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THE MRNAS ENCODING POLYHYDROXYBUTYRATE (PHB)-GRANULE ASSOCIATED PROTEINS PHAP1/2 ARE ASSOCIATED IN VIVO WITH THEIR POST-TRANSCRIPTIONAL SMALL RNA REGULATOR MMGR IN SINORHIZOBIUM MELILOTI

SMALL RNA REGULATOR MMGR IN SINORHIZOBIUM MELILOTI LAGARES (JR.), Antonio 1 | LINNE, Uwe2 | BECKER, Anke3 | VALVERDE, Claudio4

LBMIBS, UNIVERSIDAD NACIONAL DE QUILMES - CONICET / SYNMIKRO, PHILLIPS-UNIVERSITY, MARBURG 1; CHEMISTRY DEPARTMENT – MASS SPECTROMETRY, PHILLIPS-UNIVERSITY, MARBURG 2; SYNMIKRO, PHILIPPS-UNIVERSITY, MARBURG 3; LBMIBS, UNIVERSIDAD NACIONAL DE QUILMES - CONICET 4

Introduction and objectives: The highly conserved mmgR gene encodes a 77-nt non-coding small RNA (sRNA) in the N2-fixing legume symbiont Sinorhizobium meliloti. In S. meliloti, the expression of MmgR is mainly regulated at the transcriptional level by the N and C metabolism master regulators NtrC and AniA, respectively. MmgR regulates the accumulation of the major C- and reducing-power-storage polymer polyhydroxybutyrate (PHB) under conditions of N starvation and C surplus. The absence of MmgR in the S. meliloti mmgR mutant strain results in the relaxation of a post-transcriptional control over both PHB-granule associated phasins PhaP1/2. Our current model for MmgR function suggests that the sRNA is part of a regulatory loop that operates to maintain a proper structure and amount of PHB granules in S. meliloti through a fine-tuning of the intracellular levels of phasins and polymer, on the basis of the availability of N and C. Since the molecular interactions between MmgR and its specific target molecules –which ultimately determine the biological function of the sRNA- were still unknown, we planned to characterize the interactome of MmgR. Materials and methods: In order to identify the RNAs and proteins to which MmgR binds in vivo, we expressed an MS2-tagged MmgR species in the S. meliloti mmgR mutant strain as a bait to selectively purify the interactome of the small RNA by means of an affinity chromatography of the bacterial lysate in an amylosefilled column loaded with the MS2bp-MBP protein (i.e., MS2-binding protein fused to the mannose binding protein), followed by several wash steps and a final elution of the selectively retained molecules using a mannose-containing buffer. The population of RNAs and proteins extracted from the chromatographic eluates were subjected to RNA-sequencing and quantitative proteomics in order to determine MmgR RNA and protein binding partners, respectively. Results: A small set of RNAs that interact in vivo with MmgR was identified. Among them, the transcripts corresponding to the genes SMc00777 and SMc02111 (phaP1 and phaP2, encoding for PHB-granule-associated phasins 1 and 2, respectively) were of particular relevance since their finding strongly suggests that a direct MmgR-mediated regulation operates over them to impact on PHB storage metabolism. Whether base-paring between MmgR and these mRNAs accounts for the observed regulation is currently being addressed. Furthermore, Hfq (the chaperone of sRNA-mRNA interactions) was confirmed as a member of MmgR interactome. Conclusions: We uncovered the interactome of the small RNA MmgR in S. meliloti by means of the MS2-sRNA tagging strategy coupled to affinity purification and RNAseq/qProteomics, an approach that has been set up and successfully used in enterobacterial models. Remarkably, the mRNAs encoding PHB-granule associated proteins were found to be associated in vivo with their post-transcriptional sRNA regulator MmgR, which points to a direct post-transcriptional regulation of PhaP1/2 expression by MmgR.