For statistical analysis, the spss statistical package, version 16.0, was used. Deletion mutagenesis of blr1515 and blr1516 was performed by marker exchange as indicated in Fig. 1. The 5′ and 3′ flanking
regions of bdeA were PCR amplified using appropriate primers and subcloned in the pBluescript SK(+) vector (Stratagene, Afatinib supplier La Jolla, CA). Sequences of all primers used in this work are available from the authors upon request. A streptomycin-resistance cassette (Ω) excised from pBSL15Ω was inserted in between the two B. japonicum DNA fragments, yielding plasmid pRJ9587. pBSL15Ω had been constructed by cloning of the Ω cassette from pHP45Ω (Prentki & Krisch, 1984) on a 2.1-kb EcoRI fragment into the 2.6-kb backbone of EcoRI-digested plasmid pBSL15 (Alexeyev, 1995). The insert of pRJ9587 was cloned into the vector pSUP202pol6K (Zufferey et al., 1996), and the resulting plasmid pRJ9589 was then mobilized by conjugation from E. coli S17-1 (Simon et al., 1983) into B. japonicum strain 110spc4 (wild type) for marker replacement, yielding mutant strain 9589. A 6.1-kb B. japonicum genome fragment
containing the bdeAB genes including the flanking regions was PCR amplified with appropriate primers using the Phusion™ DNA Polymerase (Finnzymes, BioConcept, Allschwil, Switzerland). The PCR fragment was digested using intrinsic restriction sites for PstI and EcoRI (Fig. 1), subcloned for verification by sequencing, and eventually cloned into pSUP202pol6K. The resulting plasmid pRJ9638 was mobilized by conjugation into the B. japonicum mutant strain 9589, yielding strain 9589-38. Candidates this website with a single-recombination event were picked, and the correct integration of pRJ9638 upstream of the Ω cassette present in strain 9589 was verified by Southern blot analysis of genomic DNA. Potential growth-inhibitory compounds and antimicrobial peptides were purchased from Sigma Aldrich Co. (Buchs, Switzerland) and screened first for growth inhibition using the gradient agar plate technique (Szybalski
& Bryson, 1952). For agar plate diffusion assays, 15 ml of 0.9% PSY agar was warmed to 42 °C, inoculated with bacterial cell suspensions to 5 × 106 CFU ml−1 and quickly Mannose-binding protein-associated serine protease poured into round Petri dishes. Holes were punched into each plate using the end of a Pasteur glass pipette, and 15 μL of the test compound was pipetted into each hole. After 2 days of incubation at 30 °C, test plates were monitored for zones of growth inhibition on the bacterial lawn. The assays were repeated at least three times. Based on annotations compiled in the B. japonicum genome sequence database (http://bacteria.kazusa.or.jp/rhizobase/), genes blr1515 and blr1516 code for components of a multidrug efflux system, and their genomic organization (Fig. 1) suggests that they form an operon. Gene blr1515 is predicted to encode a protein of 397 amino acids (aa) with sequence similarity to Pseudomonas aeruginosa MexC (43%) and E. coli AcrA (40%) (Poole et al., 1993; Ma et al., 1995), for example.