Some promoters, including the DmpR-controlled ς^N-dependent Po promoter, are effectively rendered silent in cells lacking the nutritional alarmone (p)ppGpp. Here we demonstrate that four mutations within the housekeeping ς^D-factor can restore ς^N-dependent Po transcription in the absence of (p)ppGpp. Using both in vitro and in vivotranscription competition assays, we show that all the four ς^D mutant proteins are defective in their ability to compete with ς^N for available core RNA polymerase and that the magnitude of the defect reflects the hierarchy of restoration of transcription from Po in (p)ppGpp-deficient cells. Consistently, underproduction of ς^D or overproduction of the anti-ς^D protein Rsd were also found to allow (p)ppGpp-independent transcription from the ς^N-Po promoter. Together with data from the direct effects of (p)ppGpp on ς^N-dependent Po transcription and ς-factor competition, the results support a model in which (p)ppGpp serves as a master global regulator of transcription by differentially modulating alternative ς-factor competition to adapt to changing cellular nutritional demands.

The Hfq protein was discovered in Escherichia coli as a host factor for bacteriophage Qβ RNA replication. Subsequent studies indicated that Hfq is a pleiotropic regulator of bacterial gene expression. The regulatory role of Hfq is ascribed mainly to its function as an RNA-chaperone, facilitating interactions between bacterial noncoding RNA and its mRNA target. Thus, it modulates mRNA translation and stability. Nevertheless, Hfq is able to interact with DNA as well. Its role in the regulation of DNA-related processes has been demonstrated. In this mini-review, it is discussed how Hfq interacts with DNA and what is the role of this protein in regulation of DNA transactions. Particularly, Hfq has been demonstrated to be involved in the control of ColE1 plasmid DNA replication, transposition, and possibly also transcription. Possible mechanisms of these Hfq-mediated regulations are described and discussed.

The Escherichia coli proteins DksA, GreA, and GreB are all structural homologs that bind the secondary channel of RNA polymerase (RNAP) but are thought to act at different levels of transcription. DksA, with its co-factor ppGpp, inhibits rrnB P1 transcription initiation, whereas GreA and GreB activate RNAP to cleave back-tracked RNA during elongational pausing. Here, in vivo and in vitro evidence reveals antagonistic regulation of rrnB P1 transcription initiation by Gre factors (particularly GreA) and DksA; GreA activates and DksA inhibits. DksA inhibition is epistatic to GreA activation. Both modes of regulation are ppGpp-independent in vivo but DksA inhibition requires ppGpp in vitro. Kinetic experiments and studies of rrnB P1-RNA polymerase complexes suggest that GreA mediates conformational changes at an initiation step in the absence of NTP substrates, even before DksA acts. GreA effects on rrnB P1 open complex conformation reveal a new feature of GreA distinct from its general function in elongation. Our findings support the idea that a balance of the interactions between the three secondary channel-binding proteins and RNAP can provide a new mode for regulating transcription.

Production of Shiga toxins by enterohemorrhagic Escherichia coli (EHEC) which is responsible for the pathogenicity of these strains, is strictly correlated with induction of lambdoid bacteriophages present in the host’s genome, replication of phage DNA and expression of stx genes. Antibiotic treatment of EHEC infection may lead to induction of prophage into a lytic development, thus increasing the risk of severe complications. This, together with the spread of multi-drug resistance, increases the need for novel antimicrobial agents. We report here that isothiocyanates (ITC), plant secondary metabolites, such as sulforaphane (SFN), allyl isothiocyanate (AITC), benzyl isothiocynanate (BITC), phenyl isothiocyanate (PITC) and isopropyl isothiocyanate (IPRITC), inhibit bacterial growth and lytic development of stx-harboring prophages. The mechanism underlying the antimicrobial effect of ITCs involves the induction of global bacterial stress regulatory system, the stringent response. Its alarmone, guanosine penta/tetraphosphate ((p)ppGpp) affects major cellular processes, including nucleic acids synthesis, which leads to the efficient inhibition of both, prophage induction and toxin synthesis, abolishing in this way EHEC virulence for human and simian cells. Thus, ITCs could be considered as potential therapeutic agents in EHEC infections.

The bacterial nutritional and stress alarmone ppGpp and its co-factor DksA directly bind RNA polymerase to regulate its activity at certain σ⁷⁰-dependent promoters. A number of promoters that are dependent on alternative σ-factors function poorly in the absence of ppGpp. These include the Pseudomonas-derived σ⁵⁴-dependent Po promoter and several other σ⁵⁴-promoters, the transcription from which is essentially abolished in Escherichia coli devoid of ppGpp and DksA. However, ppGpp and DksA have no apparent effect on reconstituted in vitro σ⁵⁴-transcription, which suggests an indirect mechanism of control. Here we report analysis of five hyper-suppressor mutants within the β- and β′-subunits of core RNA polymerase that allow high levels of transcription from the σ⁵⁴-Po promoter in the absence of ppGpp. Using in vitro transcription and competition assays, we present evidence that these core RNA polymerase mutants are defective in one or both of two properties that could combine to explain their hyper-suppressor phenotypes: (i) modulation of competitive association with σ-factors to favor σ⁵⁴-holoenzyme formation over that with σ⁷⁰, and (ii) reduced innate stability of RNA polymerase-promoter complexes, which mimics the essential effects of ppGpp and DksA for negative regulation of stringent σ⁷⁰-promoters. Both these properties of the mutant holoenzymes support a recently proposed mechanism for regulation of σ⁵⁴-transcription that depends on the potent negative effects of ppGpp and DksA on transcription from powerful stringent σ⁷⁰-promoters, and suggests that stringent regulation is a key mechanism by which the activity of alternative σ-factors is controlled to meet cellular requirements.