Regulation of Polyhydroxybutyrate Accumulation in Sinorhizobium meliloti by the Trans-Encoded Small RNA MmgR

Differential growth yield (OD₆₀₀) between S. meliloti 2011 wild-type and mmgR^Δ33–51 mutant strains at the stationary phase of growth in RDM. (a) ○, S. meliloti 2011 wild-type strain; △, S. meliloti 2011 mmgR^Δ33–51 strain. Curves with solid or dashed lines represent the culture optical density at 600 nm (OD₆₀₀) or viable cell count kinetics, respectively. Each curve represents the average from three independent cultures ± SD. The experiment was repeated three times with essentially the same results. (b) OD₆₀₀ kinetics of cultures in which IPTG was added at an OD₆₀₀ of 1.6 (right panel) and of noninduced cultures (left panel). The arrow indicates the time point at which IPTG was added to the cultures. 2011, wild-type strain. Each curve represents the average from three independent cultures ± SD. The error bars corresponding to SD values lower than 0.1 OD₆₀₀ unit might not be visible in the plot. The experiment was repeated twice with essentially the same results.

Accumulation of higher PHB levels in the mmgR^Δ33–51 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 mmgR^Δ33–51 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 mmgR^Δ33–51 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, mmgR^Δ33–51, and mmgR^Δ33–51 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 mmgR^Δ33–51 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 mmgR^Δ33–51 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 mmgR^Δ33–51 (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, MmgR^Δ33–51, asRNA812, and Sm84 over the frequency of intracellular PHB content in an mmgR^Δ33–51 background is shown in Fig. S2 in the supplemental material.

Limitation of PHB accumulation under conditions of N starvation and surplus C set by the expression of MmgR in S. meliloti. (a) ○, S. meliloti 2011 wild-type strain; △, S. meliloti 2011 mmgR^Δ33–51 strain. Curves with solid lines or dashed lines represent the OD₆₀₀ kinetics of cultures performed in RDM with a 6× (C/N ratio, 60:1) or a 3× (C/N ratio, 30:1) overbalanced C/N ratio, respectively. Each curve represents the average from three independent cultures ± SD. (b) Cellular PHB content (arbitrary fluorescence units [AFU]) of bacteria in the exponential and stationary growth phases under balanced (10:1) or 3× (30:1) and 6× (60:1) overbalanced C/N ratios. PHB was determined by flow cytometry with Nile red as the PHB fluorescent stain. 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 experiments shown for panels a and b were repeated twice with essentially the same results.

Morphological changes accompanying differential intracellular PHB accumulation. Transmission electron microscopy of wild-type and mmgR^Δ33–51 bacteria demonstrated that under conditions of C/N overbalance, stationary-phase mmgR^Δ33–51 cells are longer and accumulate a higher number of irregularly shaped PHB granules than the wild-type cells do. The control image corresponds to wild-type bacteria during the exponential phase of growth in RDM, when almost no PHB would be expected to be synthesized. The scale bars indicate a length of 0.25 μm.

Altered PhaP2 expression in mmgR^Δ33–51 mutant cells. SDS-PAGE-based comparative proteomic profiling of the S. meliloti 2011 wild-type and mmgR^Δ33–51 strains during the stationary phase of growth in RDM is shown. PhaP2 was identified by mass spectrometry (Orbitrap) (see Materials and Methods) as the major protein component of the material extracted from the band indicated with an arrow. The image shows one representative electrophoretic profile among three repetitions with comparable results. The molecular masses of the reference bands are given on the left side of the image.