PMC

Expression of vanT during growth of the wild type and the vanU mutant. The transcriptional (a) and translational (b) vanT : : gfp reporter gene fusions were expressed from the chromosome and were measured during growth of the wild type and the vanU mutant. Growth (dotted lines) is indicated as OD₆₀₀. GFP-ASV expression (solid lines) was measured as fluorescence and is presented as relative fluorescence units (RFU), which equals fluorescence units per 10⁸ cells. Error bars indicate sd.

VanU negatively regulates the four qrr genes. The transcriptional gene fusions qrr1 : : gfp, qrr2 : : gfp, qrr3 : : gfp and qrr4 : : gfp (indicated above each graph) were expressed from the chromosome and were measured during growth of the wild type and the vanU mutant. Growth (dotted lines) is indicated as OD₆₀₀. GFP-ASV expression (solid lines) was measured as fluorescence and is presented as relative fluorescence units (RFU), which equals fluorescence units per 10⁸ cells. Error bars indicate sd.

Expression profiles of the qrr genes during growth in the wild type and the vanO mutant. For the wild type, expression profiles of the transcriptional gene fusions qrr1 : : gfp, qrr3 : : gfp and qrr4 : : gfp are presented in (a), whereas the transcriptional gene fusion qrr2 : : gfp was expressed less than the other qrr genes and is presented separately (b). For the vanO mutant (c), expression profiles of all qrr : : gfp transcriptional gene fusions are shown. For all strains, the gene fusions were expressed from the chromosome. Growth (dotted lines) is indicated as OD₆₀₀. At each time point, GFP-ASV expression (solid lines) was measured as fluorescence and presented as relative fluorescence units (RFU; ±sd, which equals fluorescence units per 10⁸ cells.

V. anguillarum quorum-sensing phosphorelay model. Solid lines with arrows and bars indicate activation and repression of gene expression, respectively. Solid lines with double arrowheads indicate the transfer of phosphoryl groups from one protein to another. Details of the model are given in the Introduction and Discussion. In this study, a model is presented suggesting that the phosphotransferase VanU phosphorylates two response regulators: one response regulator, VanO, activates Qrr sRNA expression, repressing VanT expression, and a second, predicted response regulator (RR-2) is hypothesized to repress Qrr sRNA expression, activating VanT expression. The RR-2 is predicted to be phosphorylated via a histidine kinase (HK-2) of an independent two-component pathway. Abbreviations: OM, outer membrane; IM, inner membrane.

DNA alignment of the four V. anguillarum qrr genes and their promoters. The qrr1, qrr2, qrr3 and qrr4 genes and their 5′- and 3′-flanking sequences were aligned using clustal w (). Regions of the gene sequences with 100 % identity are highlighted in grey. In each promoter region, consensus σ⁵⁴ promoter sequences were found and designated −24 and −12. The consensus σ⁵⁴ promoter sequence (YTGGCACG-N4-TTGCWNN, ) is given below the aligned sites. Putative transcriptional start sites are designated +1. Putative LuxO binding sites were found upstream of the promoters and are indicated with a box. The consensus sequence for the LuxO binding site (TTGCA-N₃-TGCAA, ) is shown below the aligned sites. Possible stem–loop transcriptional terminators are indicated with solid lines.

Qrr sRNAs modulate expression of genes regulated by VanT. (a) Western blot analyses of the extracellular proteins EmpA and Hcp. Bacterial cultures were grown for 16 h and supernatant proteins were precipitated with 5 % TCA. Supernatant proteins from equal cell numbers were separated by using 12.5 % SDS-PAGE. Western blot analysis was done using antisera raised against EmpA and Hcp. An OmpU antiserum was used as a loading/transfer control. V. anguillarum produces outer membrane vesicles and OmpU is localized within these vesicles and precipitates with the extracellular proteins (data not shown). (b) Total protease activity of extracellular proteins was determined by measuring azocasein degradation. For both (a) and (b), the strains used are indicated by their mutation. For negative controls, the empA and the hcp mutant were also included. Error bars in (b) indicate sd.

Expression of vanT mRNA and VanT protein in the wild type and various mutant strains. (a) Northern analysis of vanT mRNA. Total RNA was isolated from cultures grown to OD₆₀₀ 1.0. To detect vanT mRNA, 5 µg mRNA-containing (>200 bases) RNA fraction was separated in a denaturing agarose gel and hybridized to a DIG-labelled fragment amplified by PCR from the vanT gene. A probe to detect 16S rRNA transcripts was used as a loading/transfer control. The transcript detected on each filter is given to the left of each panel. (b) Real-time qRT-PCR of vanT mRNA. Total RNA was isolated from cultures grown to OD₆₀₀ 1.0. Real-time qRT-PCR was done as described in Methods. The vanT mRNA was normalized to 16S rRNA transcripts. P-values less than 0.001 are considered significant and are indicated by an asterisk. Error bars indicate sd. (c) Western blot analysis of VanT. Culture samples from the wild type and mutant strains were taken at OD₆₀₀ 1.0. Proteins from equal cell numbers were separated by using 12.5 % SDS-PAGE and Western blot analysis was done using a VanT antiserum. An OmpU antiserum was used as a loading/transfer control. After detection of VanT, the blot was stripped and OmpU antiserum was applied. For (a–c), the strains used are indicated by their mutation. For (a) and (c), the vanT mutant was included as a negative control.

(a, b) Expression of vanT during growth of the wild type, and the Δqrr1, Δqrr2, Δqrr3, Δqrr4 and Δqrr1–4 mutants. The transcriptional (a) and translational (b) vanT : : gfp reporter gene fusions were expressed from the chromosome and were measured during growth of each strain. Growth (dotted lines) is indicated as OD₆₀₀. At each time point, GFP-ASV expression (solid lines) was measured as fluorescence and presented as relative fluorescence units (RFU), which equals fluorescence units per 10⁸ cells. (c, d) Stability of vanT mRNA in the wild-type and the Δqrr1–4, Δhfq and ΔvanU mutant strains. All strains were grown to OD₆₀₀ 0.2 (c) and 1.0 (d). At these time points, transcription was stopped by the addition of rifampicin (200 µg ml⁻¹). Culture samples were taken at 0, 2, 5 and 10 min. The zero time point was withdrawn before rifampicin addition. Total RNA was isolated from each sample and qRT-PCR was done to determine the amount of vanT mRNA remaining at each time point. The mRNA levels at the zero time point for each sample were set at 1.0. The vanT mRNA was normalized to the 16S rRNA and each time point was normalized to the respective zero time point. The approximate half-life of the vanT mRNA in each strain is given as minutes on the right of each curve.

VanO and σ⁵⁴ positively regulate expression of the four Qrr sRNAs. (a) The transcriptional gene fusions qrr1 : : gfp, qrr2 : : gfp, qrr3 : : gfp and qrr4 : : gfp were localized to the chromosome of each strain. Expression of GFP-ASV was measured as fluorescence at early (OD₆₀₀ 0.2, black bars) and late exponential growth (OD₆₀₀ 1.0, white bars) and is presented as relative fluorescence units (RFU; ±sd), which equals fluorescence units per 10⁸ cells. The expression data for each qrr : : gfp gene fusion are given under a labelled solid line and the qrr2 : : gfp gene fusion data are presented with a different scale from that of the other three gene fusions. (b) Northern analysis of the four Qrr sRNAs. The wild type and various mutant strains were grown to OD₆₀₀ 1.0 and RNA was purified from each strain. For each qrr mRNA, 3 µg microRNA-containing (<200 bases) RNA was separated in a denaturing agarose gel. The mRNA was hybridized to a DIG-labelled PCR fragment amplified from a qrr gene. The transcript detected on each filter is given above each panel. A probe to detect 5S rRNA transcripts was used as a loading and transfer control. Strains used in both images are designated by their mutation at the bottom of the image.

Analyses of the induction of qrr1 expression by AHL signal molecules in the wild type and the vanO and vanU mutants. Fluorescence microscopy was used to determine the effect of C6-HSL on expression of the qrr1 gene in the various strains at a cell density of 100 cells ml⁻¹. Overnight cultures carrying the transcriptional gene fusion qrr1 : : gfp on the chromosome were washed and diluted in TSB-1 % to a cell density of 100 bacteria ml⁻¹. The cells were allowed to stabilize at low cell density by incubation at room temperature for 80 min, which is the half-life of the unstable GFP-ASV variant in V. anguillarum. Two cell samples were prepared for each culture. To one sample (induced), C6-HSL (10 nM) was added, whereas nothing was added to the second sample (uninduced). The bacteria were incubated at room temperature for 45 min and then mounted onto a glass slide. The same field of vision was imaged using both bright-field differential interference contrast microscopy to show all bacteria and UV light to detect GFP-ASV fluorescence, which detected qrr gene expression in the bacterial cells. Representative images are given for each strain, which are designated above each group of four images. For the induced sample, one bacterial cell was chosen and enlarged in the inset. +/−C6-HSL at the bottom of a column of images indicates the presence or absence of signal molecules. As the C6-HSL was first dissolved in acetonitrile, solvent alone was used as a negative control. Similar images were also obtained using the gene fusions for qrr2, qrr3 and qrr4 in the same strains and are presented as Supplementary Figs S1, S2 and S3.