, 2007). The RegSR system has long been known to activate the transcription of the nifA gene that encodes the key regulator for nitrogen-fixation

genes in B. japonicum (Bauer et al., 1998). Among the novel RegR target genes, we identified a putative operon (blr1515–blr1516) that encodes a predicted multidrug efflux system. Here, we report the characterization of a mutant lacking Apoptosis Compound high throughput screening this predicted transport system, now designated BdeAB, and demonstrate that it confers antibiotic resistance and is required for an efficient symbiosis specifically with soybean. Bradyrhizobium japonicum strains were routinely cultivated in a peptone–salts–yeast extract (PSY) medium supplemented with 1 g l−1l-arabinose as described elsewhere (Regensburger & Hennecke, 1983; Mesa et al., 2008). Alternatively, we used a modified Vincent’s minimal medium (Vincent, 1970; find protocol Becker et al., 2004) that was supplemented with 3 g l−1l-arabinose, 10 mM MOPS (final pH of medium adjusted to 6.8 with 2 M NH3), and trace elements as described elsewhere (Bishop et al., 1976). When appropriate, antibiotics were used at the following concentrations (μg ml−1): spectinomycin, 100; streptomycin, 50; tetracycline, 50 (solid media) or 25 (liquid media); and cycloheximide, 100. Bradyrhizobium japonicum strain 110spc4 was used as the wild type (Regensburger & Hennecke, 1983). Mutant

derivatives relevant for this work were strain 2426 (ΔregR∷Ω; Bauer et al., 1998); strain 9589 (ΔbdeAB∷Ω; see below); and strain 9589-38 (strain 9589 complemented with chromosomally inserted wild-type bdeAB genes; see below). Escherichia coli strains were grown in Luria–Bertani medium (Miller, 1972) containing the following concentrations of antibiotics for plasmid selection (μg ml−1): ampicillin, 200; streptomycin, 50; and tetracycline, 10. Strain DH5α (Bethesda Research Laboratories, Gaithersburg, MD) was the host for cloning, and S17-1 (Simon

et al., 1983) for the conjugation of plasmids into B. japonicum. Sterilization of seeds of soybean [Glycine max (L.) Merr. cv. Williams], cowpea (Vigna unguiculata), siratro (Macroptilium atropurpureum), and mungbean (Vigna radiata), plant growth conditions, and measurement of nitrogenase activity were performed as described previously (Göttfert et al., 1990; Gourion O-methylated flavonoid et al., 2009; Koch et al., 2010). At least 107 cells of B. japonicum were added as inoculum. For bacteroid isolation, all nodules from individual soybean plants infected by either the wild type or the ΔbdeAB strain 9589 were collected and weighed. Nodule material was then crushed in PSY medium, and serial dilutions of the bacteroid suspension were spotted in four parallels on PSY agar plates containing spectinomycin and cycloheximide. After a 1-week incubation at 30 °C, the number of CFU per milligram of nodule wet weight was determined.