Sivaramesh Wigneshweraraj

Opening and Closing of the Bacterial RNA Polymerase Clamp

Clamping Down Crystal structures of RNA polymerase show that a “clamp” region which surrounds the DNA binding site can adopt conformations ranging from a closed to an open state. Chakraborty et al. (p. 591) used single-molecule fluorescence energy transfer experiments to detect the clamps conformational changes in solution during the transcription cycle. The results support a model in which a clamp opening allows DNA to be loaded into the active-center cleft and unwound. Direct interactions with DNA likely trigger clamp closure upon formation of a catalytically competent transcription initiation complex. Single-molecule fluorescence measurements define the clamp conformation during transcription initiation and elongation. Using single-molecule fluorescence resonance energy transfer, we have defined bacterial RNA polymerase (RNAP) clamp conformation at each step in transcription initiation and elongation. We find that the clamp predominantly is open in free RNAP and early intermediates in transcription initiation but closes upon formation of a catalytically competent transcription initiation complex and remains closed during initial transcription and transcription elongation. We show that four RNAP inhibitors interfere with clamp opening. We propose that clamp opening allows DNA to be loaded into and unwound in the RNAP active-center cleft, that DNA loading and unwinding trigger clamp closure, and that clamp closure accounts for the high stability of initiation complexes and the high stability and processivity of elongation complexes.

Modus operandi of the bacterial RNA polymerase containing the σ54 promoter‐specificity factor

Bacterial sigma (σ) factors confer gene specificity upon the RNA polymerase, the central enzyme that catalyses gene transcription. The binding of the alternative σ factor σ54 confers upon the RNA polymerase special functional and regulatory properties, making it suited for control of several major adaptive responses. Here, we summarize our current understanding of the interactions the σ54 factor makes with the bacterial transcription machinery.

Phage T7 Gp2 inhibition of Escherichia coli RNA polymerase involves misappropriation of σ70 domain 1.1

Significance After infection of Escherichia coli by bacteriophage T7, the host RNA polymerase (RNAP) produces early phage transcription products that encode the phages own RNAP (that transcribes subsequent phage genes) as well as Gp2, an essential inhibitor of the host RNAP. X-ray crystal structures of E. coli RNAP define the structure and location of the RNAP σ70 subunit domain 1.1 inside the RNAP active site channel, where it must be displaced by the DNA upon formation of the transcription complex. Gp2 binds inside the RNAP active site channel and also interacts with , preventing from exiting the RNAP active site channel. Gp2 thus misappropriates a domain of the RNAP, , to inhibit the function of the enzyme. Bacteriophage T7 encodes an essential inhibitor of the Escherichia coli host RNA polymerase (RNAP), the product of gene 2 (Gp2). We determined a series of X-ray crystal structures of E. coli RNAP holoenzyme with or without Gp2. The results define the structure and location of the RNAP σ70 subunit domain 1.1 inside the RNAP active site channel, where it must be displaced by the DNA upon formation of the open promoter complex. The structures and associated data, combined with previous results, allow for a complete delineation of the mechanism for Gp2 inhibition of E. coli RNAP. In the primary inhibition mechanism, Gp2 forms a protein–protein interaction with , preventing the normal egress of from the RNAP active site channel. Gp2 thus misappropriates a domain of the RNAP holoenzyme, , to inhibit the function of the enzyme.

Nitrogen stress response and stringent response are coupled in Escherichia coli

Assimilation of nitrogen is an essential process in bacteria. The nitrogen regulation stress response is an adaptive mechanism used by nitrogen-starved Escherichia coli to scavenge for alternative nitrogen sources and requires the global transcriptional regulator NtrC. In addition, nitrogen-starved E. coli cells synthesize a signal molecule, guanosine tetraphosphate (ppGpp), which serves as an effector molecule of many processes including transcription to initiate global physiological changes, collectively termed the stringent response. The regulatory mechanisms leading to elevated ppGpp levels during nutritional stresses remain elusive. Here, we show that transcription of relA, a key gene responsible for the synthesis of ppGpp, is activated by NtrC during nitrogen starvation. The results reveal that NtrC couples these two major bacterial stress responses to manage conditions of nitrogen limitation, and provide novel mechanistic insights into how a specific nutritional stress leads to elevating ppGpp levels in bacteria.

What role does the quorum-sensing accessory gene regulator system play during Staphylococcus aureus bacteremia?

Staphylococcus aureus is a major cause of bacteremia, which frequently results in serious secondary infections such as infective endocarditis, osteomyelitis, and septic arthritis. The ability of S. aureus to cause such a wide range of infections has been ascribed to its huge armoury of different virulence factors, many of which are under the control of the quorum-sensing accessory gene regulator (Agr) system. However, a significant fraction of S. aureus bacteremia cases are caused by agr-defective isolates, calling into question the role of Agr in invasive staphylococcal infections. This review draws on recent work to define the role of Agr during bacteremia and explain why the loss of this major virulence regulator is sometimes a price worth paying for S. aureus.

Structural and Mechanistic Basis for the Inhibition of Escherichia coli RNA Polymerase by T7 Gp2

Summary The T7 phage-encoded small protein Gp2 is a non-DNA-binding transcription factor that interacts with the jaw domain of the Escherichia coli (Ec) RNA polymerase (RNAp) β′ subunit and inhibits transcriptionally proficient promoter-complex (RPo) formation. Here, we describe the high-resolution solution structure of the Gp2-Ec β′ jaw domain complex and show that Gp2 and DNA compete for binding to the β′ jaw domain. We reveal that efficient inhibition of RPo formation by Gp2 requires the amino-terminal σ70 domain region 1.1 (R1.1), and that Gp2 antagonizes the obligatory movement of R1.1 during RPo formation. We demonstrate that Gp2 inhibits RPo formation not just by steric occlusion of the RNAp-DNA interaction but also through long-range antagonistic effects on RNAp-promoter interactions around the RNAp active center that likely occur due to repositioning of R1.1 by Gp2. The inhibition of Ec RNAp by Gp2 thus defines a previously uncharacterized mechanism by which bacterial transcription is regulated by a viral factor.

Nitrogen and Carbon Status Are Integrated at the Transcriptional Level by the Nitrogen Regulator NtrC In Vivo

ABSTRACT Nitrogen regulation in Escherichia coli is a model system for gene regulation in bacteria. Growth on glutamine as a sole nitrogen source is assumed to be nitrogen limiting, inferred from slow growth and strong NtrB/NtrC-dependent gene activation. However, we show that under these conditions, the intracellular glutamine concentration is not limiting but 5.6-fold higher than in ammonium-replete conditions; in addition, α-ketoglutarate concentrations are elevated. We address this glutamine paradox from a systems perspective. We show that the dominant role of NtrC is to regulate glnA transcription and its own expression, indicating that the glutamine paradox is not due to NtrC-independent gene regulation. The absolute intracellular NtrC and GS concentrations reveal molecular control parameters, where NtrC-specific activities were highest in nitrogen-starved cells, while under glutamine growth, NtrC showed intermediate specific activity. We propose an in vivo model in which α-ketoglutarate can derepress nitrogen regulation despite nitrogen sufficiency. IMPORTANCE Nitrogen is the most important nutrient for cell growth after carbon, and its metabolism is coordinated at the metabolic, transcriptional, and protein levels. We show that growth on glutamine as a sole nitrogen source, commonly assumed to be nitrogen limiting and used as such as a model system for nitrogen limitation, is in fact nitrogen replete. Our integrative quantitative analysis of key molecules involved in nitrogen assimilation and regulation reveal that glutamine is not necessarily the dominant molecule signaling nitrogen sufficiency and that α-ketoglutarate may play a more important role in signaling nitrogen status. NtrB/NtrC integrates α-ketoglutarate and glutamine signaling—sensed by the UTase (glnD) and PII (glnB), respectively—and regulates the nitrogen response through self-regulated expression and phosphorylation-dependent activation of the nitrogen (ntr) regulon. Our findings support α-ketoglutarate acting as a global regulatory metabolite. Nitrogen is the most important nutrient for cell growth after carbon, and its metabolism is coordinated at the metabolic, transcriptional, and protein levels. We show that growth on glutamine as a sole nitrogen source, commonly assumed to be nitrogen limiting and used as such as a model system for nitrogen limitation, is in fact nitrogen replete. Our integrative quantitative analysis of key molecules involved in nitrogen assimilation and regulation reveal that glutamine is not necessarily the dominant molecule signaling nitrogen sufficiency and that α-ketoglutarate may play a more important role in signaling nitrogen status. NtrB/NtrC integrates α-ketoglutarate and glutamine signaling—sensed by the UTase (glnD) and PII (glnB), respectively—and regulates the nitrogen response through self-regulated expression and phosphorylation-dependent activation of the nitrogen (ntr) regulon. Our findings support α-ketoglutarate acting as a global regulatory metabolite.

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.

Molecular insights into the control of transcription initiation at the Staphylococcus aureus agr operon.

The accessory gene regulatory (agr) operon of the opportunistic human pathogen Staphylococcus aureus is a prime pathogenesis factor in this bacterium. The agr operon consists of two transcription units, RNAII and RNAIII, which are transcribed from divergent promoters, P2 and P3, respectively. RNAII encodes a quorum-sensing system, including AgrA, the master transcription activator of the agr operon. RNAIII is the effector RNA molecule that regulates the expression of many virulence genes. Owing to the atypical spacer lengths of P2 and P3, it is widely considered that transcription from P2 and P3 only occurs in a strictly AgrA-dependent manner. Here, using a fully native S. aureus in vitro transcription system, we provide the first molecular and mechanistic characterisation of the regulation of transcription initiation at the agr operon. Surprisingly, the results demonstrate that RNA polymerase (RNAp) can interact with P2 and P3 equally well in the absence of AgrA. However, formation of a transcription-competent open promoter complex (RPo) occurs more readily at P2 than at P3 when AgrA is absent. Reducing the atypical P3 spacer region length to the optimal length of 17 nucleotides significantly improves promoter activity by facilitating the isomerisation of the initial RNAp-P3 complexes to RPo, and the extended -10-like element of P3 is required for optimal promoter activity. AgrA increases the occupancy of both promoters by RNAp and thereby increases the amount of transcription initiated at P2 and P3. However, the AgrA-mediated effect on transcription initiation is more prominent at P3 that at P2. The effect of AgrA at P2 and P3 appears to be restricted to events leading to the formation of RPo. The relevance of AgrA-independent and AgrA-dependent transcription initiation at P2 and P3 is presented in the context of our current understanding of the role of the agr operon in the pathobiology of S. aureus.

A role for the conserved GAFTGA motif of AAA+ transcription activators in sensing promoter DNA conformation

Transcription from σ54-dependent bacterial promoters can be regarded as a second paradigm for bacterial gene transcription. The initial σ54-RNA polymerase (RNAP)·promoter complex, the closed complex, is transcriptionally silent. The transcriptionally proficient σ54-RNAP·promoter complex, the open complex, is formed upon remodeling of the closed complex by actions of a specialized activator protein that belongs to the AAA (ATPases associated with various cellular activities) protein family in an ATP hydrolysis-dependent reaction. The integrity of a highly conserved signature motif in the AAA activator (known as the GAFTGA motif) is important for the remodeling activity of the AAA activator and for open complex formation. We now provide evidence that the invariant threo-nine residue of the GAFTGA motif plays a role in sensing the DNA downstream of the σ54-RNAP-binding site and in coupling this information to σ54-RNAP via the conserved regulatory Region I domain of σ54 during open complex formation.