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 ...

An Overview of Virulence-Associated Factors of Gram-Negative Fish Pathogenic Bacteria

“…Besides, the TonB1 gene cluster was shown to specifically contribute to haemin and haemoglobin utilization in this fish pathogen . In the same way, two different iron-regulated TonB mechanisms were identified in L. anguillarum (Stork et al, 2004). The analysis of these systems revealed that TonB2 protein but not TonB1 is involved in the transport of the siderophores anguibactin and enterobactin and that TonB2 protein is essential for virulence of L. anguillarum, since the LD 50 value for the tonB2 mutant strain was more than 100-fold higher than that of the parental strain (Stork et al, 2004).…”

Identification and cloning of immunogenic Aliivibrio salmonicida Pal-like protein present in profiled outer membrane and secreted subproteome

“…Other candidate genes and systems are, for example, iron acquisition systems linked to virulence mechanisms in several vibrios (Stork et al 2004). Iron acquisition in A. salmonicida is proposed to constitute a temperature-sensitive virulence factor, as siderophore production is only detected at ≤10°C.…”

“…Intriguingly, in contrast to the majority of pathogenic bacteria, A. salmonicida has not been shown to induce any extracellular protease activity or cytotoxicity in cell models although numerous experiments have been performed (Hjelmeland et al 1988, present study data not presented). Other candidate genes and systems are, for example, iron acquisition systems linked to virulence mechanisms in several vibrios (Stork et al 2004). Iron acquisition in A. salmonicida is proposed to constitute a temperature-sensitive virulence factor, as siderophore production is only detected at ≤10°C.…”

Plasmid-Encoded Iron Uptake Systems

“…It has a plug domain that blocks the open channel and extracellular loops that are needed for the initial binding to the siderophore (187). FatA then facilitates the transport over the outer membrane via an energy-dependent mechanism that requires the TonB system (16,188). The genes of the TonB system are encoded on the chromosome (16,188).…”