A Sporulation-Specific sRNA Bvs196 Contributing to the Developing Spore in Bacillus velezensis

Many putative sRNAs have been characterized using bioinformatic analysis and high-throughput sequencing in Gram-positive Bacillus strains, but there are only a few functional studies on the sRNAs involved in the spore formation developmental process. In particular, there is no sRNA confirmed experimentally to regulate the late stages of sporulation. Bvs196 is an sRNA with a length of 294 nucleotides that is abundantly expressed in the stationary phase of several media and independently transcribed in Bacillus velezensis strain PEBA20, as validated by RNA-seq and Northern blot,. It is also confirmed, by qRT-PCR, that Bvs196 is transcribed abundantly throughout the intermediate and late stages of sporulation. Using the gfpmut3a gene transcriptional reporter demonstrates that Bvs196 is expressed specifically in the forespore during sporulation and controlled by σ^F and σ^G (mainly by σ^G). This was observed by fluorescence microscopy and multi-function microplate reader. Further evolutionary conservation analysis found that Bvs196 is widely present in Bacillus with a strongly conserved and stable secondary structure. Resistance phenotypic assays of spores formed from the Bvs196 deletion mutant, the overexpressed Bvs196 mutant, and the wild-type strain revealed that the absence of Bvs196 led to reduced heat and UV resistance and enhanced formaldehyde resistance. We determined, by MST analysis, that Bvs196 can directly interact with spo0A and sspN-tlp mRNAs in vitro, and that short incomplete complementary paired bases affect the binding affinity of Bvs196 to target mRNAs. Our results suggest that Bvs196 is a novel sporulation-specific sRNA of B. velezensis, 294 nt in length, independently transcribed under the control of σ^F and σ^G in the forespore during sporulation, and that it affects spore resistance, and is able to directly interact with spo0A and sspN-tlp mRNAs. The remarkable conservation and impressive expression level of Bvs196 imply that it acts as an important conservative regulator, presumably by interacting with many other unknown targets in the forespore, and therefore contributing to spore properties. This work provides new clues for further understanding of the spore formation regulatory network.