Article Figures & Data
Figures
- FIG. 1.
Schematic representation of the tonB1 and tonB2 loci in V. anguillarum. The sizes of the genes and the corresponding proteins are shown above the tonB cluster genes. Also shown is the site of insertion of the Tn10 Kmr transposon in the tolR homologue. The lines under the loci represent the DNA fragments cloned in the pACYC177 plasmid generating pMS789, used in complementing studies, and the riboprobes used in the RPAs. AA, amino acids.
- FIG. 2.
Amino acid sequence alignment of the TonB2 proteins of V. anguillarum and V. cholerae (TonB2va and TonB2vc, respectively) and the TonB protein of E. coli (TonBec). We used the sequence analysis software package of the University of Wisconsin Genetics Computer Group. Underlined are the transmembrane domain, the proline-rich region, and the putative site of interaction with outer membrane proteins as determined for E. coli TonB. Identical amino acids are highlighted in gray.
- FIG. 3.
The tonB1 and tonB2 clusters are transcribed as separate operons. RT-PCR was performed with a downstream primer for the RT reaction in exbD1 and in tonB2 for the tonB1 and tonB2 clusters, respectively. In the subsequent PCR, specific primer sets for each gene were used to identify their presence in the same cDNA sample. (A) Lanes: 1, tonB1 RT-PCR product; 2, exbB1 RT-PCR product; 3, exbD1 RT-PCR product; 4, control with no reverse transcriptase; 5, H2O control; 6, molecular weight marker. (B) Lanes: 1, exbB2 RT-PCR product; 2, exbD2 RT-PCR product; 3, tonB2 RT-PCR product; 4, control with no reverse transcriptase; 5, H2O control; 6, molecular weight marker. (C) Lanes: 1, tolR RT-PCR product; 2, control with no reverse transcriptase; 3, molecular weight marker.
- FIG. 4.
Iron regulation of the two tonB systems. RPAs were performed with the MAXIscript kit and the RPA III RNase Protection kit, both from Ambion. RPAs for tonB1 (A) and tonB2 (B) were performed with RNA isolated from V. anguillarum 775 cultures grown under iron-rich (lanes 1 and 4) or iron-limiting (lanes 2 and 5) conditions. The aroC control is included in lanes 4 and 5. The riboprobes are shown in lanes 3 and 6.
Tables
- TABLE 1.
Bioassay results
Strain Result with following Fe source: FACa Ang.b Ent.c Fer.d Heme V. anguillarum 775 +e + + + + H775-3h + −f + + + MS533 (tonB1) + + + + + MS801 (tonB2) + − − + + MS658 (tonB1 tonB2) + − − − − MS801/pMS789i + + + + + MS658/pMS789 + + + + + E. coli W3110 + NDg + + ND E. coli KP1032 + ND − − ND E. coli KP1032/pMS789 + ND − − ND ↵ a FAC, ferric ammonium citrate.
↵ b Ang.: anguibactin from V. anguillarum 775.
↵ c Ent.: enterobactin from E. coli HB101.
↵ d Fer., ferrichrome.
↵ e +, positive, forming a zone of growth around the iron source.
↵ f −, negative, no zone of growth around iron source.
↵ g ND, not determined.
↵ h Plasmidless derivative of V. anguillarum 775 lacking the anguibactin transport system.
↵ i Plasmid containing the V. anguillarum tonB2, exbB2, and exbD2 genes.
