Transcriptional downregulation of agr expression in Staphylococcus aureus during growth in human serum can be overcome by constitutively active mutant forms of the sensor kinase AgrC

The temporal and cell density-dependent regulation of expression of virtually all the Staphylococcus aureus virulon is under the control of the agr (accessory gene regulatory) operon. The expression of the agr operon is subject to transcriptional regulation by the AgrA/C two-component response regulator/sensor kinase pair. During bacteraemia, a frequent syndrome caused by methicillin-resistant S. aureus (MRSA), the transcriptional downregulation of agr expression has been attributed to the sequestration of the quorum-signalling molecule auto-inducing peptide (AIP) by the human serum component apolipoprotein B as part of an innate immune response to infection. However, it is not known whether transcriptional downregulation of agr expression during growth in human serum is additionally subjected to regulation by transcription regulatory proteins that either directly or indirectly affect transcription from the agr operon promoters. Here, using chromosomal fluorescence reporters of agr expression in S. aureus, we show that the transcriptional downregulation of agr expression in human serum can be overcome using constitutive active mutant forms of AgrC. Therefore, it seems that the sequestration of the AIP is likely to be the only mechanism by which the host innate immune response limits agr expression at the transcriptional level to maintain the host–pathogen balance towards a noninvasive outcome

The Xp10 bacteriophage protein P7 inhibits transcription by the major and major variant forms of the host RNA polymerase via a common mechanism

The σ factor is a functionally obligatory subunit of the bacterial transcription machinery, the RNA polymerase. Bacteriophage-encoded small proteins that either modulate or inhibit the bacterial RNAP to allow the temporal regulation of bacteriophage gene expression often target the activity of the major bacterial σ factor, σ70. Previously, we showed that during Xanthomonas oryzae phage Xp10 infection, the phage protein P7 inhibits the host RNAP by preventing the productive engagement with the promoter and simultaneously displaces the σ70 factor from the RNAP. In this study, we demonstrate that P7 also inhibits the productive engagement of the bacterial RNAP containing the major variant bacterial σ factor, σ54, with its cognate promoter. The results suggest for the first time that the major variant form of the host RNAP can also be targeted by bacteriophage-encoded transcription regulatory proteins. Since the major and major variant σ factor interacting surfaces in the RNAP substantially overlap, but different regions of σ70 and σ54 are used for binding to the RNAP, our results further underscore the importance of the σ–RNAP interface in bacterial RNAP function and regulation and potentially for intervention by antibacterials

Phenotypic consequences of RNA polymerase dysregulation in Escherichia coli

Many bacterial adaptive responses to changes in growth conditions due to biotic and abiotic factors involve reprogramming of gene expression at the transcription level. The bacterial RNA polymerase (RNAP), which catalyzes transcription, can thus be considered as the major mediator of cellular adaptive strategies. But how do bacteria respond if a stress factor directly compromises the activity of the RNAP? We used a phage-derived small protein to specifically perturb bacterial RNAP activity in exponentially growing Escherichia coli. Using cytological profiling, tracking RNAP behavior at single-molecule level and transcriptome analysis, we reveal that adaptation to conditions that directly perturb bacterial RNAP performance can result in a biphasic growth behavior and thereby confer the ‘adapted’ bacterial cells an enhanced ability to tolerate diverse antibacterial stresses. The results imply that while synthetic transcriptional rewiring may confer bacteria with the intended desirable properties, such approaches may also collaterally allow them to acquire undesirable traits

Staphylococcus aureus inactivates daptomycin by releasing membrane phospholipids

Daptomycin is a bactericidal antibiotic of last resort for serious infections caused by methicillin-resistant Staphylococcus aureus (MRSA)1,2. Although resistance is rare, treatment failure can occur in more than 20% of cases3,4 and so there is a pressing need to identify and mitigate factors that contribute to poor therapeutic outcomes. Here, we show that loss of the Agr quorum-sensing system, which frequently occurs in clinical isolates, enhances S. aureus survival during daptomycin treatment. Wild-type S. aureus was killed rapidly by daptomycin, but Agr-defective mutants survived antibiotic exposure by releasing membrane phospholipids, which bound and inactivated the antibiotic. Although wild-type bacteria also released phospholipid in response to daptomycin, Agr-triggered secretion of small cytolytic toxins, known as phenol soluble modulins, prevented antibiotic inactivation. Phospholipid shedding by S. aureus occurred via an active process and was inhibited by the β-lactam antibiotic oxacillin, which slowed inactivation of daptomycin and enhanced bacterial killing. In conclusion, S. aureus possesses a transient defence mechanism that protects against daptomycin, which can be compromised by Agr-triggered toxin production or an existing therapeutic antibiotic

The Rsb phosphoregulatory network controls availability of the primary sigma factor in Chlamydia trachomatis and influences the kinetics of growth and development

Chlamydia trachomatis is the leading cause of both bacterial sexually transmitted infection and infection-derived blindness world-wide. No vaccine has proven protective to date in humans. C. trachomatis only replicates from inside a host cell, and has evolved to acquire a variety of nutrients directly from its host. However, a typical human immune response will normally limit the availability of a variety of essential nutrients. Thus, it is thought that the success of C. trachomatis as a human pathogen may lie in its ability to survive these immunological stress situations by slowing growth and development until conditions in the cell have improved. This mode of growth is known as persistence and how C. trachomatis senses stress and responds in this manner is an important area of research. Our report characterizes a complete signaling module, the Rsb network, that is capable of controlling the growth rate or infectivity of Chlamydia. By manipulating the levels of different pathway components, we were able to accelerate and restrict the growth and development of this pathogen. Our results suggest a mechanism by which Chlamydia can tailor its growth rate to the conditions within the host cell. The disruption of this pathway could generate a strain incapable of surviving a typical human immune response and would represent an attractive candidate as an attenuated growth vaccine

A role for the RNA polymerase gene specificity factor sigma(54) in the uniform colony growth of uropathogenic escherichia coli

The canonical function of a bacterial sigma (σ) factor is to determine the gene specificity of the RNA polymerase (RNAP). In several diverse bacterial species, the σ54 factor uniquely confers distinct functional and regulatory properties on the RNAP. A hallmark feature of the σ54-RNAP is the obligatory requirement for an activator ATPase to allow transcription initiation. Different activator ATPases couple diverse environmental cues to the σ54-RNAP to mediate adaptive changes in gene expression. Hence, the genes that rely upon σ54 for their transcription have a wide range of different functions suggesting that the repertoire of functions performed by genes, directly or indirectly affected by σ54, is not yet exhaustive. By comparing the growth patterns of prototypical enteropathogenic, uropathogenic, and nonpathogenic Escherichia coli strains devoid of σ54, we uncovered that the absence of σ54 results in two differently sized colonies that appear at different times specifically in the uropathogenic E. coli (UPEC) strain. Notably, UPEC bacteria devoid of individual activator ATPases of the σ54-RNAP do not phenocopy the σ54 mutant strain. Thus, it seems that σ54’s role as a determinant of uniform colony appearance in UPEC bacteria represents a putative non-canonical function of σ54 in regulating genetic information flow

A bacteriophage transcription regulator inhibits bacterial transcription initiation by Sigma-factor displacement

Bacteriophages (phages) appropriate essential processes of bacterial hosts to benefit their own development. The multisubunit bacterial RNA polymerase (RNAp) enzyme, which catalyses DNA transcription, is targeted by phage-encoded transcription regulators that selectively modulate its activity. Here, we describe the structural and mechanistic basis for the inhibition of bacterial RNAp by the transcription regulator P7 encoded by Xanthomonas oryzae phage Xp10. We reveal that P7 uses a two-step mechanism to simultaneously interact with the catalytic β and β’ subunits of the bacterial RNAp and inhibits transcription initiation by inducing the displacement of the σ70-factor on initial engagement of RNAp with promoter DNA. The new mode of interaction with and inhibition mechanism of bacterial RNAp by P7 underscore the remarkable variety of mechanisms evolved by phages to interfere with host transcription

A bacteriophage DNA mimic protein employs a non-specific strategy to inhibit the bacterial RNA polymerase

DNA mimicry by proteins is a strategy that employed by some proteins to occupy the binding sites of the DNA-binding proteins and deny further access to these sites by DNA. Such proteins have been found in bacteriophage, eukaryotic virus, prokaryotic, and eukaryotic cells to imitate non-coding functions of DNA. Here, we report another phage protein Gp44 from bacteriophage SPO1 of Bacillus subtilis, employing mimicry as part of unusual strategy to inhibit host RNA polymerase. Consisting of three simple domains, Gp44 contains a DNA binding motif, a flexible DNA mimic domain and a random-coiled domain. Gp44 is able to anchor to host genome and interact bacterial RNA polymerase via the β and β′ subunit, resulting in bacterial growth inhibition. Our findings represent a non-specific strategy that SPO1 phage uses to target different bacterial transcription machinery regardless of the structural variations of RNA polymerases. This feature may have potential applications like generation of genetic engineered phages with Gp44 gene incorporated used in phage therapy to target a range of bacterial hosts

Dissipation of Proton Motive Force is not Sufficient to Induce the Phage Shock Protein Response in Escherichia coli

Phage shock proteins (Psp) and their homologues are found in species from the three domains of life: Bacteria, Archaea and Eukarya (e.g. higher plants). In enterobacteria, the Psp response helps to maintain the proton motive force (PMF) of the cell when the inner membrane integrity is impaired. The presumed ability of ArcB to sense redox changes in the cellular quinone pool and the strong decrease of psp induction in ΔubiG or ΔarcAB backgrounds suggest a link between the Psp response and the quinone pool. The authors now provide evidence indicating that the physiological signal for inducing psp by secretin-induced stress is neither the quinone redox state nor a drop in PMF. Neither the loss of the H+-gradient nor the dissipation of the electrical potential alone is sufficient to induce the Psp response. A set of electron transport mutants differing in their redox states due to the lack of a NADH dehydrogenase and a quinol oxidase, but retaining a normal PMF displayed low levels of psp induction inversely related to oxidised ubiquinone levels under microaerobic growth and independent of PMF. In contrast, cells displaying higher secretin induced psp expression showed increased levels of ubiquinone. Taken together, this study suggests that not a single but likely multiple signals are needed to be integrated to induce the Psp response

BosR (BB0647) Controls the RpoN-RpoS Regulatory Pathway and Virulence Expression in Borrelia burgdorferi by a Novel DNA-Binding Mechanism

In Borrelia burgdorferi (Bb), the Lyme disease spirochete, the alternative σ factor σ54 (RpoN) directly activates transcription of another alternative σ factor, σS (RpoS) which, in turn, controls the expression of virulence-associated membrane lipoproteins. As is customary in σ54-dependent gene control, a putative NtrC-like enhancer-binding protein, Rrp2, is required to activate the RpoN-RpoS pathway. However, recently it was found that rpoS transcription in Bb also requires another regulator, BosR, which was previously designated as a Fur or PerR homolog. Given this unexpected requirement for a second activator to promote σ54-dependent gene transcription, and the fact that regulatory mechanisms among similar species of pathogenic bacteria can be strain-specific, we sought to confirm the regulatory role of BosR in a second virulent strain (strain 297) of Bb. Indeed, BosR displayed the same influence over lipoprotein expression and mammalian infectivity for strain Bb 297 that were previously noted for Bb strain B31. We subsequently found that recombinant BosR (rBosR) bound to the rpoS gene at three distinct sites, and that binding occurred despite the absence of consensus Fur or Per boxes. This led to the identification of a novel direct repeat sequence (TAAATTAAAT) critical for rBosR binding in vitro. Mutations in the repeat sequence markedly inhibited or abolished rBosR binding. Taken together, our studies provide new mechanistic insights into how BosR likely acts directly on rpoS as a positive transcriptional activator. Additional novelty is engendered by the facts that, although BosR is a Fur or PerR homolog and it contains zinc (like Fur and PerR), it has other unique features that clearly set it apart from these other regulators. Our findings also have broader implications regarding a previously unappreciated layer of control that can be involved in σ54–dependent gene regulation in bacteria