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

Abstract

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. In Sinorhizobium meliloti, over 400 sRNAs are expressed under different stimuli. The sRNA MmgR (standing for Makes more granules Regulator) 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 in S. meliloti 2011 by characterizing the effect of a deletion of the internal conserved core of mmgR (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-type mmgR copy but not with unrelated sRNA genes. Furthermore, the expression of mmgR 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 in S. meliloti. IMPORTANCE High-throughput RNA sequencing has recently uncovered an overwhelming number of trans-encoded small RNAs (sRNAs) in diverse prokaryotes. In the nitrogen-fixing alphaproteobacterial symbiont of alfalfa root nodules Sinorhizobium 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 gene mmgR in S. meliloti. Our results strongly suggest that mmgR encodes a negative regulator of the accumulation of polyhydroxybutyrate, the major carbon and reducing power storage polymer in S. meliloti cells growing under conditions of C/N overbalance.

Riboregulation in Nitrogen-Fixing Endosymbiotic Bacteria

“…As revealed by the loss-of-function phenotype, MmgR preserves synthesis of both PHB-related phasin proteins (PhaP1 and PhaP2) under conditions of C surplus, thereby limiting intracellular accumulation of PHB granules. Together, these data suggest that mmgR expression depends on cellular N and C status [72,73]. However, lack of the PHB synthase coding gene phbC has different effect depending on rhizobial species.…”

“…However, in most cases expression in nodules concurs with stress-induced accumulation of the sRNA in free-living rhizobia. This is the case of some of the functionally characterized S. meliloti trans-sRNAs described above, e.g., AbcR1, MmgR, EcpR1 or GspR1, whose loss-of-function has none or modest impact in symbiosis [62,72,87,89].…”

A conserved α‐proteobacterial small RNA contributes to osmoadaptation and symbiotic efficiency of rhizobia on legume roots

“…It is known that the S. meliloti reference strain Rm1021 and its close relative Rm2011 express hundreds of sRNAs of the trans-acting class (Schl€ uter et al, 2010;Sallet et al, 2013;Schl€ uter et al, 2013). However, only five have been functionally characterized to date namely, EcpR1, RcsR1, MmgR, and the homologous AbcR1 and AbcR2 sRNAs (Torres-Quesada et al, 2013;Baumgardt et al, 2015;Robledo et al, 2015;Borella et al, 2016;Lagares et al, 2017). These sRNAs are widely conserved in related a-proteobacteria and, with the exception of AbcR1, they accumulate in the cell under stress conditions.…”

“…EcpR1 and RcsR1 target cell cycle and quorum sensing related mRNAs, respectively, in an Hfq-independent manner (Baumgardt et al, 2015;Robledo et al, 2015). AbcR1/AbcR2 and MmgR are all Hfq-dependent sRNAs functionally related to nutrient uptake and carbon metabolism, respectively (Torres-Quesada et al, 2013;Borella et al, 2016;Lagares et al, 2017). However, none of these riboregulators is required for the establishment of an efficient symbiosis.…”

A Genome-Wide Prediction and Identification of Intergenic Small RNAs by Comparative Analysis in Mesorhizobium huakuii 7653R

“…MmgR is another trans-encoded small RNA, and highly conserved among the a-proteobacteria. MmgR regulates the cellular Polyhydroxybutyrate accumulation and is controlled by the cellular nitrogen status in S. meliloti (Ceizel Borella et al, 2016 ; Lagares et al, 2016 , 2017 ). In cyanobacteria, a nitrogen stress-induced RNA (NsiR4) was shown to be involved in the regulation of glutamine synthetase, a key enzyme required for biological nitrogen assimilation (Klahn et al, 2015 ).…”