Global Annotation, Expression Analysis, and Stability of Candidate sRNAs in Group B Streptococcus

Abstract

ABSTRACT Small, noncoding RNAs (sRNAs) are being increasingly identified as important regulatory molecules in prokaryotes. Due to the prevalence of next-generation sequencing-based techniques, such as RNA sequencing (RNA-seq), there is potential for increased discovery of sRNAs within bacterial genomes; however, these elements are rarely included in annotation files. Consequently, expression values for sRNAs are omitted from most transcriptomic analyses, and mechanistic studies have lagged behind those of protein regulators in numerous bacteria. Two previous studies have identified sRNAs in the human pathogen group B Streptococcus (GBS). Here, we utilize the data from these studies to create updated genome annotation files for the model GBS strains NEM316 and COH1. Using the updated COH1 annotation file, we reanalyze publicly available GBS RNA-seq whole-transcriptome data from GenBank to monitor GBS sRNA expression under a variety of conditions and genetic backgrounds. This analysis generated expression values for 232 putative sRNAs that were overlooked in previous transcriptomic analyses in 21 unique comparisons. To demonstrate the utility of these data, we identify an sRNA that is upregulated during vaginal colonization and demonstrate that overexpression of this sRNA leads to increased bacterial invasion into host epithelial cells. Finally, to monitor RNA degradation, we perform a transcript stability assay to identify highly stable sRNAs and compare stability profiles of sRNA- and protein-coding genes. Collectively, these data provide a wealth of transcriptomic data for putative sRNAs in GBS and a platform for future mechanistic studies. IMPORTANCE In recent years, sRNAs have emerged as potent regulatory molecules in bacteria, including numerous streptococcal species, and contribute to diverse processes, including stress response, metabolism, housekeeping, and virulence regulation. Improvements in sequencing technologies and in silico analyses have facilitated identification of these regulatory molecules as well as improved attempts to determine the location of sRNA genes on the genome. However, despite these advancements, sRNAs are rarely included in genome annotation files. Consequently, these molecules are often omitted from transcriptomic data analyses and are commonly repeat identified across multiple studies. Updating current genomes to include sRNA genes is therefore critical for better understanding bacterial regulation.

Keywords

• RNA stability
• Streptococcus agalactiae
• group B streptococcus
• regulatory RNA
• sRNA
• virulence