Abstract
Vibrio vulnificus is a halophilic estuarine bacterium that causes fatal septicemia and necrotizing wound infections in humans. Virulent V. vulnificus isolates produce a catechol siderophore called vulnibactin, made up of one residue of 2, 3-dihydroxybenzoic acid (2, 3-DHBA) and two residues of salicylic acid (SA). Vulnibactin biosynthetic genes (VV2_0828 to VV2_0844) are clustered at one locus of chromosome 2, expression of which is significantly up-regulated in vivo. In the present study, we decipher the biosynthetic network of vulnibactin, focusing specifically on genes around SA and 2, 3-DHBA biosynthetic steps. Deletion mutant of isochorismate pyruvate lyase (VV2_0839) or 2, 3-dihydroxybenzoate-2, 3-dehydrogenase (VV2_0834) showed retarded growth under iron-limited conditions though the latter showed more significant growth defect than the former, suggesting a dominant role of 2, 3-DHBA in the vulnibactin biosynthesis. A double deletion mutant of VV2_0839 and VV2_0834 manifested additional growth defect under iron limitation. Though the growth defect of respective single deletion mutants could be restored by exogenous SA or 2, 3-DHBA, only 2, 3-DHBA could rescue the double mutant when supplied alone. However, double mutant could be rescued with SA only when hydrogen peroxide was supplied exogenously, suggesting a chemical conversion of SA to 2, 3-DHBA. Assembly of two SA and one 2, 3-DHBA into vulnibactin was mediated by two AMP ligase genes (VV2_0836 and VV2_0840). VV2_0836 deletion mutant showed more significant growth defect under iron limitation, suggesting its dominant function. In conclusion, using molecular genetic analytical tools, we confirm that vulnibactin is assembled of both 2, 3-DHBA and SA. However, conversion of SA to 2, 3-DHBA in presence of hydrogen peroxide and growth profile of AMP ligase mutants suggest a plausible existence of yet unidentified alternative siderophore that may be composed solely of 2, 3-DHBA.
Highlights
Vibrio vulnificus is an opportunistic Gram-negative bacterial pathogen that causes fatal septicemia and necrotizing wound infections in susceptible individuals with high serum iron levels (Strom and Paranjpye, 2000)
V. vulnificus CMCP6 was grown in 2.5% NaCl heart infusion (HI) medium while Escherichia coli strains were grown in Luria-Bertani (LB) medium supplemented appropriately with antibiotics
To confirm the regulatory effect of Fur, we developed a Fur deletion mutant of V. vulnificus and checked the expression of the four selected genes in this mutant grown in 2.5% NaCl-HI without any DP (Figure 1B)
Summary
Vibrio vulnificus is an opportunistic Gram-negative bacterial pathogen that causes fatal septicemia and necrotizing wound infections in susceptible individuals with high serum iron levels (Strom and Paranjpye, 2000). The low molecular weight compound called siderophore binds iron with high affinity (Braun and Killmann, 1999) and is an important virulence factor produced by V. vulnificus (Morris et al, 1987; Litwin et al, 1996). Virulent V. vulnificus produces a phenolate (catechol) siderophore called vulnibactin that enables it to acquire iron from highly iron saturated host proteins such as transferrin (Stelma et al, 1992). The chemical structure of vulnibactin is closely related to the Vibrio cholerae catechol siderophore, vibriobactin. Vibriobactin was reported to be composed of three residues of 2, 3-DHBA, and like vulnibactin its biosynthesis was found to be dependent on non-ribosomal peptide synthatases (NRPS) (Griffiths et al, 1984; Keating et al, 2000). The most important precursor in NRPS-dependent siderophore biosynthetic pathway is chorismate. 2, 3-DHBA could be formed from chorismate by a three step reaction catalyzed by isochorismate synthase (Liu et al, 1990), isochorismatase
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