, 2011). Collectively, these results indicate that although there is a general requirement for the VLCFA during infection, there are also species-specific differences in the role of the VLCFA during symbiosis. In R. leguminosarum bv. viciae, it has been shown that isolates of an acpXL mutant recovered from pea plant nodules [ex-nodule (EN) isolates] were restored in their tolerance to detergents, hyperosmotic and acid stress, despite the fact that their lipid A did not regain the VLCFA modification (Vedam et al., 2006). EN isolates of an acpXL mutant of R. leguminosarum bv. phaseoli were also resistant to detergent and hyperosmotic

stress (Brown et al., 2011); however, a mechanism has not been defined. Previously, we used a gusA transcriptional fusion to show that ropB is not expressed above background levels in Epacadostat cost a fabF2XL, F1XL mutant of R. leguminosarum bv. viciae 3841 (Foreman et al., 2010). RopB is an see more outer membrane protein found in the Rhizobiales that is important for outer membrane stability as demonstrated by the increased sensitivity of ropB mutants to detergent stress, hyperosmotic stress, and acidic pH (de Maagd et al., 1989; Foreman et al., 2010). Therefore, the lack of ropB expression may contribute to the membrane stress-related

phenotypes observed in the fabF2XL, fabF1XL mutant. The objective of this study was to use a genetic approach to further characterize the significance of ropB repression on the phenotypes of VLCFA-deficient mutants in free-living conditions and during symbiosis. Strains and plasmids used in the study are summarized in Table 1. Escherichia coli strains were cultured using Luria–Bertani medium (Sambrook et al., 1989), supplemented

as necessary with the following concentrations of antibiotics (μg mL−1): spectinomycin, 100; and tetracycline, 10. R. leguminosarum cells were cultured using tryptone yeast Interleukin-2 receptor (TY) (Beringer, 1974) or Vincent’s minimal medium with 10 mM mannitol (VMM) (Vincent, 1970), supplemented as required with the following concentrations of antibiotics (μg mL−1): kanamycin, 100; gentamicin, 30; neomycin, 100; tetracycline, 5; and streptomycin, 500. RNA was extracted using a modification of the method supplied with TRIzol® reagent (Invitrogen; Vanderlinde et al., 2011). RT reactions were carried out according to a previously described protocol (Manzon et al., 2007), with modifications described by Vanderlinde et al. (2009, 2011). PCRs were performed as described by Vanderlinde et al. (2009) with the following primer sets: AcpXLF2 (ACAAGGAATTCGGCATCAAG) and FabF2R2 (ACCGGATAGGGCTTGAACTT), AcpXLF4 (TTGCCGACATTATTGCAGAA) and AcpXLR4 (TTGAGCTCGTCGATCTTGG), and FD1 and RD1 (Weisburg et al., 1991). Detergent and hyperosmotic sensitivity assays were performed as described previously, (Gilbert et al., 2007; Vanderlinde et al., 2009). Acid stress sensitivity was determined by inoculating overnight cultures of R.