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In Escherichia coli and Salmonella, many genes silenced by the nucleoid structuring protein H-NS are activated upon inhibiting Rho-dependent transcription termination. This response is poorly understood and difficult to reconcile with the view that H-NS acts mainly by blocking transcription initiation. Here we have analyzed the basis for the up-regulation of H-NS–silenced Salmonella pathogenicity island 1 (SPI-1) in cells depleted of Rho-cofactor NusG. Evidence from genetic experiments, semiquantitative 50 rapid amplification of complementary DNA ends sequencing (5’ RACE-Seq), and chromatin immunoprecipitation sequencing (ChIP-Seq) shows that transcription originating from spurious antisense promoters, when not stopped by Rho, elongates into a H-NS–bound regulatory region of SPI-1, displacing H-NS and rendering the DNA accessible to the master regulator HilD. In turn, HilD’s ability to activate its own transcription triggers a positive feedback loop that results in transcriptional activation of the entire SPI-1. Significantly, single-cell analyses revealed that this mechanism is largely responsible for the coexistence of two subpopulations of cells that either express or do not express SPI-1 genes. We propose that cell-to-cell differences produced by stochastic spurious transcription, combined with feedback loops that perpetuate the activated state, can generate bimodal gene expression patterns in bacterial populations. Agence Nationale de la Recherche de Francia - ANR-15-CE11-0024-03 Ministerio de Ciencia e Innovación y Agencia Estatal de Investigación, de España y Fondo Regional Europeo - PID2020-116995RB-I00

Evolutionarily conserved NusG protein enhances bacterial RNA polymerase processivity but can also promote transcription termination by binding to, and stimulating the activity of, Rho factor. Rho terminates transcription upon anchoring to cytidine-rich motifs, the so-called Rho utilization sites (Rut) in nascent RNA. Both NusG and Rho have been implicated in the silencing of horizontally-acquired A/T-rich DNA by nucleoid structuring protein H-NS. However, the relative roles of the two proteins in H-NS-mediated gene silencing remain incompletely defined. In the present study, a Salmonella strain carrying the nusG gene under the control of an arabinose-inducible repressor was used to assess the genome-wide response to NusG depletion. Results from two complementary approaches, i) screening lacZ protein fusions generated by random transposition and ii) transcriptomic analysis, converged to show that loss of NusG causes massive upregulation of Salmonella pathogenicity islands (SPIs) and other H-NS-silenced loci. A similar, although not identical, SPI-upregulated profile was observed in a strain with a mutation in the rho gene, Rho K130Q. Surprisingly, Rho mutation Y80C, which affects Rho’s primary RNA binding domain, had either no effect or made H-NS-mediated silencing of SPIs even tighter. Thus, while corroborating the notion that bound H-NS can trigger Rho-dependent transcription termination in vivo, these data suggest that H-NS-elicited termination occurs entirely through a NusG-dependent pathway and is less dependent on Rut site binding by Rho. We provide evidence that through Rho recruitment, and possibly through other still unidentified mechanisms, NusG prevents pervasive transcripts from elongating into H-NS-silenced regions. Failure to perform this function causes the feedforward activation of the entire Salmonella virulence program. These findings provide further insight into NusG/Rho contribution in H-NS-mediated gene silencing and underscore the importance of this contribution for the proper functioning of a global regulatory response in growing bacteria. The complete set of transcriptomic data is freely available for viewing through a user-friendly genome browser interface., Author summary Most virulence genes of pathogenic Enterobacteria lie within genomic islands acquired through horizontal transfer in the course of evolution. While essential during infection, expression of many of these genes is detrimental for growth outside the host. Hence, mechanisms have evolved that prevent transcription of this foreign DNA, except when needed. A major mechanism uses Histone-like protein H-NS which binds to AT-rich nucleation sites, a hallmark of horizontally acquired DNA, forming oligomeric structures that silence transcription over extended regions. This silencing activity is all the more important as A/T-rich sequences are intrinsically prone to spurious transcription initiation events. Nonetheless, H-NS-silenced regions remain exposed to invading transcripts that either escape repression or originate from neighboring DNA. Results presented here, support the proposal that NusG, a conserved transcription elongation factor, plays a key role in ensuring that elongation complexes come to a stop when running into a patch of oligomerized H-NS. NusG performs this function by stimulating the activity of transcription termination factor Rho. We show that alteration of this mechanism in Salmonella has profound physiological consequences, most likely because unstopped transcripts invade H-NS-silenced pathogenicity islands and trigger a feed-forward regulatory cascade leading to the gratuitous activation of these islands and other co-regulated loci.