Sinorhizobium meliloti RNase III: Catalytic Features and Impact on Symbiosis

Abstract

Members of the ribonuclease (RNase) III family of enzymes are metal-dependent double-strand specific endoribonucleases. They are ubiquitously found and eukaryotic RNase III-like enzymes include Dicer and Drosha, involved in RNA processing and RNA interference. In this work, we have addressed the primary characterization of RNase III from the symbiotic nitrogen-fixing α-proteobacterium Sinorhizobium meliloti. The S. meliloti rnc gene does encode an RNase III-like protein (SmRNase III), with recognizable catalytic and double-stranded RNA (dsRNA)-binding domains that clusters in a branch with its α–proteobacterial counterparts. Purified SmRNase III dimerizes, is active at neutral to alkaline pH and behaves as a strict metal cofactor-dependent double-strand endoribonuclease, with catalytic features distinguishable from those of the prototypical member of the family, the Escherichia coli ortholog (EcRNase III). SmRNase III prefers Mn2+ rather than Mg2+ as metal cofactor, cleaves the generic structured R1.1 substrate at a site atypical for RNase III cleavage, and requires higher cofactor concentrations and longer dsRNA substrates than EcRNase III for optimal activity. Furthermore, the ultraconserved E125 amino acid was shown to play a major role in the metal-dependent catalysis of SmRNase III. SmRNase III degrades endogenous RNA substrates of diverse biogenesis with different efficiency, and is involved in the maturation of the 23S rRNA. SmRNase III loss-of-function neither compromises viability nor alters morphology of S. meliloti cells, but influences growth, nodulation kinetics, the onset of nitrogen fixation and the overall symbiotic efficiency of this bacterium on the roots of its legume host, alfalfa, which ultimately affects plant growth. Our results support an impact of SmRNase III on nodulation and symbiotic nitrogen fixation in plants.

Ribonucleases control distinct traits of Pseudomonas putida lifestyle

“…These observations are consistent with what was already observed for other bacteria namely, E . coli , Salmonella and Sinorhizobium meliloti where disruption of the rnc gene caused a slow growth‐phenotype (Studier, 1975; Takiff et al ., 1989; Babitzke et al ., 1993; Nicholson, 1999; Viegas et al ., 2007; Saramago et al ., 2018). This slow‐growth phenotype has been pointed out as a possible result of a reduction in the kinetics of rRNA processing, leading to less functional ribosomes and protein synthesis (Babitzke et al ., 1993; Mattatall and Sanderson, 1998; Amblar et al ., 2004).…”

Transcription attenuation-derived small RNA rnTrpL regulates tryptophan biosynthesis gene expression in trans

“…To address the role of ribonucleases in the rnTrpL-mediated decrease of trpDC mRNA levels, the lacZ’-rnTrpL production was induced for 10 min in the RNase mutants 2011 rne (27,62) and 2011 Δrnc (63) using plasmid pSRKTc-rnTrpL, and changes in the levels of trpC mRNA were analyzed. In contrast to the parental strain 2011, in which trpC was confirmed to be decreased, the trpC levels were increased in both mutants (Figure 3E).…”

Tailor-made sRNAs: a toolbox to control metabolic targets

“…MicC scaffold was generated and 32 P-labelled by in vitro transcription from a synthetic DNA template (MicC_T7, Table S2) with a commercial T7 Promoter similarly to (Milligan et al 1987, Saramago et al 2018). Riboprobe synthesis and oligoprobe labelling were performed as described (Viegas et al 2007).…”