Complete genome sequence of sp. strain AH8042_DFU013_CI05 isolated from a diabetes-related foot ulcer.

This study presents the complete genome sequence of . strain AH8042_DFU013_CI05, isolated from a diabetes-related foot ulcer at The Queen Elizabeth Hospital in Adelaide, Australia. The genome comprises a 1,752,963 bp circular chromosome and a 14,073 bp plasmid, with G+C contents of 32% and 29%, respectively.

Structural insights into S-lignin O-demethylation via a rare class of heme peroxygenase enzymes.

The O-demethylation of lignin aromatics is a rate-limiting step in their bioconversion to higher-value compounds. A recently discovered cytochrome P450, SyoA, demethylates the S-lignin aromatic syringol. In this work, we solve high-resolution X-ray crystal structures of substrate-free and substrate-bound SyoA and evaluate demethylation of para-substituted S-lignin aromatics via monooxygenase and peroxide shunt pathways.

When push comes to shove - RNA polymerase and DNA-bound protein roadblocks.

In recent years, transcriptional roadblocking has emerged as a crucial regulatory mechanism in gene expression, whereby other DNA-bound obstacles can block the progression of transcribing RNA polymerase (RNAP), leading to RNAP pausing and ultimately dissociation from the DNA template. In this review, we discuss the mechanisms by which transcriptional roadblocks can impede RNAP progression, as well as how RNAP can overcome these obstacles to continue transcription. We examine different DNA-binding proteins involved in transcriptional roadblocking and their biophysical properties that determine their effectiveness in blocking RNAP progression.

Efficient -demethylation of lignin monoaromatics using the peroxygenase activity of cytochrome P450 enzymes.

A crucial reaction in harnessing renewable carbon from lignin is -demethylation. We demonstrate the selective -demethylation of syringol and guaiacol using different cytochrome P450 enzymes. These can efficiently use hydrogen peroxide which, when compared to nicotinamide cofactor-dependent monooxygenases and synthetic methods, allows for cheap and clean -demethylation of lignin-derived aromatics.

Analysis of Infection Time Courses Shows CII Levels Determine the Frequency of Lysogeny in Phage 186.

Engineered phage with properties optimised for the treatment of bacterial infections hold great promise, but require careful characterisation by a number of approaches. Phage-bacteria infection time courses, where populations of bacteriophage and bacteria are mixed and followed over many infection cycles, can be used to deduce properties of phage infection at the individual cell level. Here, we apply this approach to analysis of infection of by the temperate bacteriophage 186 and explore which properties of the infection process can be reliably inferred.

The pIT5 Plasmid Series, an Improved Toolkit for Repeated Genome Integration in .

We describe a new set of tools for inserting DNA into the bacterial chromosome. The system uses site-specific recombination reactions carried out by bacteriophage integrases to integrate plasmids at up to eight phage attachment sites in . MG1655.

Simplified heavy-atom derivatization of protein structures via co-crystallization with the MAD tetragon tetrabromoterephthalic acid.

The phase problem is a persistent bottleneck that impedes the structure-determination pipeline and must be solved to obtain atomic resolution crystal structures of macromolecules. Although molecular replacement has become the predominant method of solving the phase problem, many scenarios still exist in which experimental phasing is needed. Here, a proof-of-concept study is presented that shows the efficacy of using tetrabromoterephthalic acid (B4C) as an experimental phasing compound.

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening.

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction phase problem. Novel structures that lack a suitable homology model are often derivatized with heavy atoms to provide experimental phase information. The presented protocol efficiently generates derivatized protein crystals by combining random microseeding matrix screening with derivatization with a heavy atom molecule I3C (5-amino-2,4,6-triiodoisophthalic acid).

The loopometer: a quantitative in vivo assay for DNA-looping proteins.

Proteins that can bring together separate DNA sites, either on the same or on different DNA molecules, are critical for a variety of DNA-based processes. However, there are no general and technically simple assays to detect proteins capable of DNA looping in vivo nor to quantitate their in vivo looping efficiency. Here, we develop a quantitative in vivo assay for DNA-looping proteins in Escherichia coli that requires only basic DNA cloning techniques and a LacZ assay.

Instability of CII is needed for efficient switching between lytic and lysogenic development in bacteriophage 186.

The CII protein of temperate coliphage 186, like the unrelated CII protein of phage λ, is a transcriptional activator that primes expression of the CI immunity repressor and is critical for efficient establishment of lysogeny. 186-CII is also highly unstable, and we show that in vivo degradation is mediated by both FtsH and RseP. We investigated the role of CII instability by constructing a 186 phage encoding a protease resistant CII.

A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186.

The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs.

Advanced Resistance Studies Identify Two Discrete Mechanisms in to Overcome Antibacterial Compounds that Target Biotin Protein Ligase.

Biotin protein ligase (BPL) inhibitors are a novel class of antibacterial that target clinically important methicillin-resistant () In BPL is a bifunctional protein responsible for enzymatic biotinylation of two biotin-dependent enzymes, as well as serving as a transcriptional repressor that controls biotin synthesis and import. In this report, we investigate the mechanisms of action and resistance for a potent anti-BPL, an antibacterial compound, biotinyl-acylsulfamide adenosine (BASA). We show that BASA acts by both inhibiting the enzymatic activity of BPL in vitro as well as functioning as a transcription co-repressor.

Combining random microseed matrix screening and the magic triangle for the efficient structure solution of a potential lysin from bacteriophage P68.

Two commonly encountered bottlenecks in the structure determination of a protein by X-ray crystallography are screening for conditions that give high-quality crystals and, in the case of novel structures, finding derivatization conditions for experimental phasing. In this study, the phasing molecule 5-amino-2,4,6-triiodoisophthalic acid (I3C) was added to a random microseed matrix screen to generate high-quality crystals derivatized with I3C in a single optimization experiment. I3C, often referred to as the magic triangle, contains an aromatic ring scaffold with three bound I atoms.

RNA polymerase pausing at a protein roadblock can enhance transcriptional interference by promoter occlusion.

Convergent promoters exert transcriptional interference (TI) by several mechanisms including promoter occlusion, where elongating RNA polymerases (RNAPs) block access to a promoter. Here, we tested whether pausing of RNAPs by obstructive DNA-bound proteins can enhance TI by promoter occlusion. Using the Lac repressor as a 'roadblock' to induce pausing over a target promoter, we found only a small increase in TI, with mathematical modelling suggesting that rapid termination of the stalled RNAP was limiting the occlusion effect.

Positive and Negative Control of Enhancer-Promoter Interactions by Other DNA Loops Generates Specificity and Tunability.

Enhancers are ubiquitous and critical gene-regulatory elements. However, quantitative understanding of the role of DNA looping in the regulation of enhancer action and specificity is limited. We used the Escherichia coli NtrC enhancer-σ54 promoter system as an in vivo model, finding that NtrC activation is highly sensitive to the enhancer-promoter (E-P) distance in the 300-6,000 bp range.

Native mass spectrometry identifies an alternative DNA-binding pathway for BirA from Staphylococcus aureus.

An adequate supply of biotin is vital for the survival and pathogenesis of Staphylococcus aureus. The key protein responsible for maintaining biotin homeostasis in bacteria is the biotin retention protein A (BirA, also known as biotin protein ligase). BirA is a bi-functional protein that serves both as a ligase to catalyse the biotinylation of important metabolic enzymes, as well as a transcriptional repressor that regulates biotin biosynthesis, biotin transport and fatty acid elongation.

Characterizing Transcriptional Interference between Converging Genes in Bacteria.

Antisense transcription is common in naturally occurring genomes and is increasingly being used in synthetic genetic circuitry as a tool for gene expression control. Mutual influence on the expression of convergent genes can be mediated by antisense RNA effects and by transcriptional interference (TI). We aimed to quantitatively characterize long-range TI between convergent genes with untranslated intergenic spacers of increasing length.

Biotin-mediated growth and gene expression in Staphylococcus aureus is highly responsive to environmental biotin.

Biotin (Vitamin B7) is a critical enzyme co-factor in metabolic pathways important for bacterial survival. Biotin is obtained either from the environment or by de novo synthesis, with some bacteria capable of both. In certain species, the bifunctional protein BirA plays a key role in biotin homeostasis as it regulates expression of biotin biosynthetic enzymes in response to biotin demand and supply.

Programmable DNA looping using engineered bivalent dCas9 complexes.

DNA looping is a ubiquitous and critical feature of gene regulation. Although DNA looping can be efficiently detected, tools to readily manipulate DNA looping are limited. Here we develop CRISPR-based DNA looping reagents for creation of programmable DNA loops.

Hidden Secrets of Sigma54 Promoters Revealed.

Bacterial sigma54 (σ) promoters are the DNA-binding motif for σ-containing RNA polymerase (RNAP) holoenzymes. A recent study using a combination of synthetic oligonucleotide library screening, biochemical characterization, and bioinformatics has uncovered a new and unexpected role for σ promoters, encoding a form of bacterial 'insulator sequence' to dampen unwanted translation.

Aspects of protein-DNA interactions: a review of quantitative thermodynamic theory for modelling synthetic circuits utilising LacI and CI repressors, IPTG and the reporter gene lacZ.

Protein-DNA interactions are central to the control of gene expression across all forms of life. The development of approaches to rigorously model such interactions has often been hindered both by a lack of quantitative binding data and by the difficulty in accounting for parameters relevant to the intracellular situation, such as DNA looping and thermodynamic non-ideality. Here, we review these considerations by developing a thermodynamically based mathematical model that attempts to simulate the functioning of an Escherichia coli expression system incorporating two of the best characterised prokaryotic DNA binding proteins, Lac repressor and lambda CI repressor.

Efficient chromosomal-scale DNA looping in Escherichia coli using multiple DNA-looping elements.

Genes are frequently regulated by interactions between proteins that bind to the DNA near the gene and proteins that bind to DNA sites located far away, with the intervening DNA looped out. But it is not understood how efficient looping can occur when the sites are very far apart. We develop a simple theoretical framework that relates looping efficiency to the energetic cost and benefit of looping, allowing prediction of the efficiency of single or multiple nested loops at different distances.

Directing traffic on DNA-How transcription factors relieve or induce transcriptional interference.

Transcriptional interference (TI) is increasingly recognized as a widespread mechanism of gene control, particularly given the pervasive nature of transcription, both sense and antisense, across all kingdoms of life. Here, we discuss how transcription factor binding kinetics strongly influence the ability of a transcription factor to relieve or induce TI.

Clonetegration Using OSIP Plasmids: One-Step DNA Assembly and Site-Specific Genomic Integration in Bacteria.

Clonetegration is a method for site-specific insertion of DNA into prokaryotic chromosomes, based on bacteriophage integrases. The method combines DNA cloning/assembly and chromosomal integration into a single step, providing a simple and rapid strategy for inserting DNA sequences into bacterial chromosomes.

The role of repressor kinetics in relief of transcriptional interference between convergent promoters.

Transcriptional interference (TI), where transcription from a promoter is inhibited by the activity of other promoters in its vicinity on the same DNA, enables transcription factors to regulate a target promoter indirectly, inducing or relieving TI by controlling the interfering promoter. For convergent promoters, stochastic simulations indicate that relief of TI can be inhibited if the repressor at the interfering promoter has slow binding kinetics, making it either sensitive to frequent dislodgement by elongating RNA polymerases (RNAPs) from the target promoter, or able to be a strong roadblock to these RNAPs. In vivo measurements of relief of TI by CI or Cro repressors in the bacteriophage λ PR-PRE system show strong relief of TI and a lack of dislodgement and roadblocking effects, indicative of rapid CI and Cro binding kinetics.