The V. parahaemolyticus strain RIMD2210633 was used as the wild type (WT) in this study (Makino et al., 2003). The non-polar aphA and opaR single-gene deletion mutants derived from WT (designated as ΔaphA and ΔopaR, respectively) were described in our previous studies (Sun et al., 2012; Zhang et al., 2012) (shown also in Supplementary Figure S2). For construction of toxR deletion mutant (ΔtoxR), the 428 and 455 bp DNA regions upstream and downstream of toxR were amplified by PCR, purified, and used as the templates to create an 879 bp deletion construct that was subsequently inserted between the Pst I and Sph I sites of pDS132 (Philippe et al., 2004). After being verified by DNA sequencing, the recombinant vector was transformed into Escherichia coli S17-bbbpir, and then transferred into WT by conjugation (Philippe et al., 2004). The mutant strain was selected using resistance to 10% sucrose and sensitivity to 5 μg/ml chloramphenicol, and further verified by PCR. All the primers used were listed in Table 1.

For complementation of the mutants (Sun et al., 2014), a PCR-generated DNA fragment containing the coding region together with an upstream synthetic ribosome binding Shine-Dalgarno (SD) sequence (AGGAGG) (Shine and Dalgarno, 1974) for each deleted gene was inserted between the Xba I and Hind III sites of the pBAD33 (Guzman et al., 1995) vector harboring an arabinose P_BAD promoter and a chloramphenicol resistance gene. After being verified by DNA sequencing, the recombinant plasmid for each gene was transformed into the corresponding mutant, yielding the complemented mutant strain ΔaphA/pBAD33-aphA, ΔopaR/pBAD33-opaR, orΔtoxR/pBAD33-toxR. For controls, the empty vector pBAD33 was also transformed into WT and each mutant to counteract the effects of arabinose and chloramphenicol on bacterial growth and physiology.

Vibrio parahaemolyticus strains were cultured in Difco marine broth 2216 (BD Biosciences) at 30°C with shaking at 200 rpm. The glyceric stock of bacterial cells were inoculated into 5 ml of M broth and incubated overnight for at least 12 h. The overnight cell cultures were diluted 1:50 into 15 ml of fresh M broth, and grown to reach at OD₆₀₀≈1.0, and then diluted 1:1000 into 15 ml of marine broth for the third-round growth, and were harvested at required cell densities. The culture medium was supplemented with 50 μg/ml gentamicin, 5 μg/ml chloramphenicol, or 0.1% arabinose where necessary. V. parahaemolyticus is a biosafety level 2 (BSL-2) pathogen, and thus all the experimental operations involving in live bacteria were done in the BSL-2 lab.

Total bacterial RNAs were extracted using the TRIzol Reagent (Invitrogen, United States). The contaminated genome DNA in the total RNAs was removed by using the Ambion’s DNA-free^TM Kit according to the manufacturer’s instructions. cDNAs were generated by using 3 ∼ 8 μg of total RNAs and 3 μg of random hexamer primers. The SYBR Green qRT-PCR assay was performed and analyzed as previously described (Gao et al., 2011). The relative mRNA levels were determined based on the standard curve of 16S rRNA (reference gene) expression for each RNA preparation.

For the primer extension assay (Sun et al., 2012; Zhang et al., 2012), an oligonucleotide primer complementary to a portion of the RNA transcript of each indicated gene was employed to synthesize cDNAs from total RNA templates. Approximately 10 μg of total RNAs were annealed with 1 pmol of 5′- ³²P-end labeled reverse oligonucleotide primer to generate cDNAs using a Primer Extension System (Promega, United States). The same labeled primer was used for sequencing with the AccuPower and Top DNA Sequencing Kit (Bioneer, South Korea). The primer extension products and sequencing materials were concentrated and analyzed in an 8 M urea-6% polyacrylamide gel electrophoresis, and the results were detected by autoradiography with the Fuji Medical X-ray film (Fuji Photo Film Co., Ltd., Japan).

For the LacZ fusion and β-galactosidase assay (Sun et al., 2012, 2014), the promoter DNA region of each indicated gene was amplified by PCR with ExTaq^TM DNA polymerase (Takara, Japan) using the genomic DNA as the template. PCR amplicons were cloned into the corresponding restriction endonuclease sites of pHRP309 plasmid harboring a promoterless lacZ reporter gene and a gentamicin resistance gene (Parales and Harwood, 1993). After being verified by DNA sequencing, the recombinant plasmid was transferred into WT and mutant strains, respectively. An empty pHRP309 plasmid was also introduced into each strain and tested as the negative control. The V. parahaemolyticus strains transformed with recombinant or empty pHRP309 plasmids were cultivated as above to measure the β-galactosidase activity in cellular extracts using a β-Galactosidase Enzyme Assay System (Promega, United States) according to the manufacturer’s instructions.

The entire coding region of aphA, opaR, and the truncated toxR (1–528 bp, a.a.1–176) of the strain RIMD 2210633 were amplified, purified, and cloned into plasmid pET28a (Novagen, United States), respectively. The recombinant plasmid encoding His-tagged protein was transformed into E. coli BL21bbbDE3 cells for protein expression (Kleberjanke and Becker, 2000). Expression and purification of His-AphA and His-OpaR have been described previously (Sun et al., 2012; Zhang et al., 2012), while His-ToxR was the same as that of His-AphA.

For EMSA (Sun et al., 2012; Zhang et al., 2012), the 5′-ends of the promoter-proximal DNA region of each indicated gene were labeled using [γ-³²P] ATP and T4 polynucleotide kinase. DNA binding was performed in a 10 μl reaction volume containing binding buffer (1 mM MgCl₂, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl/pH 7.5, and 10 mg/ml salmon sperm DNA), labeled DNA (1000–2000 CPM/μl), and increasing amounts of His-tagged protein. Three controls were included in each EMSA experiment: (1) cold probe as specific DNA competitor (the same promoter-proximal DNA region unlabeled), (2) negative probe as non-specific DNA competitor (the unlabeled coding region of the 16S rRNA gene), and (3) non-specific protein competitor (rabbit anti-F1-protein polyclonal antibodies). After incubation at room temperature for 30 min, the products were loaded onto a native 4% (w/v) polyacrylamide gel, and electrophoresed in 0.5× TBE buffer for about 50 min at 200 V. Radioactive species were detected by autoradiography after exposure to Fuji Medical X-ray film at -20°C.

For DNase I footprinting (Sun et al., 2012; Zhang et al., 2012), the target promoter DNA regions with a single ³²P-labeled end were PCR amplified with either sense or antisense primer being end-labeled. The PCR products were purified using the QiaQuick columns (Qiagen, Germany). Increasing amounts of His-tagged protein were incubated with the purified, labeled DNA fragment (2–5 pmol) for 30 min at room temperature, in a final 10 μl reaction volume containing the binding buffer used in EMSA. Before DNA digestion, 10 μl of Ca²⁺/Mg²⁺ solution (5 mM CaCl₂ and 10 mM MgCl₂) was added, followed by incubation for 1 min at room temperature. The optimized RQ1 RNase-Free DNase I (Promega, United States) was then added to the reaction mixture, and the mixture was incubated at room temperature for 40–90 s. The reaction was quenched by adding 9 μl of stop solution (200 mM NaCl, 30 mM EDTA, and 1% SDS), followed by incubation for 1 min at room temperature. The partially digested DNA samples were extracted with phenol/chloroform, precipitated with ethanol, and analyzed in 6% polyacrylamide/8 M urea gel. Protected regions were identified by comparison with the sequence ladders. The templates for DNA sequencing were the same as the DNA fragments for DNase I footprinting assay. Radioactive species were detected by autoradiography after exposure to Fuji Medical X-ray film at -20°C.

The LacZ fusion assay and qRT-PCR were performed with at least three independent bacterial cultures and the values were expressed as mean ± standard deviation. Paired Student’s t-test was used to calculate statistically significant differences, p < 0.01 was considered to indicate statistical significance. The presented data of primer extension, EMSA, and DNase I footprinting assays were done with at least two independent biological replicates.

VP1393-1406 and VP1392-1391 (also VP1409-1407 and VP1410-1420) are adjacent but are transcribed in the opposite direction and they share the same intergenic DNA region (Supplementary Figure S1). Thus, the 400 bp upstream regions of VP1386-1387, VP1388-1390, VP1393-1406, VP1400-1406, and VP1409-1407 in the T6SS1 gene cluster were retrieved from the genome sequence of RIMD 2210633 with the ‘retrieve-sequence’¹. Subsequently, the DNA binding boxes of AphA (Sun et al., 2012), OpaR (Zhang et al., 2012), and ToxR (Goss et al., 2013) were used to statistically predict the presence of AphA/OpaR/ToxR box-like sequences within the above target upstream regions by using the matrix-scan toolaaa. The analysis generated the weight scores for each target upstream region. The higher score values represented the higher probability of regulatory protein and upstream region association. When the weight score of six was taken as the cutoff value, the OpaR box-like sequences were found for VP1388-1390, VP1400-1406 and VP1409-1407, while the ToxR box-like sequences were found for VP1386-1387 and VP1409-1407 (Table 2). However, the AphA box-like sequences were not found in all of the upstream regions tested. Thus, the first genes of VP1388-1390, VP1393-1406, VP1400-1406 and VP1409-1407 were selected for the following gene regulation studies.

The mRNA levels of aphA, opaR, toxR, VP1388 and VP1393 (hcp1) were measured in WT grown at different cell densities by the primer extension assay (Figure 1). The aphA and toxR mRNA levels decreased considerably with the increasing of cell density, and the highest transcription appeared at an OD₆₀₀ value of 0.05 to 0.2; when the OD₆₀₀ value was higher than 0.4, the mRNAs of both aphA and toxR were undetectable. On the contrary, the opaR mRNA level was increased but then reduced with the increasing of cell density, and the highest transcription occurred at an OD₆₀₀ value of 0.4 to 0.6; when the OD₆₀₀ value was lower than 0.2, the opaR mRNA was undetectable. The highest transcription level of hcp1 emerged at an OD₆₀₀ value of 0.6; when the OD₆₀₀ value was lower than 0.4 or higher than 0.8, the hcp1 mRNA was undetectable. However, the VP1388 mRNAs were detected at all cell densities, and the highest transcription occurred at an OD₆₀₀ value of 0.8. Thus, the bacterial cells were harvested at an OD₆₀₀ value of about 0.15 and 0.4–0.6 for characterizing AphA/ToxR- and OpaR-mediated gene regulation, respectively. In addition, the cell density-dependent transcription of VP1388 and hcp1 suggests T6SS1 expression would be under control of QS.

As determined by the qRT-PCR assay (Figure 2A), the mRNA level of all the four genes, i.e., VP1388, hcp1, VP1400, and VP1409, was enhanced in ΔaphA relative to WT. The primer extension assay (Figure 2B) further indicated that the mRNA levels of the four genes were enhanced in ΔaphA relative to WT. The recombinant lacZ fusion plasmid that contains the indicated promoter-proximal region and promoterless lacZ gene was transformed into ΔaphA and WT, respectively, to test the action of AphA on the promoter activity of the above four genes. The results disclosed a significantly enhanced promoter activity of each of the four genes in ΔaphA relative to WT (Figure 2C). The promoter DNA regions of the above four genes were amplified, purified, radioactively labeled, and then subjected to EMSA with the purified His-AphA (Figure 2D). The results showed that His-AphA was unable to bind to the upstream DNA fragment of each target promoters, these were consistent with the predict results (Table 2). Taken together, AphA appears to negatively regulate the transcription of VP1388-1390, VP1393-1406, VP1400-1406, and VP1409-1407 in an indirect manner.

The qRT-PCR and primer extension assays indicated that the transcription of all the four target genes VP1388, hcp1, VP1400, and VP1409 increased in ΔopaR relative to WT (Figures 3A,B). The lacZ fusion results showed that the promoter activity of each of the four operons in ΔopaR was much higher relative to that in WT (Figure 3C). The EMSA results showed that His-OpaR was able to bind to the upstream DNA fragment of VP1388, VP1400, and VP1409 in a dose dependent manner, but a negative EMSA result was observed for hcp1 (Figure 3D). His-OpaR at all amounts used could not bind to the 16S rDNA fragment as the negative control (Figure 3D). As further determined by DNA footprinting (Figure 3E), His-OpaR protected two different DNA regions upstream of VP1388 and VP1409 against DNase I digestion that were considered as the OpaR sites, while only a single OpaR site was detected for VP1400. Taken together, OpaR represses the transcription of VP1388-1390, VP1400-1406, and VP1409-1407 in a direct manner, but it appears to negatively regulate the transcription of VP1393-1406 in an indirect manner.

As determined by the qRT-PCR assay (Figure 4A), the mRNA level of each target gene was greatly increased in ΔtoxR relative to WT. The primer extension assay detected a single ToxR-repressed transcription start site for each of the four target operons (Figure 4B). The lacZ fusion results showed that the promoter activity of each of the four operons in ΔtoxR was significantly enhanced than that in WT (Figure 4C). The EMSA results showed that His-ToxR was able to bind to the upstream DNA fragment of VP1400 and VP1409 in a dose dependent manner, but negative EMSA results were observed for VP1388 and hcp1 (Figure 4D). As further determined by DNA footprinting (Figure 4E), His-ToxR protected one or more DNA regions upstream of VP1409 or VP1400 against DNase I digestion that were considered as the ToxR sites. Taken together, ToxR represses the transcription of VP1400-1406 and VP1409-1407 in a direct manner, but it appears to negatively regulate the transcription of VP1388-1390 and VP1393-1406 in an indirect manner.

Collection of data of translation/transcription start sites, promoter -10 and -35 (or -12 and -24) elements, AphA/OpaR/ToxR sites, AphA/OpaR/ToxR box-like sequences, Shine-Dalgarno (SD) sequences (ribosomal binding sites) enabled us to depict the organization of VP1388, hcp1, VP1400 and VP1409 promoters characterized herein (Figure 5).