Accumulation of higher PHB levels in the mmgRΔ33–51 strain than in the wild-type strain during the stationary phase of growth under C/N overbalance in RDM. (a) Cellular dry weight and cellular protein, glycogen, and PHB contents of wild-type and mmgRΔ33–51 cells at the stationary phase of growth. The results are the average of the data from three independent cultures ± SD. The asterisk indicates that the average value for the mmgRΔ33–51 strain differs significantly from that for the wild-type strain at a P value of <0.05. (b) Cellular PHB contents (arbitrary fluorescence units [AFU]) of the wild-type, mmgRΔ33–51, and mmgRΔ33–51 p-sRNA complemented strains at the stationary phase of growth, as determined by flow cytometric analysis with Nile red as a PHB fluorescent stain. Induction with IPTG of the complemented strains was carried out with cultures having an OD600 of 1.6. The results are the average of the data from three independent cultures ± SD. The values were analyzed statistically by Tukey's multiple-comparison test. Different letters over the bars indicate that the average values differ significantly at a P value of <0.05. The experiment was repeated twice with essentially the same results. (c) Northern blot analysis confirming that the MmgR level in the mmgRΔ33–51 p-MmgR strain is restored to nearly wild-type levels in the stationary growth phase (after 90 h of growth) after IPTG addition at an OD600 of 1.6 (after ca. 30 h of growth). The relative abundance of MmgR (normalized to the signal for the 5S rRNA) in the mmgRΔ33–51 p-MmgR strain relative to that in the wild-type strain is indicated over the image. (d) Frequency distribution of the intracellular PHB contents of S. meliloti 2011 (solid line) and mmgRΔ33–51 (dashed line) cells at the stationary growth phase in RDM (C/N ratio, 30:1). Stationary S. meliloti 2011 cells in RDM with a balanced C/N ratio (10:1) were used as a negative control for PHB accumulation (shaded histogram). The effect of induction of plasmid-borne MmgR, MmgRΔ33–51, asRNA812, and Sm84 over the frequency of intracellular PHB content in an mmgRΔ33–51 background is shown in Fig. S2 in the supplemental material.