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Bacteriophage exclusion ('BREX') systems are multi-protein complexes encoded by a variety of bacteria and archaea that restrict phage by an unknown mechanism. One BREX factor, termed BrxL, has been noted to display sequence similarity to various AAA+ protein factors including Lon protease. In this study we describe multiple CryoEM structures of BrxL that demonstrate it to be a chambered, ATP-dependent DNA binding protein. The largest BrxL assemblage corresponds to a dimer of heptamers in the absence of bound DNA, versus a dimer of hexamers when DNA is bound in its central pore. The protein displays DNA-dependent ATPase activity, and ATP binding promotes assembly of the complex on DNA. Point mutations within several regions of the protein-DNA complex alter one or more in vitro behaviors and activities, including ATPase activity and ATP-dependent association with DNA. However, only the disruption of the ATPase active site fully eliminates phage restriction, indicating that other mutations can still complement BrxL function within the context of an otherwise intact BREX system. BrxL displays significant structural homology to MCM subunits (the replicative helicase in archaea and eukaryotes), implying that it and other BREX factors may collaborate to disrupt initiation of phage DNA replication., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bacteriophage exclusion ('BREX') phage restriction systems are found in a wide range of bacteria. Various BREX systems encode unique combinations of proteins that usually include a site-specific methyltransferase; none appear to contain a nuclease. Here we describe the identification and characterization of a Type I BREX system from Acinetobacter and the effect of deleting each BREX ORF on growth, methylation, and restriction. We identified a previously uncharacterized gene in the BREX operon that is dispensable for methylation but involved in restriction. Biochemical and crystallographic analyses of this factor, which we term BrxR ('BREX Regulator'), demonstrate that it forms a homodimer and specifically binds a DNA target site upstream of its transcription start site. Deletion of the BrxR gene causes cell toxicity, reduces restriction, and significantly increases the expression of BrxC. In contrast, the introduction of a premature stop codon into the BrxR gene, or a point mutation blocking its DNA binding ability, has little effect on restriction, implying that the BrxR coding sequence and BrxR protein play independent functional roles. We speculate that elements within the BrxR coding sequence are involved in cis regulation of anti-phage activity, while the BrxR protein itself plays an additional regulatory role, perhaps during horizontal transfer., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

To overcome CRISPR-Cas defense systems, many phages and mobile genetic elements (MGEs) encode CRISPR-Cas inhibitors called anti-CRISPRs (Acrs). Nearly all characterized Acrs directly bind Cas proteins to inactivate CRISPR immunity. Here, using functional metagenomic selection, we describe AcrIIA22, an unconventional Acr found in hypervariable genomic regions of clostridial bacteria and their prophages from human gut microbiomes. AcrIIA22 does not bind strongly to SpyCas9 but nonetheless potently inhibits its activity against plasmids. To gain insight into its mechanism, we obtained an X-ray crystal structure of AcrIIA22, which revealed homology to PC4-like nucleic acid-binding proteins. Based on mutational analyses and functional assays, we deduced that acrIIA22 encodes a DNA nickase that relieves torsional stress in supercoiled plasmids. This may render them less susceptible to SpyCas9, which uses free energy from negative supercoils to form stable R-loops. Modifying DNA topology may provide an additional route to CRISPR-Cas resistance in phages and MGEs., Competing Interests: The authors have declared that no competing interests exist.

Protein engineering has enabled the design of molecular scaffolds that display a wide variety of sizes, shapes, symmetries and subunit compositions. Symmetric protein-based nanoparticles that display multiple protein domains can exhibit enhanced functional properties due to increased avidity and improved solution behavior and stability. Here we describe the creation and characterization of a computationally designed circular tandem repeat protein (cTRP) composed of 24 identical repeated motifs, which can display a variety of functional protein domains (cargo) at defined positions around its periphery. We demonstrate that cTRP nanoparticles can self-assemble from smaller individual subunits, can be produced from prokaryotic and human expression platforms, can employ a variety of cargo attachment strategies and can be used for applications (such as T-cell culture and expansion) requiring high-avidity molecular interactions on the cell surface.

The wine yeast Saccharomyces cerevisiae forms an integral part of wine production by converting relatively 'neutral' flavoured grape must into varietal aromatic wines. Additionally, non-Saccharomyces strains can be used with S. cerevisiae for the production of wines with more complexity. Yeast strains, to varying extents, produce and/or mediate the release of a whole range of key metabolites, which in turn contribute to enhanced aroma and flavour of the final wine, especially Sauvignon blanc. These metabolites viz. thiols are dependent on yeast-expressed enzymes during fermentation. Inoculation with an appropriate yeast will, therefore, lead to more commercial wine sales due to resultant wines with sought-after aroma and flavour. Likewise, inoculation with the incorrect yeast will have a negative effect on sales. It is also important to have quality control measures in place to ensure that the inoculated yeast strain quickly dominate, and is present throughout the fermentation process. Traditionally, the laborious contour clamped homogeneous electric field (CHEF) DNA karyotyping technique was shown to be reliable in this regard, however cutting-edge matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) biotyping is proving to be a faster alternative. As both methods have advantages and disadvantages, they should be used in complementary as opposed to competitively. Standard chemical and descriptive sensory analyses of wine also serve as evaluation and/or characterisation tools of yeast starter cultures. Additionally, metabolomic and proteomic profiling using gas chromatography (GC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of final wines was shown to be instrumental yeast evaluation tools. Therefore, this review highlights the importance and practicality of a more inclusive approach to evaluate and characterise novel yeast strains used for winemaking by deploying traditional and modern chemical and organoleptic evaluation techniques of wines in conjunction with cutting-edge omics approaches towards enhancing white wine varietal aroma., (Copyright © 2019 Elsevier B.V. All rights reserved.)

CRISPR-Cas systems protect bacteria and archaea from phages and other mobile genetic elements, which use small anti-CRISPR (Acr) proteins to overcome CRISPR-Cas immunity. Because Acrs are challenging to identify, their natural diversity and impact on microbial ecosystems are underappreciated. To overcome this discovery bottleneck, we developed a high-throughput functional selection to isolate ten DNA fragments from human oral and fecal metagenomes that inhibit Streptococcus pyogenes Cas9 (SpyCas9) in Escherichia coli . The most potent Acr from this set, AcrIIA11, was recovered from a Lachnospiraceae phage. We found that AcrIIA11 inhibits SpyCas9 in bacteria and in human cells. AcrIIA11 homologs are distributed across diverse bacteria; many distantly-related homologs inhibit both SpyCas9 and a divergent Cas9 from Treponema denticola . We find that AcrIIA11 antagonizes SpyCas9 using a different mechanism than other previously characterized Type II-A Acrs. Our study highlights the power of functional selection to uncover widespread Cas9 inhibitors within diverse microbiomes., Competing Interests: KF, IB, DS, KJ, KD, BS, IF, BK, HM No competing interests declared, (© 2019, Forsberg et al.)

LAGLIDADG meganucleases are DNA cleaving enzymes used for genome engineering. While their cleavage specificity can be altered using several protein engineering and selection strategies, their overall targetability is limited by highly specific indirect recognition of the central four base pairs within their recognition sites. In order to examine the physical basis of indirect sequence recognition and to expand the number of such nucleases available for genome engineering, we have determined the target sites, DNA-bound structures, and central four cleavage fidelities of nine related enzymes. Subsequent crystallographic analyses of a meganuclease bound to two noncleavable target sites, each containing a single inactivating base pair substitution at its center, indicates that a localized slip of the mutated base pair causes a small change in the DNA backbone conformation that results in a loss of metal occupancy at one binding site, eliminating cleavage activity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

LAGLIDADG homing endonucleases ("meganucleases") are highly specific DNA cleaving enzymes that are used for genome engineering. Like other enzymes that act on DNA targets, meganucleases often display binding affinities and cleavage activities that are dominated by one protein domain. To decipher the underlying mechanism of asymmetric DNA recognition and catalysis, we identified and characterized a new monomeric meganuclease (I-SmaMI), which belongs to a superfamily of homologous enzymes that recognize divergent DNA sequences. We solved a series of crystal structures of the enzyme-DNA complex representing a progression of sequential reaction states, and we compared the structural rearrangements and surface potential distributions within each protein domain against their relative contribution to binding affinity. We then determined the effects of equivalent point mutations in each of the two enzyme active sites to determine whether asymmetry in DNA recognition is translated into corresponding asymmetry in DNA cleavage activity. These experiments demonstrate the structural basis for "dominance" by one protein domain over the other and provide insights into this enzyme's conformational switch from a nonspecific search mode to a more specific recognition mode., (Copyright © 2015 Elsevier Ltd. All rights reserved.)