Termination factor Rho mediates transcriptional reprogramming of Bacillus subtilis stationary phase

Abstract

Transcription termination factor Rho is known for its ubiquitous role in suppression of pervasive, mostly antisense, transcription. In the model Gram-positive bacterium Bacillus subtilis, de-repression of pervasive transcription by inactivation of rho revealed the role of Rho in the regulation of post-exponential differentiation programs. To identify other aspects of the regulatory role of Rho during adaptation to starvation, we have constructed a B. subtilis strain (Rho+) that expresses rho at a relatively stable high level in order to compensate for its decrease in the wild-type cells entering stationary phase. The RNAseq analysis of Rho+, WT and Δrho strains (expression profiles can be visualized at http://genoscapist.migale.inrae.fr/seb_rho/) shows that Rho over-production enhances the termination efficiency of Rho-sensitive terminators, thus reducing transcriptional read-through and antisense transcription genome-wide. Moreover, the Rho+ strain exhibits global alterations of sense transcription with the most significant changes observed for the AbrB, CodY, and stringent response regulons, forming the pathways governing the transition to stationary phase. Subsequent physiological analyses demonstrated that maintaining rho expression at a stable elevated level modifies stationary phase-specific physiology of B. subtilis cells, weakens stringent response, and thereby negatively affects the cellular adaptation to nutrient limitations and other stresses, and blocks the development of genetic competence and sporulation. These results highlight the Rho-specific termination of transcription as a novel element controlling stationary phase. The release of this control by decreasing Rho levels during the transition to stationary phase appears crucial for the functionality of complex gene networks ensuring B. subtilis survival in stationary phase.

Complex sporulation-specific expression of transcription termination factor Rho highlights its involvement inBacillus subtiliscell differentiation

“…In stationary phase, rho expression remained at a basal level. A low-level expression of rho and its down-regulation during the transition to stationary phase were reported previously (Ingham et al, 1999; Nicolas et al, 2012; Bidnenko et al, 2017; 2023). The inactivation of Rho in RM cells did not alter the kinetics of rho expression, but rather increased its level approximately threefold, suggesting a release from autoregulation (Fig 1A).…”

“…Two spore phenotypes, a decreased UV-resistance and a structurally altered outer coat, resulting from the complete inactivation of rho , or following the inhibition of rho expression in the forespores and in the mother cells, respectively, highlight the importance of the compartmentalized Rho-mediated transcription termination for spore development. Our previous transcriptome analysis of RM cells in stationary phase revealed disordered and untimely expression of many sporulation genes via their extensive sense or antisense read-through transcription or, more rarely, unscheduled activation of promoters (Bidnenko et al, 2023). The relevant gene expression changes are compiled in S1 Table and can be visualized at http://genoscapist.migale.inrae.fr/seb_rho/ (Bidnenko et al, 2023; Dérozier et al, 2021).…”

“…Our previous transcriptome analysis of RM cells in stationary phase revealed disordered and untimely expression of many sporulation genes via their extensive sense or antisense read-through transcription or, more rarely, unscheduled activation of promoters (Bidnenko et al, 2023). The relevant gene expression changes are compiled in S1 Table and can be visualized at http://genoscapist.migale.inrae.fr/seb_rho/ (Bidnenko et al, 2023; Dérozier et al, 2021). We posit that at least some of the sporulation genes abnormally expressed in the stationary RM cells are subject to Rho-mediated regulation during sporulation.…”

“…Interestingly, the expression levels of cotG are strongly upregulated in RM cells, due to the transcription upshift at, or close to, the SigK-dependent cotG promoter and the read-through transcription initiated at the upstream alsR gene (Nicolas et al, 2012; Bidnenko et al, 2023; S1 Table). The same read-through transcript appears antisense to the oppositely oriented neighboring genes cotB and cotH .…”

“…Analysis of promoters' activity using luciferase fusions was performed as described previously (Mirouze et al, 2011) with minor modifications detailed in (Bidnenko et al, 2023). Cells were grown in LB medium to mid-exponential phase (OD600 0,4-0,5), cultures were centrifuged and resuspended to OD 1.0 in fresh LB or DSM, to follow expression of the fusions during growth or sporulation, respectively.…”