Fig 8

Potential roles and RT-PCR validation of predicted excludons. (a) GO enrichment analysis of responder genes in convergent excludons. Orange and blue bars represent enriched annotations for upregulated and downregulated antisense transcripts, respectively. Numbers on the bars represent the number of genes with the given annotation, and numbers in parentheses represent the ratio of genes to all genes with the annotation. (b) Representative responder gene in convergent excludon, cycA2, in a convergent gene pair with ETPO_02324. The first (upper) panel shows the positions and orientations of genes. The second panel shows DEGs: orange, upregulated; blue, downregulated. The third panel shows DEATs: purple, downregulated. The bottom three panels show the mean log2 fragments per bins per million mapped reads in 10 bp bins mapped to the Watson (blue) and Crick (red) strands in control bacterial RNAs (gray bar), MNP-enriched RNAs (orange bar), and centrifuge-enriched RNAs (pink bar). (c) RNA from broth-cultured bacteria was used for RT-PCR confirmation of overlapping transcription of predicted convergent and divergent excludons using primers designed within 100 bp upstream of the stop codons or 100 bp downstream of the start codons for convergent or divergent gene pairs, respectively. An internal fragment of the 16S rRNA gene was used as an RT control. Templates in PCR reaction: RT, RT reaction with enzyme; NR, RT reaction with no enzyme; +, 1 ng gDNA; and −, sterile water.