Single cross-over recombinants were selected on VMM with streptomycin and gentamicin. There was no statistically significant difference
in the gusA activity of the chromosomal fusions and the plasmid fusions. The enzyme assays for β-glucuronidase activity were carried out based on the β-galactosidase activity method of Miller (1972), with modifications described by Yost et al. (2004). To measure the gusA activity of the pEV65, pEV60, pEV58, and pDF4 fusions from bacteroids, 5–10 nodules were placed into 500 μL of sterile 0.25 M mannitol, 0.05 M Tris-HCl, pH 8.0, and crushed with a sterile inoculating stick. The nodule debris was allowed to settle for several minutes, and the supernatant was used in a standard GusA assay as described previously. It was recently shown in S. meliloti that the acpXL gene, located upstream of fabZXL, is part of an operon with fabZXL, fabF2XL, and fabF1XL, buy Roxadustat while the adh2XL and lpxXL genes comprise a second operon (Haag et al., 2011). We used RT-PCR to determine the operon structure of the acpXL gene in R. leguminosarum. Primers binding within the acpXL and
fabF2XL genes did not amplify a PCR product following RT of R. leguminosarum 3841 mRNA, indicating that these genes are not cotranscribed (Fig. 1). Primers binding within the acpXL gene confirmed expression of the acpXL gene, and primers binding to the 16S rRNA gene were used to confirm the quality of the mRNA and the success of the RT reactions. The GusA activity observed from the gusA transcriptional fusion with the upstream DNA from fabZXL (pEV60) further confirms the CP868596 presence of a promoter between the acpXL and fabZXL genes. The DNA sequences immediately upstream of the fabZXL gene from a number of different species
within the Rhizobiaceae were aligned to investigate the difference in operon structure between the rhizobial strains. While the acpXL and fabZXL gene sequences are ≥ 88% and ≥ 90% identical, Ixazomib molecular weight respectively, the intergenic region between acpXL and fabZXL is heterogeneous among the different species. There is also variability in the length of the intergenic sequence between the acpXL and fabZ genes. In R. leguminosarum bv. viciae 3841 and R. leguminosarum bv. trifolii WSM1325, the sequence is 205 and 204 bp, respectively. In S. meliloti, the sequence is 172 bp, and in Agrobacterium tumefaciens str C58, the intergenic sequence is further reduced to 90 bp. These differences in length of the intergenic region can be partially explained by a unique 72-bp insertion found in R. leguminosarum bv. viciae 3841 and R. leguminosarum bv. trifolii WSM1325 (Fig. 1). These results demonstrate that while the individual genes for biosynthesis of the VLCFA are homologous between different rhizobial species, the arrangement of those genes into operons has not been as stringently conserved.