Abstract
ABSTRACTRfaH activates horizontally acquired operons that encode lipopolysaccharide core components, pili, toxins, and capsules. Unlike its paralog NusG, which potentiates Rho-mediated silencing, RfaH strongly inhibits Rho. RfaH is recruited to its target operons via a network of contacts with an elongating RNA polymerase (RNAP) and a specific DNA element called ops to modify RNAP into a pause- and NusG-resistant state. rfaH null mutations confer hypersensitivity to antibiotics and detergents, altered susceptibility to bacteriophages, and defects in virulence. Here, we carried out a selection for suppressors that restore the ability of a ΔrfaH mutant Escherichia coli strain to grow in the presence of sodium dodecyl sulfate. We isolated rho, rpoC, and hns suppressor mutants with changes in regions previously shown to be important for their function. In addition, we identified mutants with changes in an unstructured region that connects the primary RNA-binding and helicase domains of Rho. The connector mutants display strong defects in vivo, consistent with their ability to compensate for the loss of RfaH, and act synergistically with bicyclomycin (BCM), which has been recently shown to inhibit Rho transformation into a translocation-competent state. We hypothesize that the flexible connector permits the reorientation of Rho domains and serves as a target for factors that control the motor function of Rho allosterically. Our results, together with the existing data, support a model in which the connector segment plays a hitherto overlooked role in the regulation of Rho-dependent termination.
Highlights
Escherichia coli RfaH is a sequence-specific paralog of the housekeeping transcription elongation factor NusG
Consistent with these reports, we found that the ⌬rfaH mutant MG1655 strain was more sensitive to bile salts, novobiocin, and nalidixic acid than the wild-type (WT) isogenic strain
On the basis of previous results with Salmonella (10) and the primary mode of RfaH action as an antagonist of Rho (9), we expected to identify suppressors in rho, rpoBC, and perhaps nusG that alleviate Rho-mediated polarity in waa, in which a polar mini-Tn5 insertion phenocopies the effects of rfaH (13)
Summary
Escherichia coli RfaH is a sequence-specific paralog of the housekeeping transcription elongation factor NusG. RfaH is thought to recruit the ribosome to mRNAs and subsequently couple transcription to translation via direct interactions with ribosomal protein S10 (7); the coupled ribosome will shield the nascent mRNA from Rho (8) Together, these activities essentially abrogate Rho-mediated polarity; e.g., the expression of the rfb operon, which is silenced by Rho, increases ~300-fold in the presence of RfaH (9). We carried out a selection for suppressors that restored the ability of ΔrfaH mutant E. coli strain MG1655 to grow in the presence of sodium dodecyl sulfate (SDS) This selection could identify several classes of suppressors that (i) reduce Rho-dependent termination, (ii) enable RfaH-independent ribosome recruitment, (iii) reduce the intracellular concentration of SDS, and (iv) increase the transcription of RfaH-dependent operons, e.g., by activating their promoter.
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