Padraig Deighan

Regulation of gene expression by histone-like proteins in bacteria.

Histone-like proteins in bacteria contribute to the control of gene expression, as well as participating in other DNA transactions such as recombination and DNA replication. They have also been described, somewhat vaguely, as contributors to the organization of the bacterial nucleoid. Our view of how these proteins act in the cell is becoming clearer, particularly in the cases of Fis, H-NS and HU, three of the most intensively studied members of the group. Especially helpful have been studies of the contributions of these proteins to the regulation of specific genes such as the gal operon, and genes coding for stable RNA species, topoisomerases, and the histone-like proteins themselves. Recent advances have also been assisted by insights into the effects the histone-like proteins exert on DNA structure not only at specific promoters but throughout the genome.

A role for the Escherichia coli H‐NS‐like protein StpA in OmpF porin expression through modulation of micF RNA stability

When a wild‐type strain of Escherichia coli and its stpA, hns and stpA hns mutant derivatives were compared by two‐dimensional protein gel electrophoresis, the levels of expression of several proteins were found to vary. One of these was identified as the outer membrane porin protein, OmpF. In the stpA hns double mutant, the level of OmpF was downregulated dramatically, whereas in hns or stpA single mutants, it was affected only slightly. Transcription from the ompF promoter was reduced by 64% in the double mutant; however, the level of ompF mRNA was reduced by 96%. This post‐transcriptional expression was found to result from a strong reduction in the half‐life of ompF message in the double mutant. The micF antisense RNA was shown to be involved in OmpF regulation by StpA using a strain deleted for micF. Moreover, micF antisense RNA accumulated considerably in an stpA hns background. Transcriptional data from a micF–lacZ fusion and measurements of micF RNA half‐life confirmed that this was caused by transcriptional derepression of micF as a result of the hns lesion and increased micF RNA stability due to the absence of StpA (a known RNA chaperone). These data suggest a novel facet to the regulation of OmpF expression, namely destabilization of micF RNA by StpA.

Mycobacterial mistranslation is necessary and sufficient for rifampicin phenotypic resistance

Significance Although error rates in protein translation, particularly under stress, are high, it is not known if these errors are inherent to the system, or if they may have an adaptive function. Here, we provide evidence that specific mistranslation of mycobacterial proteins is important for phenotypic resistance to the antibiotic rifampicin. This raises the possibility that errors in protein translation play a role in adapting to stress through increasing proteomic diversity. Errors are inherent in all biological systems. Errors in protein translation are particularly frequent giving rise to a collection of protein quasi-species, the diversity of which will vary according to the error rate. As mistranslation rates rise, these new proteins could produce new phenotypes, although none have been identified to date. Here, we find that mycobacteria substitute glutamate for glutamine and aspartate for asparagine at high rates under specific growth conditions. Increasing the substitution rate results in remarkable phenotypic resistance to rifampicin, whereas decreasing mistranslation produces increased susceptibility to the antibiotic. These phenotypic changes are reflected in differential susceptibility of RNA polymerase to the drug. We propose that altering translational fidelity represents a unique form of environmental adaptation.

Three-way interactions among the Sfh, StpA and H-NS nucleoid-structuring proteins of Shigella flexneri 2a strain 2457T

Shigella flexneri 2a strain 2457T has been found to express Sfh, a new member of the H‐NS‐like family of nucleoid‐structuring proteins. With H‐NS and its paralogue, StpA, this brings to three the number of these proteins expressed in this bacterium. This raises the possibility that three‐way interactions may occur in S. flexneri among these proteins and between the proteins and each others genes. Such three‐way interactions among H‐NS‐like proteins have not been described previously. The expression of the sfh, stpA and hns genes was studied at the transcriptional and post‐transcriptional levels. The Sfh protein displays growth phase‐dependent regulation that distinguishes it from both H‐NS and StpA. Like H‐NS and StpA, Sfh can bind to its own promoter region, it negatively autoregulates transcription of its own gene, and when overexpressed all three proteins cross‐repress transcription of each others genes. The presence of highly conserved oligomerization domains within these molecules suggested the possibility of protein–protein interactions. Like H‐NS and StpA, the purified Sfh protein forms homodimers in solution. Using the yeast two‐hybrid assay we show that each of the three proteins also forms homodimers in vivo and, additionally, each protein can form heterodimers with either of its homologues. This raises the possibility that Sfh may modulate the activities of H‐NS and StpA, and vice versa.

Shigella flexneri 2a strain 2457T expresses three members of the H-NS-like protein family: characterization of the Sfh protein

Abstract Shigella flexneri 2a is known to express the H-NS nucleoid-structuring protein and the paralogous protein StpA. Using bioinformatic analysis we have now discovered a third member of the H-NS protein family, Sfh (Shigella flexneri H-NS-like protein), in strain 2457T. This protein is encoded by the sfh gene, which is located on a high-molecular-mass plasmid that is closely related to the self-transmissible plasmid R27. When expressed in Escherichia coli, the Sfh protein can complement an hns null mutation, restoring wild-type Bgl, porin protein, and mucoidy phenotypes, and wild-type expression of the fliC and proU genes. While a knockout mutation in the sfh gene alone had no effect on the expression of virulence genes in S. flexneri, an additive effect on virulence gene derepression was seen when the sfh lesion was combined with a mutation in hns. Over-expression of the sfh gene repressed expression of the VirB virulence regulatory protein and transcription of a VirB-dependent structural gene promoter. The purified Sfh protein bound specifically to DNA sequences containing the promoters of the virF and virB virulence regulatory genes. These findings show that Sfh has the ability to influence genetic events beyond the genetic element that encodes it, including the expression of the S. flexneri virulence genes. They raise the possibility of a triangular relationship among three closely related proteins with broad consequences for genetic events in the bacterium that harbours them.

Requirement for the molecular adapter function of StpA at the Escherichia coli bgl promoter depends upon the level of truncated H-NS protein.

Truncated derivatives of the Escherichia coli nucleoid‐associated protein H‐NS that lack the DNA‐binding domain remain competent for silencing of the cryptic bgl operon in vivo. Previous studies have provided evidence for the involvement of either the homologous nucleoid protein StpA or the alternative sigma factor RpoS in this unusual silencing mechanism. Here, we rationalize this apparent discrepancy. We show that two hns alleles (hns‐205::Tn10 and hns60), which produce virtually identical amino‐terminal fragments of H‐NS, have very different requirements for StpA to mediate bgl silencing. The hns60 allele produces a high level of truncated H‐NS, which can overcome the absence of StpA, whereas the lower level expressed by hns‐205::Tn10 requires StpA for silencing. Reversing the relative levels of the two H‐NS fragments reverses their requirement for StpA to silence bgl transcription. This suggests that the amino‐terminal fragment of H‐NS can be targeted to DNA to mediate silencing by multiple protein–protein interactions. The high‐specificity interaction with StpA can function at low levels of truncated H‐NS, whereas an alternative mechanism, perhaps involving lower specificity interactions with another protein(s), is only functional when truncated H‐NS is abundant. These findings have important implications for the involvement of other proteins in H‐NS‐dependent transcriptional repression.

The bacteriophage λ Q antiterminator protein contacts the β-flap domain of RNA polymerase

The multisubunit RNA polymerase (RNAP) in bacteria consists of a catalytically active core enzyme (α2ββ′ω) complexed with a σ factor that is required for promoter-specific transcription initiation. During early elongation the stability of interactions between σ and core decreases, in part because of the nascent RNA-mediated destabilization of an interaction between region 4 of σ and the flap domain of the β-subunit (β-flap). The nascent RNA-mediated destabilization of the σ region 4/β-flap interaction is required for the bacteriophage λ Q antiterminator protein (λQ) to engage the RNAP holoenzyme. Here, we provide an explanation for this requirement by showing that λQ establishes direct contact with the β-flap during the engagement process, thus competing with σ70 region 4 for access to the β-flap. We also show that λQs affinity for the β-flap is calibrated to ensure that λQ activity is restricted to the λ late promoter PR′. Specifically, we find that strengthening the λQ/β-flap interaction allows λQ to bypass the requirement for specific cis-acting sequence elements, a λQ-DNA binding site and a RNAP pause-inducing element, that normally ensure λQ is recruited exclusively to transcription complexes associated with PR′. Our findings demonstrate that the β-flap can serve as a direct target for regulators of elongation.

Efficient and specific gene knockdown by small interfering RNAs produced in bacteria

Synthetic small interfering RNAs (siRNAs) are an indispensable tool to investigate gene function in eukaryotic cells and may be used for therapeutic purposes to knock down genes implicated in disease. Thus far, most synthetic siRNAs have been produced by chemical synthesis. Here we present a method to produce highly potent siRNAs in Escherichia coli. This method relies on ectopic expression of p19, an siRNA-binding protein found in a plant RNA virus. When expressed in E. coli, p19 stabilizes an ∼21-nt siRNA-like species produced by bacterial RNase III. When mammalian cells are transfected by them, siRNAs that were generated in bacteria expressing p19 and a hairpin RNA encoding 200 or more nucleotides of a target gene reproducibly knock down target gene expression by ∼90% without immunogenicity or off-target effects. Because bacterially produced siRNAs contain multiple sequences against a target gene, they may be especially useful for suppressing polymorphic cellular or viral genes.

A regulator from Chlamydia trachomatis modulates the activity of RNA polymerase through direct interaction with the β subunit and the primary σ subunit

The obligate intracellular human pathogen Chlamydia trachomatis undergoes a complex developmental program involving transition between two forms: the infectious elementary body (EB), and the rapidly dividing reticulate body (RB). However, the regulators controlling this development have not been identified. To uncover potential regulators of transcription in C. trachomatis, we screened a C. trachomatis genomic library for sequences encoding proteins that interact with RNA polymerase (RNAP). We report the identification of one such protein, CT663, which interacts with the beta and sigma subunits of RNAP. Specifically, we show that CT663 interacts with the flap domain of the beta subunit (beta-flap) and conserved region 4 of the primary sigma subunit (sigma(66) in C. trachomatis). We find that CT663 inhibits sigma(66)-dependent (but not sigma(28)-dependent) transcription in vitro, and we present evidence that CT663 exerts this effect as a component of the RNAP holoenzyme. The analysis of C. trachomatis-infected cells reveals that CT663 begins to accumulate at the commencement of the RB-to-EB transition. Our findings suggest that CT663 functions as a negative regulator of sigma(66)-dependent transcription, facilitating a global change in gene expression. The strategy used here is generally applicable in cases where genetic tools are unavailable.

The bacteriophage λQ anti‐terminator protein regulates late gene expression as a stable component of the transcription elongation complex

The Q protein of bacteriophage λ (λQ) is a transcription anti‐terminator required for the expression of the phages late genes under the control of promoter PR′. To effect terminator read‐through, λQ must gain access to RNA polymerase (RNAP) via a promoter‐restricted pathway. In particular, λQ modifies RNAP by binding a specific DNA site embedded in PR′ and interacting with RNAP in the context of a specific paused early elongation complex. The resultant λQ‐modified transcription elongation complex is competent to read through downstream termination signals. Here we use a chromatin‐immunoprecipitation assay to test the hypothesis that λQ functions as a stable component of the transcription elongation complex. Our results indicate that, in vivo, the λQ‐modified transcription elongation complex contains Q as a stably associated subunit. Furthermore, we find that in the physiologically relevant context of an induced λ lysogen, Q remains stably associated with RNAP as it transcribes at least 22 kb of the phage late operon. Thus, our findings suggest that the promoter‐specific pathway leading to λQ‐mediated terminator read‐through results in the formation of a highly stable λQ‐containing transcription elongation complex capable of traversing the entire late operon.