Fig. 4

Metatranscriptomics support IS-mediated transcription within B. caccae. IS614 contains a putative outward-facing promoter. The relative contribution of the IS promoter to transcription was determined by comparing RNA sequencing read depths of genes upstream and downstream of it. a In timepoint B, which is dominated by ancestral strains without the promoter, RNA sequencing read coverage depth (relative transcript abundance) is relatively equal on both sides of the integration site. In timepoint C, which is dominated by strains with IS614 with its putative outwardly directed promoter, the transcription of the downstream gene norM is much higher than that of the upstream gene yidC. The relative transcript abundance of all neighboring genes increase in timepoint C relative to B, but this increase is 10-fold greater in genes immediately downstream of the introduced outward promoter. In later timepoints C and D, dominant strains harbor an introduced IS promoter positioned to upregulate norM. This is supported by read pairs spanning between the IS promoter and norM. This difference in coverage and domination by strains with this promoter both persist through timepoint D. Conversely, the earlier timepoint B is dominated by strains with no IS in this region. b PCR with primers flanking the above integration instance of IS614 yields amplicons without the insertion sequence in earlier timepoints A and B (400 bp), and with the insertion in later timepoints C and D (1.9 kb)