The catabolic gene organization in A1501 lacks the catR and pcaK genes, a feature that is not observed in other Pseudomonas strains. Figure 2 Organization of benzoate (A) or 4-hydroxybenzoate (B) degradation gene clusters of A1501 and comparison with equivalent clusters from other bacteria. Two vertical lines indicate that the genes are not adjacent in the genome. Numbers beneath the arrows indicate the percentage of amino acid sequence identity between the encoded

gene product and the equivalent product from A1501. Functional characterization of the β-ketoadipate pathway A1501 grew well on 4 mM benzoate and reached an OD600 of 0.5 after 24 h of incubation, whereas no Thiazovivin manufacturer growth was observed in the presence of 8 mM benzoate. A1501 grow poorly on 0.4 mM 4-hydroxybenzoate, while 4-hydroxybenzoate at concentrations above RG7112 supplier 0.8 mM completely inhibited bacterial growth

(Figure 3). Further investigation of the β-ketoadipate pathway was made by constructing and characterizing three mutants: benR mutant A1601, pcaR mutant A1602 and pcaD mutant A1603 (Table 1). When the wild type and mutants were cultured in media containing lactate, their growth rates were not affected (data not shown). As expected, the benR mutant failed to grow on benzoate, and the pcaR and pcaD mutants failed to grow on 4-hydroxybenzoate as the sole buy Vistusertib carbon source. Furthermore, Methane monooxygenase both the pcaR and pcaD mutants

lost their ability to utilize benzoate as a carbon source. We constructed three complementary plasmids containing the entire pcaD, pcaR and benR genes for further growth complementation assays. Complementation of the three mutants with the corresponding complementary plasmids restored the catabolic activity, and the three corresponding complementary strains grew on benzoate as the sole carbon source (data not shown). Results from gene disruption analyses and genetic complementation tests demonstrate that the three genes are required for the growth of A1501 on benzoate. Table 1 Strains and plasmids used in this study Strains or plasmids Relevant characteristic(s)a Source or reference Strains     P.