NusG prevents transcriptional invasion of H-NS-silenced genes

Abstract

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.

Regulatory interplay between small RNAs and transcription termination factor Rho

“…Furthermore, the S. aureus Rho protein can functionally replace E. coli Rho in complementation assays [89] and the Rho proteins from both S. aureus and B. subtilis are sensitive to bicyclomycin [89,93]. Finally, recent transcriptomic studies conducted in B. subtilis [27], S. aureus [30], and M. tuberculosis [29] have revealed that rho inactivation causes genome-wide pervasive (mostly antisense) transcription in these species, a pattern remarkably similar to that found in E. coli [26] and Salmonella [28] despite significantly divergent genome organizations/compositions (e.g., GC contents of ~33% for S. aureus, ~43% for B. subtilis, 50-52% for E. coli and Salmonella, and ~66% for M. tuberculosis).…”

“…In E. coli, Rho negatively regulates transcription of its own gene by terminating transcription at multiple sites within the 5' UTR and the initial translated portion of rho mRNA [84]. A similar feedback mechanism operates in Salmonella where finding that strains with rho mutations overproduce Rho protein suggests that a substantial level of termination occurs constitutively during the growth of wild-type cells [28]. A recent study identified rho as a regulatory target of the small RNA SraL in Salmonella.…”

“…Whether this arrangement confers some special features to the expression/regulation of head-on transcription units is currently unknown. The specialization toward targeting antisense transcription, either produced by bone-fide promoters or by spurious promoter-like sequences within coding regions, is a prominent feature of Rho activity in the bacterial cell [26][27][28][29][30][31].…”

“…For instance, protein Hfq appears dispensable for sRNA-mediated regulation in Firmicutes [100,101]. The DNA chaperone H-NS, which contributes to Rho-dependent termination at multiple loci in E. coli and Salmonella [26,28] is absent in Firmicutes [102]. The contribution of NusG to Rho-dependent termination could also be different in the Gram-positive group.…”

Global transcriptional regulation by cell-free sup of S. Typhimurium peptide transporter mutant leads to inhibition of intraspecies biofilm initiation

“…Abundance of FlgM correlates with the absence of agella in the ΔyjiY supernatant treated WT cells. Since, NusG works synergistically with the global transcriptional regulator H-NS that binds speci cally to AT-rich SPIs in Salmonella genome and represses the genes 46,47 . Interestingly, in E. coli, H-NS has been linked to cell cycle, since the cells attempt to optimize H-NS concentration by maintaining a constant ratio of H-NS to chromosomal DNA in the cell 48 , which might explain the increase in cell length after ΔyjiY supernatant treatment.…”