, 2006). In this strain, 44% of the total phospholipids corresponded to phosphatidylcholine, followed by phosphatidylethanolamine, phosphatidylglycerol and cardiolipin, and it was suggested that phosphatidylcholine may be involved in the bacterial response to environmental conditions (Medeot et al., 2007).

The present work was designed to identify the genes involved in phosphatidylcholine biosynthesis as well as to isolate and mutate the homologous pmtA this website gene in SEMIA 6144 in order to elucidate the role of phosphatidylcholine in free-living bacteria and during symbiosis with peanut plants. Table 1 lists the bacterial strains and plasmids used in this work. Escherichia coli was grown on Luria–Bertani medium (Miller, 1972) at 37 °C. Sinorhizobium meliloti 1021 was grown on tryptone yeast medium (Beringer, 1974). Bradyrhizobium japonicum USDA 110spc4 and SEMIA 6144 were grown on B− medium (van Brussel et al., 1977) or on yeast extract mannitol

(YEM) medium (Somasegaran & Hoben, 1994), all at 28 °C. Antibiotics were added at the following concentrations (μg mL−1) Tyrosine Kinase Inhibitor Library research buy when required: carbenicillin 100, tetracycline 10, spectinomycin 200, kanamycin 50, gentamicin 10 for E. coli, and nalidixic acid 40, spectinomycin 100 and gentamicin 40 for SEMIA 6144. Plasmids pBBR1MCS-5, pK18mobsacB and their derivatives were mobilized from E. coli S17-1 into the receptor strain SEMIA 6144 (Simon et al., 1983). To Metformin supplier obtain cell-free protein crude extracts, cells were ruptured using a French press as described previously (Martínez-Morales et al., 2003). Pmt activity was determined according to de Rudder et al. (1997). To detect minor Pcs activities, the assay

developed originally for S. meliloti (Sohlenkamp et al., 2000) was performed according to the modifications described by Martínez-Morales et al. (2003). DNA manipulations were performed according to standard procedures (Sambrook & Russell, 2001). DNA and derived protein sequences were analysed using the NCBI blast network server (Altschul et al., 1997). Probes for pmt or pcs genes were obtained from plasmids indicated in Table 1. DNA probes were labelled with the DIG-DNA labelling kit (Roche Molecular Biochemicals, Germany). Chemiluminescent detection of the DIG label was performed using CSPD (Roche Molecular Biochemicals). To obtain a DNA fragment containing the pmtA gene, a size-selected genomic library of SEMIA 6144 was constructed. SEMIA 6144 genomic DNA was digested with HindIII, and DNA fragments with sizes of around 2.5 kb were cloned into pUC18. Clones carrying the gene of interest were selected by colony hybridization using pmtA of B. japonicum USDA 110 (Minder et al., 2001) as a probe. A positive clone (pDBM01) was selected and its insert was sequenced.