Figure 1.
Quorum sensing systems in V. harveyi/V. parahaemolyticus.
The mode for signal transduction during QS in V. harveyi has been described in the text. The feedback regulatory loops are shown with dotted lines. Since all the components of V. harveyi quorum sensing appears to be intact in the V. parahaemolyticus genome [16], the QS signal transduction cascades should be conserved in V. harveyi and V. parahaemolyticus.
Table 1.
Oligonucleotide primers used in this study.
Figure 2.
Phylogenetic tree of OpaR and its orthologs.
Protein sequences were derived from V. alginolyticus ZJ-51 [60], V. parahaemolyticus RIMD 2210633 [16], V. harveyi ATCC BAA-1116 [61], V. vulnificus YJ016 [62], V. tubiashii RE22 [63], V. anguillarum 775 [64], V. cholera N16961 [65], and V. fischeri MJ11 [66]. The a.a. sequences were aligned by the CLUSTALW [67] web server at http://align.genome.jp/. The aligned sequences were then used to construct an unrooted neighbor-joining tree using the MEGA version 5.0 [68] with a bootstrap iteration number of 1000. Shown on the branch points of phylogenic tree were the bootstrap values (%).
Figure 3.
(a) The sequence logo representation of the binding sites of OpaR and its orthologs (Table 2) was generated by the WebLogo tool [27]. The 20-bp consensus box TATTGATAAA-TTTATCAATA was annotated as an inverted repeat sequence. (b) A position frequency matrix describes the alignment of the binding sites, and denotes the frequency of each nucleotide at each position.
Table 2.
Known or predicted direct targets of OpaR or its orthologs.
Figure 4.
A two-round design of bacterial seed cultivation was employed: first, the glyceric stock of bacteria was inoculated into 15 ml of the MR or HI broth for growing at 30°C for 24 h with shaking at 200 rpm, and the cell culture was subsequently diluted to an OD600 value of about 1.0; second, the resulting culture was then 50-fold diluted into 15 ml of corresponding fresh MR or HI broth, and allowed to grow to reach an OD600 value of about 1.2 to 1.4. The bacterial seeds were 50-fold diluted into 15 ml of corresponding fresh MR or HI broth for further cultivation, and the OD600 values were monitored for each culture with a 1 h interval. Experiments were done in triplicate.
Figure 5.
Repression of its own gene by OpaR.
a) Primer extension. An oligonucleotide primer was designed to be complementary to the RNA transcript of opaR. The primer extension products were analyzed with 8 M urea-6% acrylamide sequencing gel. Lanes C, T, A, and G represent the Sanger sequencing reactions. The transcription start site of opaR was underlined in the DNA sequence. b) EMSA. The radioactively labeled DNA fragment from the 300th bp upstream to the 34th bp downstream of opaR was incubated with increasing amounts of purified His-OpaR protein, and then subjected to 4% (w/v) polyacrylamide gel electrophoresis. The band of free DNA disappeared with increasing amounts of His-OpaR protein, and a retarded DNA band with decreased mobility turned up, which presumably represented the DNA-OpaR complex. Shown on the lower side of the figure was the schematic representation of the EMSA design. c) DNase I footprinting. Labeled coding or non-coding DNA probes were incubated with increasing amounts of purified His-OpaR (Lanes 1, 2, 3, and 4 containing 0, 6, 9, and 12 pmol, respectively), and subjected to DNase I footprinting assay. Lanes G, A, T, and C represented the Sanger sequencing reactions. The footprint regions were indicated with vertical bars. The negative or positive numbers indicated the nucleotide positions upstream or downstream of opaR, respectively.
Figure 6.
For primer extension (a1, b1, and c1), an oligonucleotide primer was designed to be complementary to the RNA transcript of each of qrr2–4. For EMSA (a2, b2, and c2) and DNase I footprinting (a3, b3, and c3), the upstream DNA fragments of qrr2–4 were radioactively labeled, and then incubated with increasing amounts of purified His-OpaR protein. The experiments were done as described in Fig. 5. The transcription start sites of qrr2–4 were underlined in the DNA sequence. Lanes G, A, T, and C represented the Sanger sequencing reactions. The footprint regions were indicated with vertical bars. The negative or positive numbers indicated the nucleotide positions upstream or downstream of relevant qrr gene, respectively.
Figure 7.
For primer extension (a), an oligonucleotide primer was designed to be complementary to the RNA transcript of aphA. For EMSA (b) and DNase I footprinting (c), the DNA fragment from the 380th to 161th bp upstream of aphA was incubated with increasing amounts of purified His-OpaR protein. The experiments were done as described in Fig. 5. The transcription start site of aphA were underlined in the DNA sequence. Lanes G, A, T, and C represented the Sanger sequencing reactions. The footprint regions were indicated with vertical bars.. The minus numbers indicated the nucleotide positions upstream of aphA.
Figure 8.
Organization of promoter DNA regions.
DNA sequences were derived from V. alginolyticus ZJ-51 [60], V. parahaemolyticus RIMD 2210633 [16], V. harveyi ATCC BAA-1116 [61], V. vulnificus YJ016 [62], V. tubiashii RE22 [63], V. anguillarum 775 [64], V. cholera N16961 [65], and V. fischeri MJ11 [66]. Shown were translation and transcription starts, SD sequences, MQSR box-like sequences, and −10/−12 and −35/−24 core promoter elements.