Two tonB Systems Function in Iron Transport in Vibrio anguillarum, but Only One Is Essential for Virulence

Abstract

We have identified two functional tonB systems in the marine fish pathogen Vibrio anguillarum, tonB1 and tonB2. Each of the tonB genes is transcribed in an operon with the cognate exbB and exbD genes in response to iron limitation. Only tonB2 is essential for transport of ferric anguibactin and virulence.

Outer membrane receptors for ferric siderophores require energy to internalize the complexed iron. This energy is transduced from the proton motive force in the inner membrane to the outer membrane receptor by the TonB protein in complex with the ExbB and ExbD proteins (4, 10). The presence of tonB has been associated with bacterial virulence in bacteria such as Vibrio cholerae (16), Shigella dysenteriae (15), and Bordetella pertussis (13).

To be able to cause an infection in a vertebrate fish host, the marine fish pathogen Vibrio anguillarum requires an active iron uptake mechanism (2, 6, 8, 17, 21) mediated by the siderophore anguibactin (1); however, it can also acquire iron via transport of heme and siderophores secreted by other microorganisms, like ferrichrome and enterobactin (5). Anguibactin is synthesized via a nonribosomal peptide synthetase mechanism and secreted to the extracellular environment (9). Once bound to iron, the ferric siderophore is transported back into the cell cytosol through the specific outer membrane receptor FatA and a transport complex consisting of the periplasmic binding lipoprotein FatB and the integral membrane proteins FatC and FatD (3, 7, 12).

Ferric-anguibactin transport via the FatA receptor requires energy, suggesting the existence of a tonB complex in V. anguillarum (7).

The tonB, exbB, and exbD cluster in V. anguillarum 775 also includes the heme transport genes (Fig. 1). This tonB system shows homology to the V. cholerae tonB1 cluster: TonB1, 48% (GenBank accession number NP_233295.1); ExbB1, 74% (NP_233296.1); ExbD1, 72% (NP_233297.1). When the tonB1 gene was replaced with a chloramphenicol resistance cassette (MS533), no phenotypic difference in iron uptake could be detected by bioassays with different iron sources (Table 1). This indicates that at least one other tonB system must be present in V. anguillarum. By Tn10 mutagenesis (11) of MS533, the tonB1 knockout, we identified a mutant, MS570, that was unable to transport any of the iron sources tested (Table 1). The Tn10 insertion was cloned and sequenced, showing that it occurred at bp 935 of an open reading frame homologous (66% identity) to open reading frame 1547 (tolR) of the V. cholerae genome (accession no. AF047974.1). This V. anguillarum tolR homologue is located upstream of a second tonB cluster (Fig. 1), which shares homology with the tonB2 system in V. cholerae: TonB2, 68% (NP_231186.1); ExbB2, 87% (NP_231185.1); ExbD2, 62% (NP_231184.1). We then generated a tonB2 knockout strain by inserting the kanamycin resistance (Km^r) cassette (18) into the chromosomal locus of tonB2, with the suicide vector pTW-MEV (19) in the wild-type V. anguillarum strain, generating MS801. We repeated this mutagenesis with the tonB1 mutant strain to obtain the tonB1-tonB2 double mutant MS658. We complemented the mutants with a construct harboring the tonB2 operon under the control of the Km^r gene promoter in pACYC177, pMS789. The mutants and complemented mutants were used in bioassays (Table 1). While the tonB1 mutant does not show any changes with respect to the wild type, the tonB1-tonB2 double mutant is impaired in transport of all of the iron sources tested. TonB2, but not TonB1, functions in the transport of anguibactin and enterobactin, while both TonB proteins can operate in the transport of ferrichrome and heme. We also transformed plasmid pMS789, harboring the tonB2 system, into Escherichia coli tonB mutant KP1032, showing that tonB2 from V. anguillarum cannot complement the tonB mutation in KP1032 (Table 1), even though the tonB2 operon of V. anguillarum is expressed in E. coli as determined by reverse transcription (RT)-PCR (data not shown). This was somewhat surprising since the V. cholerae tonB2 gene can complement this E. coli strain and the TonB2 proteins from V. anguillarum and V. cholerae share high homology, as is apparent in the alignment of these two proteins (Fig. 2). Furthermore, as is also shown in Fig. 2, comparison with E. coli TonB does not lead to an obvious answer for the lack of complementation.

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 Km^r 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.

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

^aFAC, ferric ammonium citrate.

^bAng.: anguibactin from V. anguillarum 775.

^cEnt.: enterobactin from E. coli HB101.

^dFer., ferrichrome.

^e+, positive, forming a zone of growth around the iron source.

^f−, negative, no zone of growth around iron source.

^gND, not determined.

^hPlasmidless derivative of V. anguillarum 775 lacking the anguibactin transport system.

ⁱPlasmid containing the V. anguillarum tonB2, exbB2, and exbD2 genes.

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.

We also analyzed the transcription of the two V. anguillarum tonB clusters by RT-PCR to determine whether the two tonB genes are transcribed in an operon with the respective exbB and exbD genes. As shown in Fig. 3, a band of the expected size was detected for all three genes, indicating that each one of the two tonB systems is indeed transcribed as an operon. Since the Tn10 mutant has the same phenotype as tonB2 mutant MS801, it may be that the tolR homologue is also part of the tonB2 operon and that the phenotype comes from a polar effect of the transposon on downstream genes. Therefore we performed an RT-PCR with a primer in exbB2 for the RT reaction and a primer set in tolR for the PCR. The results in Fig. 3C demonstrate that tolR is indeed also part of the tonB2 operon.

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.

To determine whether expression of the tonB1 and tonB2 systems is regulated by the iron concentration in the cell, we performed an RNase protection assay (RPA) with labeled riboprobes to detect either the tonB1- or the tonB2-specific mRNAs on total RNA obtained from cultures grown under iron-rich and iron-limiting conditions. Figure 4 shows the RPAs for tonB1 (panel A) and tonB2 (panel B). Comparison of lanes 1 (iron rich) and 2 (iron limiting) demonstrates that both tonB operons are iron regulated.

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.

The ability of V. anguillarum to cause infection has been correlated with the ability to synthesize anguibactin (2, 6, 8, 20, 21). We therefore investigated if the transport of anguibactin, mediated by the TonB2 protein, is essential for virulence. We performed 50% lethal dose (LD₅₀) experiments with fish, and the results are listed in Table 2. The tonB2 mutant is severely attenuated in virulence, more than 100-fold, while the tonB1 mutant shows only a 10-fold decrease in virulence. The tonB1-tonB2 double mutant is twofold more attenuated in virulence than the tonB2 mutant, possibly because the tonB2 mutant still has the ability to take up heme via TonB1. Complementation of the tonB2 and tonB1-tonB2 mutants with the wild-type tonB2 gene results in restoration of virulence to a level close to that of the wild type. Our results demonstrate that a functional tonB2 system is essential for ferric-anguibactin transport and virulence in V. anguillarum.

TABLE 2.

LD₅₀ determination in rainbow trout (Salmo gairdnerii)

Strain	LD50a	Fold attenuationb
V. anguillarum 775	1.2 × 104	1
MS533 (tonB1)	1.3 × 105	10
MS801 (tonB2)	1.3 × 106	103
MS658 (tonB1 tonB2)	2.9 × 106	234
MS801/pMS789c	7.1 × 104	6
MS658/pMS789	2.3 × 104	2

^aLD₅₀ calculated by the method of Reed and Muench (14).

^bFold attenuation normalized to wild-type V. anguillarum 775.

^cPlasmid containing the V. anguillarum tonB2 exbB2 and exbD2 genes.

Nucleotide sequence accession numbers.

The nucleotide sequences of the tonB1 and tonB2 clusters from V. anguillarum strain 775 have been deposited in the GenBank sequence library and assigned accession numbers AJ496544 and AY644719, respectively.