Immobilised Metal Affinity Chromatography (IMAC) is widely used to purify his-tagged recombinant proteins from Escherichia coli. However, endogenous contaminants with histidine clusters, such as GFAT and PDH E1 proteins, are often co-purified with the target protein. The low background strain LOBSTR-RIL has been previously engineered with mutated forms of SlyD and ArnA that exhibit reduced binding to Ni2+ resin. In this study, the LOBSTR-RIL strain was further modified to produce IMACulate(DE3), where we altered the glmS (encoding GFAT protein) and aceE (encoding PDH E1 protein) genes to reduce surface histidines. Proteins purified from this strain show reduced levels of GFAT contamination. No statistically significant difference was observed in the abundance levels of PDH E1 protein in the BL21(DE3)-RIL, LOBSTR-RIL and IMACulate(DE3) strains. The use of IMACulate(DE3) increases the purity of recombinant his-tagged protein preparations with no additional effort or expense.

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. We find that SyoA demethylates S-lignin aromatics exclusively using the peroxide shunt pathway. The atomic-resolution structures reveal the position of non-canonical residues in the I-helix (Gln252, Glu253). Mutagenesis of this amide-acid pair in SyoA shows they are critical for catalytic activity. This work expands the enzymatic toolkit for improving the capacity to funnel lignin derived aromatics towards higher value compounds and defines the chemistry within the active site of the enzyme that enables peroxygenase activity. These insights provide a framework for engineering peroxygenase activity in other heme enzymes to generate easier to use biocatalysts.

The sex chromosomes of egg-laying mammals (monotremes), which lack the sex determining gene SRY, evolved independently to those of all therian mammals. Here we characterise the candidate monotreme sex determining gene, the Y-localised anti-Müllerian hormone gene (AMHY) and trace its expression during the period of sexual differentiation. Monotreme AMHX and AMHY gametologues have significant sequence divergence at the promoter, gene and protein level, likely following an original allele inversion in the common monotreme ancestor but retain conserved features of TGF-β molecules. Expression of sexual differentiation genes in the echidna fetal gonad were significantly different from that of therian mammals. AMHY expression was seen exclusively in the male gonad during sexual differentiation, whereas AMHX was expressed in both sexes. Experimental ectopic expression of platypus AMHX or AMHY in the chicken embryo did not masculinise the female urogenital system, a possible result of mammalian specific changes to AMH proteins preventing function in the chicken. Our results provide fundamental insight into the first steps of monotreme sex chromosome evolution and sex determination with developmental expression data strongly supporting AMHY as the primary male sex determination gene.

This study presents the complete genome sequence of Anaerococcus sp. 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.

The O -demethylation of lignin aromatics is a rate-limiting step in their bioconversion to high-value compounds. A recently discovered cytochrome P450 enzyme SyoA was found to demethylate the sinapyl alcohol-derived (S-lignin) aromatic syringol. In this work, we solved high-resolution X-ray crystal structures of SyoA in the substrate-free and substrate-bound states and evaluate the demethylation of para -substituted S-lignin aromatics via the monooxygenase pathway and peroxide shunt pathway. We found that SyoA demethylates S-lignin aromatics with the following activity: 4-methylsyringol > syringaldehyde > syringol exclusively using the peroxide shunt pathway. The atomic-resolution structure of SyoA reveals the position of the non-canonical residues in the I-helix (Gln252 and Glu253). Site-directed mutagenesis of this amide-acid pair of a homologous CYP255 enzyme GcoA, which can catalyze the O-demethylation of guaiacol using both monooxygenase and peroxygenase activity, showed the amide-acid pair is critical for both pathways. This work expands the enzymatic toolkit for improving the capacity to funnel lignin towards high-value compounds, and defines the new chemistry within the active site of the enzyme that enables efficient peroxygenase activity. These insights provide a framework for engineered peroxygenase activity in other cytochrome P450 enzymes, with the potential for more facile catalysis, relative to traditional P450 monooxygenases which require difficult to handle redox partners and expensive nicotinamide cofactors. ### Competing Interest Statement The authors have declared no competing interest.

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. The catalytically dead CRISPR-Cas (dCas) protein is used as an example of an engineered programmable roadblock, and the current literature in understanding the polarity of dCas roadblocking is also discussed. Finally, we delve into a stochastic model of transcriptional roadblocking and highlight the importance of transcription factor binding kinetics and its resistance to dislodgement by an elongating RNAP in determining the strength of a roadblock.

Staphylococcus aureus colonizes 30% of the human population, but only a few clones cause severe infections. S. aureus’ virulence varies and partly depends on the presence of prophages, viral DNA embedded in the S. aureus core genome, such as hlb-converting prophage (ϕSa3int). Human-adapted S. aureus often harbours a ϕSa3int group of prophages preferentially integrated into their β-hemolysin ( hlb ) gene that encodes human immune evasion cluster (IEC) genes. Exotoxins and immune modulatory molecules encoded by this prophage can inhibit human innate immunity increasing S. aureus pathogenicity. This study aims to investigate the genomic and phenotypic plasticity of S. aureus and changes in its extracellular proteome after the acquisition of ϕSa3int prophage.To achieve this, we used S. aureus strains isolated from the sinus cavities of a patient with severe chronic rhinosinusitis (CRS) at two different time points ( S. aureus SA222 and S. aureus SA333) and hybrid sequenced the strains using short-read Illumina and long-read Oxford nanopore technology. In silico analysis showed the presence of a ϕSa3int prophage in the later isolate but not in the earlier isolate while most of the core genes remained identical. Using mitomycin C, we induced the ϕSa3int prophage, and transduced it into the Sa3int-prophage-free SA222 isolate to obtain a laboratory generated ‘double lysogen’. We confirmed the successful lysogenisation with culture methods (spot assay, blood agar) and also by sequencing. We compared growth kinetics, biofilm biomass and metabolic activity between parent and the lysogen by establishing growth curves, crystal violet and resazurin assays. Exoproteins were identified and quantified using mass spectrophotometry.Integration of ϕSa3int prophage in SA222 down-regulated the beta-hemolysin expression of the lysogen . In silico analysis of the S. aureus genome confirmed the insertion of a ∼43.8 kb ϕSa3int prophage into hlb gene. Insertion of prophage DNA did not alter the growth kinetics, biofilm formation, adhesion to primary human nasal epithelial cells and the metabolic activity in a biofilm. However, the acquisition of ϕSa3int prophage significantly changed the expression of various secreted proteins, both bacterial and prophage-encoded. Altogether, thirty-eight exoproteins were significantly differentially regulated in the laboratory created lysogen, compared to its recipient strain SA222. Among these proteins, there was significant upregulation of 21 exoproteins (55.3 %) including staphylokinase (sak), SCIN (scn), and intercellular adhesion protein B (icaB) and downregulation of 17 exoproteins (44.7 %), including β-hemolysin (hlb/sph) and outer membrane porin (phoE). Most of the upregulated proteins were involved in immunomodulation that help S. aureus escape human innate immunity and help cause chronic infection. These findings may contribute to the development of novel approaches to render S. aureus susceptible to the immune response by blocking prophage-associated defence mechanisms.Highlights ### Competing Interest StatementThe authors have declared no competing interest.* ### Glossary Active lysogen : bacterial strain harboring identifiable prophage sequence and releasing reinfecting phage particles. Double lysogen : bacterial cell harbouring two complete/intact prophages Lysogen : bacterial cell containing one or more prophages within its genome Lysogenic conversion : phenotypic change in a host bacterium caused by the integration of a prophage. Lysogeny : state of phage integration into the bacterial genome. Non-lysogen : bacterial strains lacking any identifiable prophage sequence in their genome Passive lysogen : bacterial strains harboring identifiable prophage sequence but not releasing actively reinfecting phage particles Productive induction : excision of prophage from the bacterial chromosome followed by release of phage particles either spontaneously or under external stress Prophage : temperate phage DNA integrated in the bacterial genome. Single lysogen : bacterial cell harbouring only one complete/intact prophage Temperate phage : bacterial virus that can integrate into a bacterial genome (or be maintained extra-chromosomally), become stabilized in this way, and, upon receiving a cue, can excise and propagate. Transduction : process of horizontal gene transfer, wherein a region of a bacterial genome is packaged into phage particles that, upon release and entrance into a new host, is inserted into the genome of the latter.

AbstractStaphylococcus aureuscolonizes 30% of the human population, but only a few clones cause severe infections.S. aureus’virulence varies and partly depends on the presence of prophages, viral DNA embedded in theS. aureuscore genome, such as hlb-converting prophage (ϕSa3int). Human-adaptedS. aureusoften harbours a ϕSa3int group of prophages preferentially integrated into their β-hemolysin (hlb) gene that encodes human immune evasion cluster (IEC) genes. Exotoxins and immune modulatory molecules encoded by this prophage can inhibit human innate immunity increasingS. aureuspathogenicity. This study aims to investigate the genomic and phenotypic plasticity ofS. aureusand changes in its extracellular proteome after the acquisition of ϕSa3int prophage.To achieve this, we usedS. aureusstrains isolated from the sinus cavities of a patient with severe chronic rhinosinusitis (CRS) at two different time points (S. aureusSA222 andS. aureusSA333) and hybrid sequenced the strains using short-read Illumina and long-read Oxford nanopore technology.In silicoanalysis showed the presence of a ϕSa3int prophage in the later isolate but not in the earlier isolate while most of the core genes remained identical. Using mitomycin C, we induced the ϕSa3int prophage, and transduced it into the Sa3int-prophage-free SA222 isolate to obtain a laboratory generated ‘double lysogen’. We confirmed the successful lysogenisation with culture methods (spot assay, blood agar) and also by sequencing. We compared growth kinetics, biofilm biomass and metabolic activity between parent and the lysogen by establishing growth curves, crystal violet and resazurin assays. Exoproteins were identified and quantified using mass spectrophotometry.Integration of ϕSa3int prophage in SA222 down-regulated the beta-hemolysin expression of the lysogen. In silicoanalysis of theS. aureusgenome confirmed the insertion of a ∼43.8 kb ϕSa3int prophage intohlbgene. Insertion of prophage DNA did not alter the growth kinetics, biofilm formation, adhesion to primary human nasal epithelial cells and the metabolic activity in a biofilm. However, the acquisition of ϕSa3int prophage significantly changed the expression of various secreted proteins, both bacterial and prophage-encoded. Altogether, thirty-eight exoproteins were significantly differentially regulated in the laboratory created lysogen, compared to its recipient strain SA222. Among these proteins, there was significant upregulation of 21 exoproteins (55.3 %) including staphylokinase (sak), SCIN (scn), and intercellular adhesion protein B (icaB) and downregulation of 17 exoproteins (44.7 %), including β-hemolysin (hlb/sph) and outer membrane porin (phoE). Most of the upregulated proteins were involved in immunomodulation that helpS. aureusescape human innate immunity and help cause chronic infection. These findings may contribute to the development of novel approaches to render S.aureussusceptible to the immune response by blocking prophage-associated defence mechanisms.HighlightsA ϕSa3int prophage preferentially integrates into the β-haemolysin gene (hlb) gene thereby disrupting the beta-hemolysin function.A ∼43.8 kb ϕSa3int prophage acquisition byS. aureushas no impact on its growth kinetics, biofilm formation and adhesion to primary human nasal epithelial cells (HNECs).The presence of a ϕSa3int group prophage likely enhancesStaphylococcus aureus’human immune evasion capability as the prophage encodes a complete set of immune evasion cluster (IEC) genes.Targeted identification of virulence factors in addition to species and strain identification may lead to better-personalized therapy as not allS. aureuscarry the same virulence genes.

The high infection and mortality rate of methicillin-resistant Staphylococcus aureus (MRSA) necessitates the urgent development of new treatment strategies. Bacteriophages (phages) have several advantages compared to antibiotics for the treatment of multi-drug-resistant bacterial infections, and thus provide a promising alternative to antibiotics. Here, S. aureus phages were isolated from patients and environmental sources. Phages were characterized for stability, morphology and genomic sequence and their bactericidal activity against the biofilm form of methicillin-susceptible Staphylococcus aureus (MSSA) and MRSA was investigated. Four S. aureus phages were isolated and tested against 51 MSSA and MRSA clinical isolates and reference strains. The phages had a broad host range of 82–94% individually and of >98% when combined and could significantly reduce the viability of S. aureus biofilms. The phages had a latent period of ≤20 min and burst size of >11 plaque forming units (PFU)/infected cell. Transmission electron microscopy (TEM) identified phages belonging to the family of Myoviridae. Genomic sequencing indicated the lytic nature of all four phages, with no identified resistance or virulence genes. The 4 phages showed a high complementarity with 49/51 strains (96%) sensitive to at least 2/4 phages tested. Furthermore, the frequency of bacteriophage insensitive mutant (BIM) generation was lower when the phages were combined into the phage cocktail APTC-C-SA01 than for bacteria exposed to each of the phages alone. In conclusion, APTC-C-SA01, containing four lytic S. aureus phages has the potential for further development as a treatment against MSSA and MRSA infections.

A crucial reaction in harnessing renewable carbon from lignin is O-demethylation. We demonstrate the selective O-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 O-demethylation of lignin-derived aromatics.

On behalf of the Australian Society for Biophysics (ASB) and the Editors of this Special Issue, I would like to express our appreciation to Editor-in-Chief, Damien Hall, for arranging the publication of this Special Issue. The ASB is about five times smaller than our sister the Biophysical Society for Japan (BSJ) and tenfold smaller than the US Biophysical Society (USBS), but our meetings are notable because of the encouragement the Society gives to emerging biophysicists. It can be a terrifying experience for a PhD student to have to face a roomful of professors and senior academics, but invariably they appreciate the experience. Another feature of the ASB meetings is the inclusion of contributions from the Asian Pacific region. We now have formal ties with our New Zealand colleagues and our meetings with the BSJ contain joint sessions (see below). In 2020, despite the impact of COVID-19 (see Adam Hill's Commentary), there is a joint session with the University of California Davis. This Special Issue comprises 2 Editorials, 3 Commentaries, and 25 reviews.

This Commentary represents the first instalment of a regular feature that seeks to fulfil one of Biophysical Reviews' IUPAB mandated goals-that of assisting in the international promotion of biophysical research. Known as the 'Editors' Roundup', this Commentary feature is a multi-author collective description of recently published research from journals publishing material across the biophysical realm. Although Biophysical Reviews is published by Springer-Nature, the source of contributed articles is unrestricted and can include different commercial and society publishers. In this edition we have article descriptions from the following journals, Biophysical Reviews, Biophysics and Physicobiology and Cell Biochemistry and Biophysics.

DNA can act as a scaffold for the cooperative binding of protein oligomers. For example, the phage 186 CI repressor forms a wheel of seven dimers wrapped in DNA with specific binding sites, while phage λ CI repressor dimers bind to two well‐separated sets of operators, forming a DNA loop. Atomic force microscopy was used to measure transcription elongation by Escherichia coli RNA polymerase (RNAP) through these protein complexes. 186 CI, or λ CI, bound along unlooped DNA negligibly interfered with transcription by RNAP. Wrapped and looped topologies induced by these scaffolded, cooperatively bound repressor oligomers did not form significantly better roadblocks to transcription. Thus, despite binding with high affinity, these repressors are not effective roadblocks to transcription.

Many DNA-binding proteins induce topological structures such as loops or wraps through binding to two or more sites along the DNA. Such topologies may regulate transcription initiation and may also be roadblocks for elongating RNA polymerase (RNAP). Remarkably, a lac repressor protein bound to a weak binding site (O2) does not obstruct RNAP in vitro but becomes an effective roadblock when securing a loop of 400 bp between two widely separated binding sites. To investigate whether topological structures mediated by proteins bound to closely spaced binding sites and interacting cooperatively also represent roadblocks, we compared the effect of the lambda CI and 186 CI repressors on RNAP elongation. Dimers of lambda CI can bind to two sets of adjacent sites separated by hundreds of bp and form a DNA loop via the interaction between their C-terminal domains. The 186 CI protein can form a wheel of seven dimers around which specific DNA binding sequences can wrap. Atomic force microscopy (AFM) was used to image transcription elongation complexes of DNA templates that contained binding sites for either the lambda or 186 CI repressor. While RNAP elongated past lambda CI on unlooped DNA, as well as past 186 CI-wrapped DNA, it did not pass the lambda CI-mediated loop. These results may indicate that protein-mediated loops with widely separated binding sites more effectively block transcription than a wrapped topology with multiple, closely spaced binding sites.

Supplementary material for "Prophage Encoded Human Immune Evasion Cluster Genes are Enriched in Staphylococcus aureus Isolated from Chronic Rhinosinusitis Patients with Nasal Polyps"

non-CRS (Control) sequences for paper entitled "Prophage Encoded Human Immune Evasion Cluster Genes are Enriched in Staphylococcus aureus Isolated from Chronic Rhinosinusitis Patients with Nasal Polyps"

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). By incorporating I3C into the crystal lattice, the diffraction phase problem can be efficiently solved using single wavelength anomalous dispersion (SAD) phasing. The equilateral triangle arrangement of iodine atoms in I3C allows for rapid validation of a correct anomalous substructure. This protocol will be useful to structural biologists who solve macromolecular structures using crystallography-based techniques with interest in experimental phasing.

BACKGROUND:Staphylococcus aureus is a pathogen of major concern in both acute infections and chronic conditions such as chronic rhinosinusitis (CRS). Bacteriophage (phage) therapy has recently regained interest for its potential to treat infections caused by antibiotic resistant strains including Methicillin Resistant Staphylococcus aureus (MRSA). However, bacteria can adapt and become resistant to phages. The aim of this study is to determine the potential for antibiotics to overcome phage resistance.METHODS:The susceptibility of S. aureus clinical isolates (CIs) to phages J-Sa36, Sa83 and Sa87 alone or in combination with protein synthesis inhibitor (PSI) antibiotics clindamycin, azithromycin and erythromycin was assessed using plaque spot assays, minimum inhibitory concentration (MIC) assays, double layer spot assays and resazurin assays. The safety and efficacy of subinhibitory PSI antibiotics in combination with phage was tested in a Sprague Dawley rat model of sinusitis infected with a phage resistant S. aureus CI.RESULTS:All three antibiotics at subinhibitory concentrations showed synergy when combined with all 3 phages against S. aureus CIs in planktonic and biofilm form and could sensitize phage-resistant S. aureus to promote phage infection. The combination of topical subinhibitory clindamycin or azithromycin and phage was safe and could eradicate S. aureus sinonasal biofilms in vivo.CONCLUSION:Subinhibitory concentrations of PSI antibiotics could sensitize phage-resistant S. aureus and MRSA strains to phages in vitro and in vivo. This data supports the potential use of phage-PSI antibiotic combination therapies, in particular for difficult-to-treat infections with phage-resistant S. aureus and MRSA strains.

Abstract 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. The assay is based on loop assistance, where two binding sites for the candidate looping protein are inserted internally to a pair of operators for the E. coli LacI repressor. DNA looping between the sites shortens the effective distance between the lac operators, increasing LacI looping and strengthening its repression of a lacZ reporter gene. Analysis based on a general model for loop assistance enables quantitation of the strength of looping conferred by the protein and its binding sites. We use this ‘loopometer’ assay to measure DNA looping for a variety of bacterial and phage proteins.

DNA can act as a scaffold for the cooperative binding of protein oligomers. For example, the phage 186 CI repressor forms a wheel of seven dimers wrapped in DNA carrying specific binding sites, while the phage λ CI repressor binds in units of dimers to two well-separated sets of operators, forming a DNA loop. Atomic force microscopy was used to measure transcription elongation by E. coli RNA polymerase through these protein complexes. 186 CI or λ CI bound along unlooped DNA negligibly interfered with transcription by RNAP. More complex topologies induced by scaffolded, cooperatively bound repressor oligomers did not form significantly better roadblocks to transcription. Thus, despite binding with rather high affinity, these repressors are not effective roadblocks to transcription.