Construction and symbiosis assays of mutants in conserved genes Thirteen of the 139 conserved ORFs were chosen for further study because they are of undetermined function in S. meliloti and have no close homologs in the S. meliloti genome that might be expected to provide redundant function. Six of the longer ORFs, including SMc00911, were disrupted by cloning a small internal ORF fragment into the plasmid pJH104,
conjugating the plasmid LCZ696 order into S. meliloti 1021, and selecting for single-crossover insertion/disruption mutants. ( Additional file 2: Table S2 lists primer sequences and disruption fragment sizes and positions.) For the 6 remaining ORFs, 3 that are under 750 bp long (SMc01562, SMc01986 and SMc00135) and 3 that are all in a single operon (SMc01424, SMc01423, and SMc01422), deletion was judged to be a better strategy SCH772984 than disruption. SMc01424, SMc01423, and SMc01422 were all deleted as a single segment from the start codon of SMc01424 to the stop codon of SMc01422. The endpoints of the individual deletions
of SMc01562, SMc01986, and SMc00135 were dictated by the position of the most suitable PCR primers. ( Additional file 2: Table S2 lists primer sequences and deletion sizes and positions.) Either the disruption or the deletion strategy is expected to result in a strain that does not produce a full-length version of the protein encoded by that ORF. These ORFs and the insertion and/or deletion mutant strains of each are listed and described in Table 2. The resulting mutant strains were then tested for symbiotic proficiency on the host plant alfalfa. For the initial phenotypic Epacadostat nmr analysis, the ability of the mutants to successfully provide the plants with fixed nitrogen was determined. Alfalfa plants were inoculated with the bacterial mutants and after 5 weeks of growth, the shoot length attained on nitrogen-free medium was compared with plants inoculated with the S. meliloti 1021 wild type as the positive control and uninoculated plants as the negative control. Figure 1 shows the shoot length of
alfalfa plants inoculated with wild type S. meliloti 1021 or with disruption mutant strains of the ORFs SMb20360, SMb20431, SMc00911, SMa1344, SMc01266, and SMc03964. Alfalfa plants inoculated with these strains attain a similar average shoot length as that of the wild Liothyronine Sodium type, demonstrating that all of these strains are able to form a successful symbiosis with this host plant. Figure 2 presents the same type of assay as Figure 1 for deletion mutants in the ORFs SMc01562, SMc01986, SMc01424-22, SMc00135, and SMa0044. Additional data on the plant assays in Figures 1 and 2 is presented in Table 5. The number of plants inoculated with each strain, the average number of mature, pink nodules per plant and the average number of white pseudonodules per plant are shown. All of these mutant strains are able to mount a successful symbiosis with the host plant alfalfa.