Reactivation of the vanchrobactin siderophore system of Vibrio anguillarum by removal of a chromosomal insertion sequence originated in plasmid pJM1 encoding the anguibactin siderophore system

Abstract

A chromosomal gene cluster encoding vanchrobactin biosynthesis and transport genes was identified in the Vibrio anguillarum serotype O1 strain, 775(pJM1), harbouring the anguibactin biosynthetic genes in the pJM1 plasmid. In this strain only anguibactin is produced as the vanchrobactin chromosome cluster has a RS1 transposition insertion into vabF, one of the vanchrobactin biosynthesis genes. Removal of this RS1 generating 775(pJM1)Delta tnp, still resulted in the detection of only anguibactin in specific bioassays. Surprisingly, when the pJM1 plasmid was not present as in the plasmidless strain H775-3, removal of the RS1 resulted in the detection of only vanchrobactin. These results thus can be interpreted as if presence of the pJM1 plasmid or of anguibactin itself is associated with the lack of detection of the vanchrobactin siderophore in bioassays. As high-performance liquid chromatography (HPLC) and mass spectrometry analysis demonstrated that both vanchrobactin and anguibactin were indeed produced in 775(pJM1)Delta tnp, it is clear that the pJM1-encoded anguibactin siderophore has higher affinity for iron than the vanchrobactin system in strains in which both systems are expressed at the same time. Our results underscore the importance of the anguibactin system in the survival of V. anguillarum 775 under conditions of iron limitation.

Fig. 1. Structure of two types of siderophore produced by V. anguillarum

A. Anguibactin from O1 strain 775(pJM1) (Jalal et al., 1989). B. Vanchrobactin from O2 strain RV22 (Soengas et al., 2006).

Fig. 2. Two functional homologues of the phosphopantetheinyl transferase genes are active in the biosynthesis of both anguibactin and vanchrobactin. CAS assays: (A) anguibactin; (B) vanchrobactin; (C) anguibactin and vanchrobactin

A. Lanes 1, 775(pJM1)-pMMB; 2, H775-3-pMMB; 3, 775(pJM1)ΔangD-pMMB; 4, 775(pJM1)ΔvabDpMMB; 5, 775(pJM1)ΔangDΔvabD-pMMB; 6, 775(pJM1)ΔangDΔvabD-pMMBangD; 7, 775(pJM1)ΔangDΔvabD-pMMBvabD. B. Lane 1, 96F-pMMB; 2, 96FΔvabD-pMMB; 3, 96FΔvabD-pMMBangD; 4, 96FΔvabD-pMMBvabD. C. Lane 1, H775-3Δtnp; 2, 775(pJM1)Δtnp. Three microlitres of overnight culture of each strains was spotted on CAS plate with the addition of 1 mM IPTG. Plates were incubated for 1 day at 25°C and for 3 days at 16°C.

Fig. 3. Comparison of the chromosomal loci involved in siderophore biosynthesis and transport in V. anguillarum strains 775(pJM1) and 96F

A. The slanted line box represents the transposon RS1 inserted in the vabF gene. Black thick vertical lines correspond to the sequence of CTTGATG, which are the duplicated sequences at site of insertion. B. Detail of the transposon in vabF. The black arrow symbolizes the ORF21 corresponding to the transposase.

Fig. 4. Confirmation of vanchrobactin biosynthesis in the 775(pJM1) tnp strain

A. High-performance liquid chromatography (HPLC) chromatograms of vanchrobactin purified from 775(pJM1) tnp (left) and RV22 (right) strains. B. Positive mode electrospray ionization-mass spectra of the purified siderophores shown in (A) using 30% collision energy. The nominal masses (m/z) of the parental ion species and different fragmentation products are indicated in the spectra.

Fig. 5

High-performance liquid chromatography (HPLC) profile of vanchrobactin and anguibactin. The fractions tested in the bioassays shown in Table 2 are indicated in roman numerals.

Fig. 6

Relative affinities for Fe(III) of purified anguibactin and vanchrobactin. Chelating capability of purified anguibactin and vanchrobactin at various concentrations (10, 20 and 30 μM) measured as percentage of siderophore units as indicated in Experimental procedures. Bars: white, vanchrobactin; black, EDTA; and grey, anguibactin. Values represent mean ± SD.

Fig. 7

Detection of the FatA receptor protein in the outer membrane fraction of several strains of V. anguillarum by Western blot using an anti-FatA serum. Outer membrane proteins were prepared from 775(pJM1), 96F, 96F(pJM1), RV22 and RV22(pJM1).

Fig. 8. Identification of RS1 in chromosome and/or pJM1-like plasmid DNA

A. Southern blot hybridization of ClaI-digested DNA to assess the relative copy number of RS1 in several V. anguillarum strains. Only the transposase gene orf21 from RS1 was used as a probe to avoid problems with the bordering repeated sequences. Lane 1, 775(pJM1); 2, 775(pJM1)Δtnp; 3, H775-3; 4, H775-3Δtnp; 5, 96F; 6, 96F(pJM1); 7, RV22; 8, RV22(pJM1); 9, 531A; 10, PT77022; 11, R61. The solid arrowhead points to the location of orf21 in pJM1 of 775(pJM1), while the open arrowhead indicates the position of orf21 in vabF of 775(pJM1). B. Polymerase chain reaction to assess the site of interruption of vabF by RS1. Lane M, molecular marker; 1, 775(pJM1); 2, 775(pJM1)Δtnp; 3, H775-3; 4, H775-3Δtnp; 5, 96F; 6, 96F(pJM1); 7, RV22; 8, RV22(pJM1); 9, 531A; 10, PT77022; 11, R61.