parahaemolyticus vibrioferrin utilization Vibrio parahaemolyticu

parahaemolyticus vibrioferrin utilization. Vibrio parahaemolyticus strains, and Escherichia coli strain and plasmids used in this study are listed in Table 1, and Table S1, respectively. Vibrio parahaemolyticus VPD5, which carries a deletion in pvsB that results in no VF production, was used

as a parental strain for the construction of various mutants to avoid any effects of VF produced by the wild-type strain. Escherichia coli β2155 (Demarre et al., 2005), a diaminopimelic acid (DAP) auxotroph, was grown in Luria–Bertani (LB) medium containing 0.5% NaCl and 0.5 mM DAP. Vibrio parahaemolyticus was routinely cultured in LB medium containing 3.0% NaCl (+Fe medium). Appropriate antibiotics were added to the medium at the following concentrations: 10 μg mL−1 chloramphenicol, and 15 μg mL−1 tetracycline. When required,

V. parahaemolyticus was grown in LB medium containing 3.0% NaCl supplemented with 25 μM ethylenediamine di-o-hydroxyphenylacetic acid (EDDA; Sigma, St. Louis, MO) (−Fe medium) to impose iron limitation (Miles & Khimji, 1975). The genomic sequence information of V. parahaemolyticus RIMD2210633 (Makino et al., 2003) was obtained from the Genome Information Research Center (GIRC) at Osaka University ( A homology search was carried out using the blast program on GIRC or National Center for Biotechnology Information ( (Altschul et al., 1997). The V. parahaemolyticus cultures grown overnight in the +Fe medium were inoculated selleck inhibitor Leukotriene-A4 hydrolase into the +Fe and −Fe media at an optimal density of 0.005 at 600 nm (OD600 nm). When required, the −Fe medium was supplemented with VF (Yamamoto et al., 1994) at a final concentration of 20 μM (−Fe + VF medium). The cultures were then shaken at 70 rpm at 37 °C, and the OD600 nm was measured every 3 h for 24 h with a biophotorecorder TVS062CA

(Advantec, Tokyo, Japan). Although it was reported that EDDA is a strong chelator of ferric iron and the association constant of ferric EDDA (c. 1034) (Miles & Khimji, 1975) is higher than that of ferric VF (c. 1023) (Amin et al., 2009), growth of VF-nonproducer mutant VPD5 (i.e. ∆pvsB) repressed in the −Fe medium was restored in the –Fe + VF medium (Fig. 2). This indicates that a very small amount of ferric VF required for the growth of V. parahaemolyticus could be supplied successively by equilibrium, although almost all ferric iron would be ferric EDDA in the −Fe + VF medium. Thus, the medium prepared was successfully used to estimate growth promotion of the mutants by VF. The primers used to construct the gene-deletion fragments and confirm gene deletions in various mutants are listed in Table S2. PCR amplicons with the respective deletions in the V.

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