Table 1.
Bacterial strains used in this study.
Figure 1.
Alkaline pH-dependent utilization of Ent in VPD54.
VPD54, which is a vctA and irgA deletion mutant generated from the VPD5 vibrioferrin-deficient mutant, was grown in LB-Tris/+EDDA medium (at indicated pH) at 37°C for 18 h with shaking at 70 rpm. When required, Ent was added at 5 µM. Cultures were monitored by measuring the OD600 every 3 h. Data are shown as means ± SD from 3 separate experiments.
Figure 2.
Involvement of peuA and peuRS in Ent utilization.
The growth assay was performed as described in Figure 1. Data are shown as means ± SD from 3 separate experiments.
Figure 3.
SDS-PAGE analysis of Sarkosyl-insoluble OMPs of V. parahaemolyticus.
SDS-PAGE analysis was performed with VPD5, VPD107 (seven iron-repressible OMRs-deficient mutant derived from VPD5), VPD108 (peuA-deficient mutant derived from VPD107), VPD108/pRK415-peuA, VPD109 (peuRS-deficient mutant derived from VPD107), and VPD109/pRK415-peuRS. The OMP fractions were prepared from cells grown in LB-Tris medium at pH 7.0, LB-Tris/+EDDA media at pH 7.0 and 8.0, or LB-Tris/+EDDA/+Ent media at pH 7.0 and 8.0. Lanes 1–7 and 9–10 were loaded with 20 µg OMPs, and lane 8 was loaded with 3 µg OMPs. Electrophoresis was performed on 7.5% SDS-polyacrylamide gels (130 mm long) at a constant current of 15 mA at 4°C. The gel was stained with Coomassie Brilliant Blue. The figure shows only the relevant portions of the gel. The iron-repressible OMPs expressed by VPD5 at pH 7.0 under iron-limiting conditions are boxed in lane 2. Lane M, molecular weight marker proteins; closed arrowheads, PeuA.
Figure 4.
Genetic map and operon structure of VPA0148–VPA0156 locus.
(A) Genetic map of the peuA gene and the flanking genes. Thick arrows indicate genes and their orientations. The –35 and –10 promoter elements and putative Fur box sequences in the promoter regions of peuR (VPA0148) and peuA (VPA0150) are indicated. The transcription start sites for peuR (+1) and peuA (+1 and +39) are indicated by right-angled arrows. The putative terminator signal between the peuS and peuA genes, the predicted RBS for the peuA gene, the start codons for peuR and peuA genes, and the stop codon for peuS are also indicated. The amino acid sequence consistent with the N-terminal sequence determined for the iron-repressible OMR induced in LB-Tris/+EDDA and LB-Tris/+EDDA/+Ent media at pH 8.0 (see Figure 3) is indicated by a double underline. (B) Schematic representation of mRNAs transcribed from the VPA0148-VPA0156 genes and the primer pairs used for RT-PCR. For preparation of cDNAs by RT, VPpeuS-R and VP0156-R were used. (C) RT-PCR analysis of RT-PCR products. +RT and –RT, RT-PCR was performed with and without reverse transcriptase, respectively. M, 100-bp DNA ladder.
Figure 5.
Primer extension analyses of total RNA from VPD54 or VPD102 to determine the transcription start sites of peuR (A) and peuA (B and C).
Total RNAs were isolated from VPD54 (vctA- and irgA-deficient mutant derived from VPD5) and VPD102 (peuRS-deficient mutant derived from VPD54) grown at pH 7.0 and 8.0 in LB-Tris, LB-Tris/+EDDA, and LB-Tris/+EDDA/+Ent media. The amounts of total RNA and primers used for reverse transcription were as follows: (A) 10 µg VppeuR-PE, (B) 10 µg VppeuA-PE, and (C) 150 µg VppeuA-PE. The same primers used for primer extension analysis were used to generate the sequence ladders (A, C, G, T). The transcription start sites and putative Fur boxes are indicated at the top of panels A and B (also see Figure 3A).
Figure 6.
Relative levels of peuA mRNA, assessed by RT-qPCR.
Total RNA samples were prepared from VPD54 (vctA- and irgA-deficient mutant derived from VPD5) and VPD102 (peuRS-deficient mutant derived from VPD54) grown at pH 7.0 and 8.0 in LB-Tris, LB-Tris/+EDDA, and LB-Tris/+EDDA/+Ent media. Data are shown as means ± SD from 3 separate experiments. An asterisk indicates P<0.05 compared to other samples.
Figure 7.
Schematic representation of the +1-peuA′-flag (A) and +39-peuA′-flag (B) DNA fragments.
Each of these DNA fragments includes a nucleotide sequence corresponding to the peuA 5′-UTR from the +1 or +39 sites and the nucleotide sequence for the N-terminal 99 amino acid residues (in gray), in addition to a T7 promoter and a FLAG tag preceding the stop codon (TAA). The secondary structures of the 5′-UTRs of the +1 transcript (A) and the +39 transcript (B) of peuA are shown, both of which were predicted by the CentroidFold software (http://www.ncrna.org/centroidfold/). The RBS and start codon of peuA mRNA are boxed in the secondary structures.
Figure 8.
In vitro translation of peuA mRNA.
(A) In vitro translation analysis of the +1 and +39 peuA transcripts labeled with the FLAG tag. The +1-peuA′-flag RNA and +39-peuA′-flag RNA were first synthesized by in vitro transcription, as described in the MATERIALS AND METHODS, and a mixture containing either the +1-peuA′-flag RNA (30 pmol)/fur-flag RNA (3 pmol) or the +39-peuA′-flag RNA (30 pmol)/fur-flag RNA (3 pmol) as the template was subjected to in vitro translation. The FLAG-fused proteins translated were separated on 15% SDS-polyacrylamide gels, and were detected by western blotting using anti-FLAG IgG. (B) Confirmation of the presence of +1-peuA′-flag RNA and +39-peuA′-flag RNA in the reaction mixture for in vitro translation. These RNA fragments were detected in the reaction mixture by northern blotting using a DIG-labeled peuA probe.
Figure 9.
Distribution of the Vibrio parahaemolyticus VPA0148–VPA0156 orthologs in other Vibrio species for which whole-genomic sequences have been reported.
Arrows represent genes and their orientations. The numbers below the genes indicate percent amino acid sequence similarities to V. parahaemolyticus PeuRS and PeuA. Vp, V. parahaemolyticus; Va, V. alginolyticus; Vh, V. harveyi; Vca, V. campbellii; Vf, V. fischeri; Vs, V. splendidus; Vc, V. cholerae; Vv, V. vulnificus.