Fig 5.
Free-living ΔcbrA succinoglycan overproduction and membrane instability phenotypes are suppressed by divL and cckA alleles. (A, left and middle panels) Succinoglycan production was assessed with calcofluor-supplemented media at 30°C using a serial-dilution spot assay and visualized by UV fluorescence. Wild type is calcofluor-dim, and ΔcbrA is calcofluor-bright. ΔcbrA double mutants display a decrease in calcofluor-fluorescence relative to the ΔcbrA parent strain to varying degrees with ΔcbrA divLL454I having an intermediate phenotype. (A, right pane) Each divL and cckA single mutant displays a calcofluor-dim phenotype indistinguishable from wild type. (B, left and middle panels) Membrane stability was assessed with 10 mM DOC-supplemented media at 30°C using a serial-dilution spot assay. As a control, a replicate succinoglycan production assay with the same samples was performed (A), and no growth defects are observed in the absence of DOC. Wild type is DOC-resistant, and ΔcbrA is DOC-sensitive as seen by its inability to grow. Each ΔcbrA double mutant has increased DOC-resistance phenotype with the exception again of divLL454I, which displays an intermediate phenotype. (B, right panel) Each divL and cckA single mutant displays a DOC-resistance phenotype like wild type except for divLQ362P and divLV787M, which exhibit a severe sensitivity to DOC nearly indistinguishable from ΔcbrA. (C) Growth was assessed at 40°C to determine whether any mutants exhibit a temperature-sensitive phenotype using a serial-dilution spot assay. As a control, a replicate succinoglycan production assay with the same samples was performed at 30°C (A), and no growth defects are observed at the standard temperature for S. meliloti. ΔcbrA grows nearly as well as wild type at 40°C, as do most ΔcbrA double mutants. Two exceptions are divLV787M and to a lesser extent divLF142S. (C, right panel) Most divL and cckA single mutants grow nearly as well as wild type. Two exceptions are divLV787M and divLF142S, which have a more severe temperature-sensitive phenotype in the presence of CbrA. In addition, cckAA373S displays a mild temperature-sensitive growth defect in an otherwise wild-type background.