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

Riboregulation has a major role in the fine-tuning of multiple bacterial processes. Among the RNA players,trans -encoded untranslated small RNAs (sRNAs) regulate complex metabolic networks by tuning expression from multiple target genes in response to numerous signals. InSinorhizobium meliloti , over 400 sRNAs are expressed under different stimuli. The sRNA MmgR (standing forM akesm oreg ranulesR egulator) has been of particular interest to us since its sequence and structure are highly conserved among the alphaproteobacteria and its expression is regulated by the amount and quality of the bacterium's available nitrogen source. In this work, we explored the biological role of MmgR inS. meliloti 2011 by characterizing the effect of a deletion of the internal conserved core ofmmgR (mmgR Δ33–51 ). This mutation resulted in larger amounts of polyhydroxybutyrate (PHB) distributed into more intracellular granules than are found in the wild-type strain. This phenotype was expressed upon cessation of balanced growth owing to nitrogen depletion in the presence of surplus carbon (i.e., at a carbon/nitrogen molar ratio greater than 10). The normal PHB accumulation was complemented with a wild-typemmgR copy but not with unrelated sRNA genes. Furthermore, the expression ofmmgR limited PHB accumulation in the wild type, regardless of the magnitude of the C surplus. Quantitative proteomic profiling and quantitative reverse transcription-PCR (qRT-PCR) revealed that the absence of MmgR results in a posttranscriptional overexpression of both PHB phasin proteins (PhaP1 and PhaP2). Together, our results indicate that the widely conserved alphaproteobacterial MmgR sRNA fine-tunes the regulation of PHB storage inS. meliloti .IMPORTANCE High-throughput RNA sequencing has recently uncovered an overwhelming number oftrans -encoded small RNAs (sRNAs) in diverse prokaryotes. In the nitrogen-fixing alphaproteobacterial symbiont of alfalfa root nodulesSinorhizobium meliloti , only four out of hundreds of identified sRNA genes have been functionally characterized. Thus, uncovering the biological role of sRNAs currently represents a major issue and one that is particularly challenging because of the usually subtle quantitative regulation contributed by most characterized sRNAs. Here, we have characterized the function of the broadly conserved alphaproteobacterial sRNA genemmgR inS. meliloti . Our results strongly suggest thatmmgR encodes a negative regulator of the accumulation of polyhydroxybutyrate, the major carbon and reducing power storage polymer inS. meliloti cells growing under conditions of C/N overbalance.