar plates (left panel), in presence of 6 mM CuSO4 (middle panel) or 0.0015% H2O2 (right panel). The picture is representative of the results of at least ten experiments.
Table 4.
Antibiotic resistance of ZJ-T, △hfq-T and hfq+-T.
Fig 4.
Hfq regulates outer membrane protein expression.
A. Relative expression of OMP genes in derivative strains compared to WT. Relative expression (normalized to the WT level for each gene) was determined by qPCR as described in Materials and Methods. Error bars correspond to standard deviations from three biological replicates. Statistically significant differences are indicated (*p < 0.05, **p < 0.01, ***p <0.001). B. SDS-PAGE analysis of the outer membrane fraction from ZJ-T, Δhfq-T and hfq+-T. M: Molecular size markers. Apparent sizes of markers are indicated on the left. The positions of two bands that increase in the hfq mutant are indicated by stars.