Termination factor Rho: From the control of pervasive transcription to cell fate determination in Bacillus subtilis

Abstract

In eukaryotes, RNA species originating from pervasive transcription are regulators of various cellular processes, from the expression of individual genes to the control of cellular development and oncogenesis. In prokaryotes, the function of pervasive transcription and its output on cell physiology is still unknown. Most bacteria possess termination factor Rho, which represses pervasive, mostly antisense, transcription. Here, we investigate the biological significance of Rho-controlled transcription in the Gram-positive model bacterium Bacillus subtilis. Rho inactivation strongly affected gene expression in B. subtilis, as assessed by transcriptome and proteome analysis of a rho–null mutant during exponential growth in rich medium. Subsequent physiological analyses demonstrated that a considerable part of Rho-controlled transcription is connected to balanced regulation of three mutually exclusive differentiation programs: cell motility, biofilm formation, and sporulation. In the absence of Rho, several up-regulated sense and antisense transcripts affect key structural and regulatory elements of these differentiation programs, thereby suppressing motility and biofilm formation and stimulating sporulation. We dissected how Rho is involved in the activity of the cell fate decision-making network, centered on the master regulator Spo0A. We also revealed a novel regulatory mechanism of Spo0A activation through Rho-dependent intragenic transcription termination of the protein kinase kinB gene. Altogether, our findings indicate that distinct Rho-controlled transcripts are functional and constitute a previously unknown built-in module for the control of cell differentiation in B. subtilis. In a broader context, our results highlight the recruitment of the termination factor Rho, for which the conserved biological role is probably to repress pervasive transcription, in highly integrated, bacterium-specific, regulatory networks.

Alternative transcription cycle for bacterial RNA polymerase

“…6B, C, and D), showing that it is a feature common to datasets collected from divergent species. Furthermore, the height of the antisense initiation peak was often increased in data from a B. subtilis strain with deletion of the Rho termination factor gene, consistent with prior observations that steady-state levels of antisense transcripts are greatly increased by Rho mutation or inhibition [39][40][41][42] . Both species display a −10 box motif at sites of antisense initiation that is similar to the −10 box motif at sense initiation sites 43 , confirming that antisense initiation occurs at promoter-like sequences (Fig.…”

Alternative transcription cycle for bacterial RNA polymerase

“…Similar proximity was seen in data from B. subtilis (Figure S6B, C, and D), showing that it is a feature common to datasets collected from divergent species. Furthermore, the height of the antisense initiation peak was often increased in data from a B. subtilis strain with deletion of the Rho termination factor gene, consistent with prior observations that steady-state levels of antisense transcripts are 45 greatly increased by Rho mutation or inhibition (Bidnenko et al, 2017;Lalanne et al, 2018;Nicolas et al, 2012;Peters et al, 2012). Both species display a consensus -10 box at sites of antisense initiation that is very similar to the -10 box at sense initiation sites (Shultzaberger et al, 2007), confirming that antisense initiation occurs at promoter-like sequences ( Figure 5E, S6E; compare Dornenburg et al, 2010).…”

The torpedo effect inBacillus subtilis:RNase J1 resolves stalled transcription complexes

“…Their mechanisms of dissociating the transcription complex differ from each other and are likely also different from that of RNase J1. Rho was reported to decrease nonspecific, pervasive transcription (Bidnenko et al, 2017), and its mRNA level was decreased in the DrnjA strain. We tested the effect of its absence in UV sensitivity assay.…”

“…To prepare a DrnjA strain in a widely used B. subtilis genetic background, chromosomal DNA from a DrnjA strain (LK1191 = CCB434 in Figaro et al, 2013) was transformed into B. subtilis BaSysBio , yielding strain LK1381. Strains DrnjA Drho (LK2082) and DrnjA DhelD (LK2336) were prepared with transformation of chromosomal DNA from the DrnjA strain (LK1190) into B. subtilis Drho (LK2058) (Bidnenko et al, 2017) or DhelD (LK2329) strain .…”