Figure 1.
Model for signal transduction of QS systems in V. harveyi/V. parahaemolyticus.
The details for signal transduction during QS have been described in text. At LCD, redundant Qrr sRNAs promote AphA translation and meanwhile inhibit LuxR translation. AphA further represses the transcription of luxR/opaR, qrr2-3, and its own gene. At HCD, the cessation of Qrr sRNA production leads to no production of AphA, and LuxR/OpaR translation occurs. LuxR/OpaR in turns represses the aphA transcription, and also feeds back to inhibit its own expression. Thus, AphA acts as a master regulator of QS behaviors at LCD, and in contrast, LuxR/OpaR is the major one operating at HCD; reciprocal gradients of AphA and LuxR/OpaR are established for controlling gene expression during transition between LCD and HCD. LuxR/OpaR is also able to activate the transcription of qrr2-4 genes, leading to rapid down-regulation of luxR/opaR [47]. It should be noted that this LuxR-qrr feedback dramatically accelerates the transition HCD to LCD, but it has no effect on QS behaviors at steady-state LCD or HCD [47].The dotted lines indicated the inhibited expression of relevant regulators or the cease of relevant regulatory cascades. The blue and red lines show the regulatory cascades that were experimentally validated in V. parahaemolyticus in our previous [15] and present works, respectively.
Table 1.
Oligonucleotide primers used in this study.
Figure 2.
Phylogenetic tree of AphA orthologs.
The protein sequences were derived from V. parahaemolyticus RIMD 2210633 [14], Vibrio sp. Ex25 (Accession numbers CP001805.1, and CP001806.1), Vibrio sp. EJY3 [48], V. harveyi ATCC BAA-1116 [49], V. vulnificus YJ016 [50], V. splendidus LGP32 [51], V. anguillarum 775 [52], V. cholerae N16961 [53], V. furnissii NCTC 11218 [54], and P. profundum SS9 [25]. The a.a. sequences were aligned by the CLUSTALW [55] web server at http://align.genome.jp/. The aligned sequences were then used to construct an unrooted neighbor-joining tree using MEGA version 5.0 [56] with a bootstrap iteration number of 1000. Shown on branch points of phylogenic tree were bootstrap values (%).
Table 2.
Known or predicted direct targets of AphA.
Figure 3.
Cis-acting consensus constructs.
(a) The sequence logo representation of AphA sites (Table 2) was generated by WebLogo [24]. The 20-bp box sequence ATATGCAN6TGCATAT contained imperfect inverted repeats of ATATGCA with a 6-nt centered spacer. (b) A position frequency matrix describes the alignment of AphA sites, and denotes the frequency of each nucleotide at each position.
Figure 4.
A two-round design to prepare the bacterial seed culture was employed: firstly, the glyceric stock of bacterial cells was inoculated into 3 ml of MR broth for growing at 30°C with shaking at 200 rpm for 12 to 14 h to enter the stationary growth phase; secondly, the resulting cell culture was 50-fold diluted into 15 ml of fresh MR broth, and allowed to grow under the above conditions to reach an OD600 value of about 1.0 to 1.2. The seed culture was then 1000-fold diluted into 30 ml of fresh MR broth for the further growth under the above conditions, and the OD600 values were monitored for each culture with an 1 h interval.
Figure 5.
Transcriptional pattern of aphA during growth.
The bacterial cells were harvested at various OD600 values during growth (according to the bacterial growth curves in Fig. 4) for total RNA isolation. An oligonucleotide primer was designed to be complementary to the RNA transcript of aphA. The primer extension products were analyzed with 8 M urea-6% acrylamide sequencing gel. Lanes C, T, A, and G represented Sanger sequencing reactions. The transcription start site of aphA was underlined in the DNA sequence.
Figure 6.
Repression of its own gene by AphA. a) LacZ fusion.
The promoter-proximal DNA region of aphA was cloned into the lacZ transcriptional fusion vector pHRP309, and then transformed into WT or ΔaphA to determine the β-galactosidase activity in cellular extracts. Shown was the aphA promoter activity (Miller units) in ΔaphA or WT. b) Primer extension. An oligonucleotide primer was designed to be complementary to the RNA transcript of aphA. The primer extension products were analyzed with 8 M urea-6% acrylamide sequencing gel. Lanes C, T, A, and G represented Sanger sequencing reactions. The transcription start site of aphA was underlined in the DNA sequence. c) EMSA. The radioactively labeled DNA fragment from the 380th to 161st bp upstream of aphA was incubated with increasing amounts (lanes 1 to 4∶0, 15, 20, and 25 pmol, respectively) of purified His-AphA protein, and then subjected to 4% (w/v) polyacrylamide gel electrophoresis; the band of free DNA become weak with increasing amounts of His-AphA protein, and a retarded DNA band with decreased mobility turned up, which presumably represented the DNA-AphA complex. For lane 5, 2 pmol of cold probe, 25 pmol of His-AphA, and labeled target DNA fragment were added; the retarded DNA band disappeared due to the action of cold probe as specific DNA competitor. For lane 6, 2 pmol of negative probe, 25 pmol of His-AphA, and labeled target DNA fragment were added; the retarded DNA band occurred since the negative probe had no effect on the DNA-AphA complex. For lane 7, 20 pmol of non-specific protein competitor, and labeled target DNA fragment were added; there was no retarded DNA band observed. d) DNase I footprinting. Labeled coding or non-coding DNA probes were incubated with increasing amounts of purified His-AphA (Lanes 1, 2, 3, and 4 containing 0, 15, 20, and 25 pmol, respectively), and subjected to DNase I footprinting assay. Lanes G, A, T, and C represented Sanger sequencing reactions. The footprint regions were indicated with vertical bars. The negative numbers indicated nucleotide positions upstream of aphA.
Figure 7.
For LacZ fusion (a1, b1, and c1), the promoter-proximal fragment of each of qrr2-4 was cloned into pHRP309, and then transformed into WT or ΔaphA to determine the β-galactosidase activity (Miller units) in cellular extracts. For primer extension (a2, b2, and c2), an oligonucleotide primer was designed to be complementary to the RNA transcript of each of qrr2-4. For EMSA (a3, b3, and c3) and DNase I footprinting (c4), the promoter-proximal fragment of each of qrr2-4 were radioactively labeled, and then incubated with increasing amounts of purified His-AphA protein. The experiments were done as described in Fig 6. The transcription start sites of qrr2-4 were underlined in the DNA sequence. Lanes G, A, T, and C represented Sanger sequencing reactions. The footprint regions were indicated with vertical bars. The negative or positive numbers indicated nucleotide positions upstream or downstream of relevant qrr gene, respectively.
Figure 8.
For LacZ fusion (a), the promoter-proximal DNA fragment of opaR was cloned into pHRP309, and then transformed into WT or ΔaphA to determine the β-galactosidase activity (Miller units) in cellular extracts. For primer extension (b), an oligonucleotide primer was designed to be complementary to the RNA transcript of opaR. For EMSA (c) and DNase I footprinting (d), the promoter-proximal DNA fragment of opaR was incubated with increasing amounts of purified His-AphA protein. The experiments were done as described in Fig 6. The transcription start site of opaR was underlined in the DNA sequence. Lanes G, A, T, and C represented Sanger sequencing reactions. The footprint regions were indicated with vertical bars. The negative or positive numbers indicated nucleotide positions upstream or downstream of opaR, respectively.
Figure 9.
Organization of promoter-proximal DNA regions.
DNA sequences were derived from V. parahaemolyticus RIMD 2210633 [14], Vibrio sp. Ex25 (Accession numbers CP001805.1, and CP001806.1), Vibrio sp. EJY3 [48], V. harveyi ATCC BAA-1116 [49], V. vulnificus YJ016 [50], V. splendidus LGP32 [51], V. anguillarum 775 [52], V. cholerae N16961 [53], and V. furnissii NCTC 11218 [54]. Shown were translation and transcription starts, SD sequences, AphA or OpaR box-like sequences, and –0/−12 and –35/−24 core promoter elements. The prediction of OpaR box-like sequences were described previously [15].