The stress-related, rhizobial small RNA RcsR1 destabilizes the autoinducer synthase encoding mRNAsinIinSinorhizobium meliloti

Abstract

Quorum sensing is a cell density-dependent communication system of bacteria relying on autoinducer molecules. During the analysis of the post-transcriptional regulation of quorum sensing in the nitrogen fixing plant symbiont Sinorhizobium meliloti, we predicted and verified a direct interaction between the 5'-UTR of sinI mRNA encoding the autoinducer synthase and a small RNA (sRNA), which we named RcsR1. In vitro, RcsR1 prevented cleavage in the 5'-UTR of sinI by RNase E and impaired sinI translation. In line with low ribosomal occupancy and transcript destabilization upon binding of RcsR1 to sinI, overproduction of RcsR1 in S. meliloti resulted in lower level and shorter half-life of sinI mRNA, and in decreased autoinducer amount. Although RcsR1 can influence quorum sensing via sinI, its level did not vary at different cell densities, but decreased under salt stress and increased at low temperature. We found that RcsR1 and its stress-related expression pattern, but not the interaction with sinI homologs, are conserved in Sinorhizobium, Rhizobium and Agrobacterium. Consistently, overproduction of RcsR1 in S. meliloti and Agrobacterium tumefaciens inhibited growth at high salinity. We identified conserved targets of RcsR1 and showed that most conserved interactions and the effect on growth under salt stress are mediated by the first stem-loop of RcsR1, while its central part is responsible for the species-specific interaction with sinI. We conclude that RcsR1 is an ancient, stress-related riboregulator in rhizobia and propose that it links stress responses to quorum sensing in S. meliloti.

RNase E and RNase J are needed for S-adenosylmethionine homeostasis in Sinorhizobium meliloti

“…Growth of the pWBexpR-containing strains in the absence of IPTG led to a highly mucoid phenotype on plates (not shown), suggesting that leaky expression of expR is sufficient for functional QS [43]. This was confirmed by the similar AHL accumulation during the growth of strains 2011(pWBexpR) and Sm2B3001, the latter with a restored expR on the chromosome (data not shown; for AHL accumulation during growth of strains 2011 and Sm2B3001 see [21]). A comparison of the AHL levels of the pWBexpR-containing strains (2011, RNase mutants and mutants containing pRKrne or pRKrnj) revealed differences like those observed in the absence of pWBexpR, with the exception of strain 2011rnj (pRKrnj) at an OD 600 of 1.3 (compare Fig 1b to Fig.…”

“…The similarities between the two RNase mutants prompted us to test whether RNase J is necessary for diminishing the AHL levels upon overexpression of the sRNA RcsR1 as previously described for RNase E [21]. The AHL levels were decreased in the rcsR1-overexpressing RNase J mutant and in rcsR1-overexpressing strain 2011, but not in the rcsR1-overexpressing RNase E mutant (Fig.…”

“…The two RNases do not directly influence each other's expression (Fig. S2) and RNase J was not among the proteins, which were recently co-purified with RNase E from S. meliloti [21], suggesting that they also do not act in a protein complex. Another argument against a protein complex formed by the two RNases is provided by Fig.…”

“…Additionally, the SinR homologue ExpR senses AHLs and transcriptionally regulates sinR, sinI and other genes depending on population density, which is reflected by the AHL concentration [43,44]. As mentioned above, RNase E and the sRNA RcsR1 are involved in sinI mRNA regulation [7,21]. Furthermore, posttranscriptional regulation of sinI and expR by the RNA chaperone Hfq has also been described [45][46][47].…”

“…RNase E seems to be essential in S. meliloti and the RNase E mutant most probably expresses a truncated RNase E that has defects in degradosome formation [7]. The S. meliloti degradosome probably plays a role in the posttranscriptional regulation of sinI mRNA, since full-length RNase E is needed for degradation of this mRNA upon overexpression of the small RNA (sRNA) RcsR1 [21].…”