, 2010). Among 116 such genomic loci was an andA locus encoding anthranilate dioxygenase (see below). This locus carries an andA operon consisting of four structural genes, andAcAdAbAa. This locus also carries a divergently transcribed regulatory gene, andR, encoding a protein belonging to a AraC family of transcriptional regulators (Fig. 1a). In our IVET screening, this locus was the most repeatedly identified (51 times among the 713 IVET-positive clones) and was drastically induced (more than 100-fold induction rate) in the soil (Nishiyama et al., 2010). The gene organization and nucleotide sequence

of the ATCC 17616 andA locus are very similar to those from B. cepacia DBO1 (Chang UK-371804 research buy et al., 2003), and the deduced amino acid sequences shared

high similarities (85–96%). The study of DBO1 suggested that the AndR protein was a positive regulator of the andA promoter, as the andR mutant failed to grow on anthranilate, and that the andA promoter was upregulated by anthranilate but neither by benzoate nor by salicylate (Chang et al., 2003). However, it remained unclear whether tryptophan, a compound from which anthranilate can be formed (Fig. 1b), needs to be metabolized to induce andA promoter. The ferric uptake regulator (Fur) is a global transcriptional regulator for the iron regulon in many Gram-negative bacterial species (Faulkner & Helmann, selleck chemicals llc 2011). Our preliminary microarray analysis of ATCC 17616 revealed the transcriptional down-regulation of andAc in the fur mutant (our unpublished observation). As no canonical Fur binding site (Fur box) was located upstream of the andA operon (Yuhara et al., 2008), it was assumed that this operon is under the indirect control of Fur. We found in the present study that the ATCC 17616 andA operon is involved in the catabolism of tryptophan and anthranilate, and that the proliferation of ATCC 1716 in soil was dependent on andA. We also report the requirement of andR function and the moderate dependence of (Cornelis et al., 2009) fur function for the induction of andA promoter in

the soil. The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli cells were grown at 37 °C in Luria-Bertani (LB) broth (Maniatis et al., 1982) and B. multivorans cells at 30 °C in 1/3 LB broth (0.33% tryptone, 0.16% yeast extract, and Axenfeld syndrome 0.5% NaCl) or in M9 minimal medium (Maniatis et al., 1982). When used, succinate, tryptophan, and anthranilate were added to the media at a final concentration of 20 mM. 2,2′-Dipyridyl was added at a final concentration of 0.1 mM. Antibiotics were added to the media at the following concentrations: ampicillin at 50 μg mL−1 and kanamycin (Km) at 50 μg mL−1 for E. coli, and Km at 200 μg mL−1 and tetracycline (Tc) at 50 μg mL−1 for B. multivorans. When necessary, diaminopimelic acid (DAP) and lysine were added at 100 μg mL−1. To count LacZ+ and LacZ− colonies on agar plates, 40 μg mL−1 of X-gal was added to the media.