GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE203nnn/GSE203041/primaryOK200TranscriptomicsAzoarcus oleariusExpression profiling by high throughput sequencinghttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE203041GEOGSEfalseExtensive antisense transcription in Azoarcus olearius strain BH72Azoarcus olearius strain BH72 is a plant endophyte that can fix atmospheric nitrogen for plant assimilation. Our recent directional RNA-Seq analysis detected widespread presence of reads mapping in the antisense orientation to the annotated genes. Although this has been discovered for several bacteria, the stable expression and functional relevance of such antisense RNAs (asRNAs) are still being debated. In this study we ventured to confirm the single-cell expression and unravel the functionality of antisense transcription in strain BH72. First, from the directional RNA-Seq data we predicted 798 asRNAs that were encoded against 709 genes (18 % of the annotated genome). Using a cassette encoding for two fluorescent reporters (GFP and TdTomato), in convergent orientation with transcriptional terminators in between, we were able to confirm the single-cell co-expression of both the sense and the antisense transcripts within azo1349 in at least 90 % of the bacterial population. By modifying the above experimental setup, we observed the interference exhibited by these overlapping transcripts. Further, decreasing the ribosomal occupancy on one of the overlapping transcripts increased its repression of the complimentary transcript. However, RNA-Seq analysis of a RNase III deficient mutant indicated a minimal effect of the endoribonuclease on the regulation of sense-antisense pairs in strain BH72. Lastly, we elucidated the control mediated by RNase III and asRNA on the expression of genes in the type six secretion system-1 cluster.2026/06/30GSE203041GSM6152892GSM6152888GSM6152889GSM6152891GSM615289030254203041Azoarcus olearius