Two dual-function regulatory sRNAs in Bacillus subtilis, and their role in RNA degradation and sporulation

This study was conducted to identify a new function of the trans-encoded sRNA SR1 and to characterize a small protein encoded by the new potential dual-function sRNA SR7 in the Gram-positive model organism B. subtilis. The small RNA SR1 was found to inhibit translation of kinA mRNA. Neither the SR1 encoded peptide SR1P plays any role in the regulation of kinA nor SR1 influences the stability of kinA mRNA. The role of SR1 in sporulation was confirmed by analysing its effects on the promoters of downstream genes controlled by KinA through Spo0A. No role of Hfq or CsrA in sporulation was observed. SR1 was found to decelerate sporulation when glucose is exhausted. Moreover, SR1 impacted spore properties. Spores formed in the absence of SR1 exhibited lower resistance to stress conditions and displayed an altered composition of spore coat proteins. The sr7 gene was tagged in its native locus with a C-terminal 3x FLAG tag and the synthesis of the small protein SR7P confirmed in Western blots. Threefold increased amounts of SR7P were produced under different stress conditions. In co-elution experiments the glycolytic enzyme enolase was identified as interaction partner for SR7P. The binding of SR7P to enolase in turn improved the binding of enolase to RNase Y present in the DLN of B. subtilis. By contrast, it had no effect on the interaction of enolase with PfkA also present in the DLN. Enolase carries ten times more RNase Y in the presence of SR7P. RNA is not bridging the interaction between SR7P and enolase. SR7P does not directly bind RNase Y. In vitro RNase Y activity assays revealed a role of SR7P in modulation of the activity of Eno-bound RNase Y. The role of SR7P in modulation of the activity of Eno-bound RNase Y was confirmed in vivo for rpsO mRNA. Taken together, both small RNAs SR1 and SR7 are involved in the regulation of sporulation and modulation of DLN in B. subtilis under glucose limiting and stress conditions.