Improved N-phenylpyrrolamide inhibitors of DNA gyrase as antibacterial agents for high-priority bacterial strains.

In this work, we describe an improved series of N-phenylpyrrolamide inhibitors that exhibit potent activity against DNA gyrase and are highly effective against high-priority gram-positive bacteria. The most potent compounds show low nanomolar IC values against Escherichia coli DNA gyrase, and in addition, compound 7c also inhibits E. coli topoisomerase IV in the nanomolar concentration range, making it a promising candidate for the development of potent dual inhibitors for these enzymes.

Structures of Class I and Class II Transcription Complexes Reveal the Molecular Basis of RamA-Dependent Transcription Activation.

Transcription activator RamA is linked to multidrug resistance of Klebsiella pneumoniae through controlling genes that encode efflux pumps (acrA) and porin-regulating antisense RNA (micF). In bacteria, σ , together with activators, controls the majority of genes by recruiting RNA polymerase (RNAP) to the promoter regions. RNAP and σ form a holoenzyme that recognizes -35 and -10 promoter DNA consensus sites.

Bacterial Enhancer Binding Proteins-AAA Proteins in Transcription Activation.

Bacterial enhancer-binding proteins (bEBPs) are specialised transcriptional activators. bEBPs are hexameric AAA ATPases and use ATPase activities to remodel RNA polymerase (RNAP) complexes that contain the major variant sigma factor, σ to convert the initial closed complex to the transcription competent open complex. Earlier crystal structures of AAA domains alone have led to proposals of how nucleotide-bound states are sensed and propagated to substrate interactions.

Structural basis of transcription inhibition by the DNA mimic protein Ocr of bacteriophage T7.

Bacteriophage T7 infects and evades the host restriction/modification system. The Ocr protein of T7 was shown to exist as a dimer mimicking DNA and to bind to host restriction enzymes, thus preventing the degradation of the viral genome by the host. Here we report that Ocr can also inhibit host transcription by directly binding to bacterial RNA polymerase (RNAP) and competing with the recruitment of RNAP by sigma factors.

Mechanisms of σ-Dependent Transcription Initiation and Regulation.

Cellular RNA polymerase is a multi-subunit macromolecular assembly responsible for gene transcription, a highly regulated process conserved from bacteria to humans. In bacteria, sigma factors are employed to mediate gene-specific expression in response to a variety of environmental conditions. The major variant σ factor, σ, has a specific role in stress responses.

Structures of Bacterial RNA Polymerase Complexes Reveal the Mechanism of DNA Loading and Transcription Initiation.

Gene transcription is carried out by multi-subunit RNA polymerases (RNAPs). Transcription initiation is a dynamic multi-step process that involves the opening of the double-stranded DNA to form a transcription bubble and delivery of the template strand deep into the RNAP for RNA synthesis. Applying cryoelectron microscopy to a unique transcription system using σ (σ), the major bacterial variant sigma factor, we capture a new intermediate state at 4.

Functional Characterization of Key Residues in Regulatory Proteins HrpG and HrpV of Pseudomonas syringae pv. tomato DC3000.

The plant pathogen Pseudomonas syringae pv. tomato DC3000 uses a type III secretion system (T3SS) to transfer effector proteins into the host. The expression of T3SS proteins is controlled by the HrpL σ factor.

Evading plant immunity: feedback control of the T3SS in .

Microbes are responsible for over 10% of the global yield losses in staple crops such as wheat, rice, and maize. Understanding the decision-making strategies that enable bacterial plant pathogens to evade the host immune system and cause disease is essential for managing their ever growing threat to food security. Many utilise the needle-like type III secretion system (T3SS) to suppress plant immunity, by injecting effector proteins that inhibit eukaryotic signalling pathways into the host cell cytoplasm.

Negative Autogenous Control of the Master Type III Secretion System Regulator HrpL in Pseudomonas syringae.

Unlabelled: The type III secretion system (T3SS) is a principal virulence determinant of the model bacterial plant pathogen Pseudomonas syringae T3SS effector proteins inhibit plant defense signaling pathways in susceptible hosts and elicit evolved immunity in resistant plants. The extracytoplasmic function sigma factor HrpL coordinates the expression of most T3SS genes. Transcription of hrpL is dependent on sigma-54 and the codependent enhancer binding proteins HrpR and HrpS for hrpL promoter activation.

In vitro and in vivo methodologies for studying the Sigma 54-dependent transcription.

Here we describe approaches and methods to assaying in vitro the major variant bacterial sigma factor, Sigma 54 (σ(54)), in a purified system. We include the complete transcription system, binding interactions between σ54 and its activators, as well as the self-assembly and the critical ATPase activity of the cognate activators which serve to remodel the closed promoter complexes. We also present in vivo methodologies that are used to study the impact of physiological processes, metabolic states, global signalling networks, and cellular architecture on the control of σ(54)-dependent gene expression.

Interplay among Pseudomonas syringae HrpR, HrpS and HrpV proteins for regulation of the type III secretion system.

Pseudomonas syringae pv. tomato DC3000, a plant pathogenic gram-negative bacterium, employs the type III secretion system (T3SS) to cause disease in tomato and Arabidopsis and to induce the hypersensitive response in nonhost plants. The expression of T3SS is regulated by the HrpL extracytoplasmic sigma factor.

Determination of the self-association residues within a homomeric and a heteromeric AAA+ enhancer binding protein.

The σ(54)-dependent transcription in bacteria requires specific activator proteins, bacterial enhancer binding protein (bEBP), members of the AAA+ (ATPases Associated with various cellular Activities) protein family. The bEBPs usually form oligomers in order to hydrolyze ATP and make open promoter complexes. The bEBP formed by HrpR and HrpS activates transcription from the σ(54)-dependent hrpL promoter responsible for triggering the Type Three Secretion System in Pseudomonas syringae pathovars.

Activity map of the Escherichia coli RNA polymerase bridge helix.

Transcription, the synthesis of RNA from a DNA template, is performed by multisubunit RNA polymerases (RNAPs) in all cellular organisms. The bridge helix (BH) is a distinct feature of all multisubunit RNAPs and makes direct interactions with several active site-associated mobile features implicated in the nucleotide addition cycle and RNA and DNA binding. Because the BH has been captured in both kinked and straight conformations in different crystals structures of RNAP, recently supported by molecular dynamics studies, it has been proposed that cycling between these conformations is an integral part of the nucleotide addition cycle.

Regulation of the co-evolved HrpR and HrpS AAA+ proteins required for Pseudomonas syringae pathogenicity.

The bacterial AAA+ enhancer-binding proteins (EBPs) HrpR and HrpS (HrpRS) of Pseudomonas syringae (Ps) activate σ(54)-dependent transcription at the hrpL promoter; triggering type-three secretion system-mediated pathogenicity. In contrast with singly acting EBPs, the evolution of the strictly co-operative HrpRS pair raises questions of potential benefits and mechanistic differences this transcription control system offers. Here, we show distinct properties of HrpR and HrpS variants, indicating functional specialization of these non-redundant, tandemly arranged paralogues.

The role of the conserved phenylalanine in the sigma54-interacting GAFTGA motif of bacterial enhancer binding proteins.

sigma(54)-dependent transcription requires activation by bacterial enhancer binding proteins (bEBPs). bEBPs are members of the AAA+ (ATPases associated with various cellular activities) protein family and typically form hexameric structures that are crucial for their ATPase activity. The precise mechanism by which the energy derived from ATP hydrolysis is coupled to biological output has several unknowns.

The LysR-type transcriptional regulator CysB controls the repression of hslJ transcription in Escherichia coli.

The LysR-type transcriptional regulator (LTTR) CysB is a transcription factor in Escherichia coli cells, where as a homotetramer it binds the target promoter regions and activates the genes involved in sulphur utilization and sulphonate-sulphur metabolism, while negatively autoregulating its own transcription. The hslJ gene was found to be negatively regulated by CysB and directly correlated with novobiocin resistance of the bacterium. cysB mutants showed upregulation of the hslJ : : lacZ gene fusion and exhibited increased novobiocin resistance.

Identification of the CysB-regulated gene, hslJ, related to the Escherichia coli novobiocin resistance phenotype.

The cysB gene product is a LysR-type regulatory protein required for expression of the cys regulon. cysB mutants of Escherichia coli and Salmonella, along with being auxotrophs for the cysteine, exhibit increased resistance to the antibiotics novobiocin (Nov) and mecillinam. In this work, by using lambdaplacMu9 insertions creating random lacZ fusions, we identify a gene, hslJ, whose expression appeared to be increased in cysB mutants and needed for Nov resistance.