Identification of sigma factor 54-regulated small non-coding RNAs by employing genome-wide and transcriptome-based methods in rhizobium strains

Rhizobium-legume symbiosis is considered as the major contributor of biological nitrogen fixation. Bacterial small non-coding RNAs are crucial regulators in several cellular adaptation processes that occur due to the changes in metabolism, physiology, or the external environment. Identifying and analysing the conditional specific/sigma factor-54 regulated sRNAs provides a better understanding of sRNA regulation/mechanism in symbiotic association. In the present study, we have identified sigma factor 54-regulated sRNAs from the genome of six rhizobium strains and from the RNA-seq data of free-living and symbiotic conditions of Bradyrhizobium diazoefficiens USDA 110 to identify the novel putative sRNAs that are over expressed during the regulation of nitrogen fixation. A total of 1351 sRNAs were predicted from the genome of six rhizobium strains and 1375 sRNAs were predicted from the transcriptome data of B. diazoefficiens USDA 110. Analysis of target mRNA for these novel sRNAs was inferred to target several nodulation and nitrogen fixation genes including nodC, nodJ, nodY, nodJ, nodM, nodW, nodZ, nifD, nifN, nifQ, fixK, fixL, fdx, nolB, and several cytochrome proteins. In addition, sRNAs of B. diazoefficiens USDA 110 which targeted the regulatory genes of nitrogen fixation were confirmed by wet-lab experiments with semi-quantitative reverse transcription polymerase chain reaction. Predicted target mRNAs were functionally classified based on the COG analysis and GO annotations. The genome-wide and transcriptome-based integrated methods have led to the identification of several sRNAs involved in the nodulation and symbiosis. Further validation of the functional role of these sRNAs can help in exploring the role of sRNAs in nitrogen metabolism during free-living and symbiotic association with legumes.