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1.
Nature ; 2024 Jul 10.
Article in English | MEDLINE | ID: mdl-38987591

ABSTRACT

In all organisms, regulation of gene expression must be adjusted to meet cellular requirements and frequently involves helix-turn-helix (HTH) domain proteins1. For instance, in the arms race between bacteria and bacteriophages, rapid expression of phage anti-CRISPR (acr) genes upon infection enables evasion from CRISPR-Cas defence; transcription is then repressed by an HTH-domain-containing anti-CRISPR-associated (Aca) protein, probably to reduce fitness costs from excessive expression2-5. However, how a single HTH regulator adjusts anti-CRISPR production to cope with increasing phage genome copies and accumulating acr mRNA is unknown. Here we show that the HTH domain of the regulator Aca2, in addition to repressing Acr synthesis transcriptionally through DNA binding, inhibits translation of mRNAs by binding conserved RNA stem-loops and blocking ribosome access. The cryo-electron microscopy structure of the approximately 40 kDa Aca2-RNA complex demonstrates how the versatile HTH domain specifically discriminates RNA from DNA binding sites. These combined regulatory modes are widespread in the Aca2 family and facilitate CRISPR-Cas inhibition in the face of rapid phage DNA replication without toxic acr overexpression. Given the ubiquity of HTH-domain-containing proteins, it is anticipated that many more of them elicit regulatory control by dual DNA and RNA binding.

2.
BMC Genomics ; 25(1): 549, 2024 Jun 01.
Article in English | MEDLINE | ID: mdl-38824509

ABSTRACT

BACKGROUND: Despite Spirochetales being a ubiquitous and medically important order of bacteria infecting both humans and animals, there is extremely limited information regarding their bacteriophages. Of the genus Treponema, there is just a single reported characterised prophage. RESULTS: We applied a bioinformatic approach on 24 previously published Treponema genomes to identify and characterise putative treponemal prophages. Thirteen of the genomes did not contain any detectable prophage regions. The remaining eleven contained 38 prophage sequences, with between one and eight putative prophages in each bacterial genome. The prophage regions ranged from 12.4 to 75.1 kb, with between 27 and 171 protein coding sequences. Phylogenetic analysis revealed that 24 of the prophages formed three distinct sequence clusters, identifying putative myoviral and siphoviral morphology. ViPTree analysis demonstrated that the identified sequences were novel when compared to known double stranded DNA bacteriophage genomes. CONCLUSIONS: In this study, we have started to address the knowledge gap on treponeme bacteriophages by characterising 38 prophage sequences in 24 treponeme genomes. Using bioinformatic approaches, we have been able to identify and compare the prophage-like elements with respect to other bacteriophages, their gene content, and their potential to be a functional and inducible bacteriophage, which in turn can help focus our attention on specific prophages to investigate further.


Subject(s)
Genome, Bacterial , Genomics , Phylogeny , Prophages , Treponema , Prophages/genetics , Treponema/genetics , Treponema/virology , Genomics/methods , Computational Biology/methods , Genome, Viral , Bacteriophages/genetics , Bacteriophages/classification
3.
Curr Opin Microbiol ; 78: 102436, 2024 04.
Article in English | MEDLINE | ID: mdl-38368839

ABSTRACT

Bacteria have evolved a variety of defence mechanisms to protect against mobile genetic elements, including restriction-modification systems and CRISPR-Cas. In recent years, dozens of previously unknown defence systems (DSs) have been discovered. Notably, diverse DSs often coexist within the same genome, and some co-occur at frequencies significantly higher than would be expected by chance, implying potential synergistic interactions. Recent studies have provided evidence of defence mechanisms that enhance or complement one another. Here, we review the interactions between DSs at the mechanistic, regulatory, ecological and evolutionary levels.


Subject(s)
Bacteriophages , CRISPR-Cas Systems , Bacteria/genetics , Biological Evolution , Bacteriophages/genetics
4.
Nucleic Acids Res ; 52(3): 1313-1324, 2024 Feb 09.
Article in English | MEDLINE | ID: mdl-38038260

ABSTRACT

Type II topoisomerases effect topological changes in DNA by cutting a single duplex, passing a second duplex through the break, and resealing the broken strand in an ATP-coupled reaction cycle. Curiously, most type II topoisomerases (topos II, IV and VI) catalyze DNA transformations that are energetically favorable, such as the removal of superhelical strain; why ATP is required for such reactions is unknown. Here, using human topoisomerase IIß (hTOP2ß) as a model, we show that the ATPase domains of the enzyme are not required for DNA strand passage, but that their loss elevates the enzyme's propensity for DNA damage. The unstructured C-terminal domains (CTDs) of hTOP2ß strongly potentiate strand passage activity in ATPase-less enzymes, as do cleavage-prone mutations that confer hypersensitivity to the chemotherapeutic agent etoposide. The presence of either the CTD or the mutations lead ATPase-less enzymes to promote even greater levels of DNA cleavage in vitro, as well as in vivo. By contrast, aberrant cleavage phenotypes of these topo II variants is significantly repressed when the ATPase domains are present. Our findings are consistent with the proposal that type II topoisomerases acquired ATPase function to maintain high levels of catalytic activity while minimizing inappropriate DNA damage.


Subject(s)
DNA Topoisomerases, Type II , DNA , Humans , Adenosine Triphosphatases/genetics , Adenosine Triphosphate , DNA/genetics , DNA Topoisomerases, Type II/genetics , Etoposide/pharmacology , DNA Damage
5.
Nucleic Acids Res ; 52(4): 1909-1929, 2024 Feb 28.
Article in English | MEDLINE | ID: mdl-38113275

ABSTRACT

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a growing threat to global health, with recent efforts towards its eradication being reversed in the wake of the COVID-19 pandemic. Increasing resistance to gyrase-targeting second-line fluoroquinolone antibiotics indicates the necessity to develop both novel therapeutics and our understanding of M. tuberculosis growth during infection. ParDE toxin-antitoxin systems also target gyrase and are regulated in response to both host-associated and drug-induced stress during infection. Here, we present microbiological, biochemical, structural, and biophysical analyses exploring the ParDE1 and ParDE2 systems of M. tuberculosis H37Rv. The structures reveal conserved modes of toxin-antitoxin recognition, with complex-specific interactions. ParDE1 forms a novel heterohexameric ParDE complex, supported by antitoxin chains taking on two distinct folds. Curiously, ParDE1 exists in solution as a dynamic equilibrium between heterotetrameric and heterohexameric complexes. Conditional remodelling into higher order complexes can be thermally driven in vitro. Remodelling induces toxin release, tracked through concomitant inhibition and poisoning of gyrase activity. Our work aids our understanding of gyrase inhibition, allowing wider exploration of toxin-antitoxin systems as inspiration for potential therapeutic agents.


Subject(s)
Antitoxins , Bacterial Toxins , Mycobacterium tuberculosis , Tuberculosis , Humans , Antitoxins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , DNA Gyrase/genetics , Fluoroquinolones , Pandemics , Tuberculosis/microbiology , Bacterial Toxins/metabolism
6.
Nature ; 625(7994): 250-251, 2024 Jan.
Article in English | MEDLINE | ID: mdl-38114828

Subject(s)
Viruses , Bacteria , Virology
7.
Appl Environ Microbiol ; 89(9): e0062323, 2023 09 28.
Article in English | MEDLINE | ID: mdl-37668405

ABSTRACT

Bacteriophages (phages) outnumber bacteria ten-to-one and cause infections at a rate of 1025 per second. The ability of phages to reduce bacterial populations makes them attractive alternative antibacterials for use in combating the rise in antimicrobial resistance. This effort may be hindered due to bacterial defenses such as Bacteriophage Exclusion (BREX) that have arisen from the constant evolutionary battle between bacteria and phages. For phages to be widely accepted as therapeutics in Western medicine, more must be understood about bacteria-phage interactions and the outcomes of bacterial phage defense. Here, we present the annotated genomes of 12 novel bacteriophage species isolated from water sources in Durham, UK, during undergraduate practical classes. The collection includes diverse species from across known phylogenetic groups. Comparative analyses of two novel phages from the collection suggest they may be founding members of a new genus. Using this Durham phage collection, we determined that particular BREX defense systems were likely to confer a varied degree of resistance against an invading phage. We concluded that the number of BREX target motifs encoded in the phage genome was not proportional to the degree of susceptibility. IMPORTANCE Bacteriophages have long been the source of tools for biotechnology that are in everyday use in molecular biology research laboratories worldwide. Phages make attractive new targets for the development of novel antimicrobials. While the number of phage genome depositions has increased in recent years, the expected bacteriophage diversity remains underrepresented. Here we demonstrate how undergraduates can contribute to the identification of novel phages and that a single City in England can provide ample phage diversity and the opportunity to find novel technologies. Moreover, we demonstrate that the interactions and intricacies of the interplay between bacterial phage defense systems such as Bacteriophage Exclusion (BREX) and phages are more complex than originally thought. Further work will be required in the field before the dynamic interactions between phages and bacterial defense systems are fully understood and integrated with novel phage therapies.


Subject(s)
Bacteriophages , Bacteriophages/physiology , Phylogeny , Biological Evolution , Bacteria , England
8.
Nat Commun ; 14(1): 4644, 2023 08 17.
Article in English | MEDLINE | ID: mdl-37591829

ABSTRACT

Mycobacterium tuberculosis, the bacterium responsible for human tuberculosis, has a genome encoding a remarkably high number of toxin-antitoxin systems of largely unknown function. We have recently shown that the M. tuberculosis genome encodes four of a widespread, MenAT family of nucleotidyltransferase toxin-antitoxin systems. In this study we characterize MenAT1, using tRNA sequencing to demonstrate MenT1 tRNA modification activity. MenT1 activity is blocked by MenA1, a short protein antitoxin unrelated to the MenA3 kinase. X-ray crystallographic analysis shows blockage of the conserved MenT fold by asymmetric binding of MenA1 across two MenT1 protomers, forming a heterotrimeric toxin-antitoxin complex. Finally, we also demonstrate tRNA modification by toxin MenT4, indicating conserved activity across the MenT family. Our study highlights variation in tRNA target preferences by MenT toxins, selective use of nucleotide substrates, and diverse modes of MenA antitoxin activity.


Subject(s)
Antitoxins , Mycobacterium tuberculosis , Toxins, Biological , Humans , Antitoxins/genetics , Nucleotidyltransferases , Nucleotides , RNA, Transfer/genetics
9.
bioRxiv ; 2023 Jun 27.
Article in English | MEDLINE | ID: mdl-37425896

ABSTRACT

Type II topoisomerases effect topological changes in DNA by cutting a single duplex, passing a second duplex through the break, and resealing the broken strand in an ATP-coupled reaction. Curiously, most type II topoisomerases (topos II, IV, and VI) catalyze DNA transformations that are energetically favorable, such as the removal of superhelical strain; why ATP is required for such reactions is unknown. Here, using human topoisomerase II ß (hTOP2ß) as a model, we show that the ATPase domains of the enzyme are not required for DNA strand passage, but that their loss leads to increased DNA nicking and double strand break formation by the enzyme. The unstructured C-terminal domains (CTDs) of hTOP2ß strongly potentiate strand passage activity in the absence of the ATPase regions, as do cleavage-prone mutations that confer hypersensitivity to the chemotherapeutic agent etoposide. The presence of either the CTD or the mutations lead ATPase-less enzymes to promote even greater levels of DNA cleava in ge vitro , as well as in vivo . By contrast, the aberrant cleavage phenotypes of these topo II variants is significantly repressed when the ATPase domains are restored. Our findings are consistent with the proposal that type II topoisomerases acquired an ATPase function to maintain high levels of catalytic activity while minimizing inappropriate DNA damage.

10.
Proc Natl Acad Sci U S A ; 120(28): e2302064120, 2023 07 11.
Article in English | MEDLINE | ID: mdl-37406101

ABSTRACT

Type II topoisomerases transiently cleave duplex DNA as part of a strand passage mechanism that helps control chromosomal organization and superstructure. Aberrant DNA cleavage can result in genomic instability, and how topoisomerase activity is controlled to prevent unwanted breaks is poorly understood. Using a genetic screen, we identified mutations in the beta isoform of human topoisomerase II (hTOP2ß) that render the enzyme hypersensitive to the chemotherapeutic agent etoposide. Several of these variants were unexpectedly found to display hypercleavage behavior in vitro and to be capable of inducing cell lethality in a DNA repair-deficient background; surprisingly, a subset of these mutations were also observed in TOP2B sequences from cancer genome databases. Using molecular dynamics simulations and computational network analyses, we found that many of the mutations obtained from the screen map to interfacial points between structurally coupled elements, and that dynamical modeling could be used to identify other damage-inducing TOP2B alleles present in cancer genome databases. This work establishes that there is an innate link between DNA cleavage predisposition and sensitivity to topoisomerase II poisons, and that certain sequence variants of human type II topoisomerases found in cancer cells can act as DNA-damaging agents. Our findings underscore the potential for hTOP2ß to function as a clastogen capable of generating DNA damage that may promote or support cellular transformation.


Subject(s)
Mutagens , Neoplasms , Humans , Topoisomerase II Inhibitors/pharmacology , Etoposide/pharmacology , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , DNA Damage , DNA
11.
PLoS Genet ; 19(6): e1010784, 2023 Jun.
Article in English | MEDLINE | ID: mdl-37276233

ABSTRACT

Competitive bacteria-bacteriophage interactions have resulted in the evolution of a plethora of bacterial defense systems preventing phage propagation. In recent years, computational and bioinformatic approaches have underpinned the discovery of numerous novel bacterial defense systems. Anti-phage systems are frequently encoded together in genomic loci termed defense islands. Here we report the identification and characterisation of a novel anti-phage system, that we have termed Shield, which forms part of the Pseudomonas defensive arsenal. The Shield system comprises the core component ShdA, a membrane-bound protein harboring an RmuC domain. Heterologous production of ShdA alone is sufficient to mediate bacterial immunity against several phages. We demonstrate that Shield and ShdA confer population-level immunity and that they can also decrease transformation efficiency. We further show that ShdA homologues can degrade DNA in vitro and, when expressed in a heterologous host, can alter the organisation of the host chromosomal DNA. Use of comparative genomic approaches identified how Shield can be divided into four subtypes, three of which contain additional components that in some cases can negatively affect the activity of ShdA and/or provide additional lines of phage defense. Collectively, our results identify a new player within the Pseudomonas bacterial immunity arsenal that displays a novel mechanism of protection, and reveals a role for RmuC domains in phage defense.


Subject(s)
Bacteriophages , Bacteriophages/genetics , Pseudomonas/genetics , Bacteria/genetics , Genome
12.
Curr Opin Microbiol ; 73: 102293, 2023 06.
Article in English | MEDLINE | ID: mdl-36958122

ABSTRACT

Bacteria have evolved a broad range of defence mechanisms to protect against infection by their viral parasites, bacteriophages (phages). Toxin-antitoxin (TA) systems are small loci found throughout bacteria and archaea that in some cases provide phage defence. The recent explosion in phage defence system discovery has identified multiple novel TA systems with antiphage activity. Due to inherent toxicity, TA systems are thought to mediate abortive infection (Abi), wherein the host cell dies in response to phage infection, removing the phage, and protecting clonal siblings. Recent studies, however, have uncovered molecular mechanisms by which TA systems are activated by phages, how they mediate toxicity, and how phages escape the defences. These new models reveal dazzling complexity in phage-host interactions and provide further evidence that TA systems do not in all cases inherently perform classic Abi, suggesting an evolved conceptual definition is required.


Subject(s)
Bacteriophages , Toxin-Antitoxin Systems , Bacteriophages/genetics , Toxin-Antitoxin Systems/genetics , Bacteria/genetics
13.
Mol Microbiol ; 120(1): 45-53, 2023 07.
Article in English | MEDLINE | ID: mdl-36840376

ABSTRACT

Bacteria are continuously exposed to predation from bacteriophages (phages) and, in response, have evolved a broad range of defence systems. These systems can prevent the replication of phages and other mobile genetic elements (MGE). Defence systems are often encoded together in genomic loci defined as "defence islands", a tendency that has been extensively exploited to identify novel antiphage systems. In the last few years, >100 new antiphage systems have been discovered, and some display homology to components of the immune systems of plants and animals. In many instances, prediction tools have found domains with similar predicted functions present as different combinations within distinct antiphage systems. In this Perspective Article, we review recent reports describing the discovery and the predicted domain composition of several novel antiphage systems. We discuss several examples of similar protein domains adopted by different antiphage systems, including domains of unknown function (DUFs), domains involved in nucleic acid recognition and degradation, and domains involved in NAD+ depletion. We further discuss the potential evolutionary advantages that could have driven the independent acquisition of these domains by different antiphage systems.


Subject(s)
Bacteriophages , Animals , Bacteriophages/genetics , Bacteria/genetics , Protein Domains
14.
Nat Commun ; 13(1): 5289, 2022 09 08.
Article in English | MEDLINE | ID: mdl-36075935

ABSTRACT

Light harvesting is fundamental for production of ATP and reducing equivalents for CO2 fixation during photosynthesis. However, electronic energy transfer (EET) through a photosystem can harm the photosynthetic apparatus when not balanced with CO2. Here, we show that CO2 binding to the light-harvesting complex modulates EET in photosynthetic cyanobacteria. More specifically, CO2 binding to the allophycocyanin alpha subunit of the light-harvesting complex regulates EET and its fluorescence quantum yield in the cyanobacterium Synechocystis sp. PCC 6803. CO2 binding decreases the inter-chromophore distance in the allophycocyanin trimer. The result is enhanced EET in vitro and in live cells. Our work identifies a direct target for CO2 in the cyanobacterial light-harvesting apparatus and provides insights into photosynthesis regulation.


Subject(s)
Phycobilisomes , Synechocystis , Carbon Dioxide/metabolism , Photosynthesis , Phycobilisomes/metabolism , Phycocyanin , Receptors, Cell Surface , Synechocystis/metabolism
15.
Curr Res Struct Biol ; 4: 211-219, 2022.
Article in English | MEDLINE | ID: mdl-35783086

ABSTRACT

Bacteria are constantly challenged by bacteriophage (phage) infection and have developed multitudinous and varied resistance mechanisms. Bacteriophage Exclusion (BREX) systems protect from phage infection by generating methylation patterns at non-palindromic 6 bp sites in host bacterial DNA, to distinguish and block replication of non-self DNA. Type 1 BREX systems are comprised of six conserved core genes. Here, we present the first reported structure of a BREX core protein, BrxA from the phage defence island of Escherichia fergusonii ATCC 35469 plasmid pEFER, solved to 2.09 â€‹Å. BrxA is a monomeric protein in solution, with an all α-helical globular fold. Conservation of surface charges and structural homology modelling against known phage defence systems highlighted that BrxA contains two helix-turn-helix motifs, juxtaposed by 180°, positioned to bind opposite sides of a DNA major groove. BrxA was subsequently shown to bind dsDNA. This new understanding of BrxA structure, and first indication of BrxA biological activity, suggests a conserved mode of DNA-recognition has become widespread and implemented by diverse phage defence systems.

16.
Structure ; 30(8): 1129-1145.e3, 2022 08 04.
Article in English | MEDLINE | ID: mdl-35660158

ABSTRACT

Human topoisomerase II beta (TOP2B) modulates DNA topology using energy from ATP hydrolysis. To investigate the conformational changes that occur during ATP hydrolysis, we determined the X-ray crystallographic structures of the human TOP2B ATPase domain bound to AMPPNP or ADP at 1.9 Å and 2.6 Å resolution, respectively. The GHKL domains of both structures are similar, whereas the QTK loop within the transducer domain can move for product release. As TOP2B is the clinical target of bisdioxopiperazines, we also determined the structure of a TOP2B:ADP:ICRF193 complex to 2.3 Å resolution and identified key drug-binding residues. Biochemical characterization revealed the N-terminal strap reduces the rate of ATP hydrolysis. Mutagenesis demonstrated residue E103 as essential for ATP hydrolysis in TOP2B. Our data provide fundamental insights into the tertiary structure of the human TOP2B ATPase domain and a potential regulatory mechanism for ATP hydrolysis.


Subject(s)
Adenosine Triphosphatases , Adenosine Triphosphate , DNA Topoisomerases, Type II , Adenosine Diphosphate/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphate/metabolism , Adenylyl Imidodiphosphate , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/genetics , Humans , Hydrolysis , Poly-ADP-Ribose Binding Proteins
17.
Nucleic Acids Res ; 50(9): 5191-5207, 2022 05 20.
Article in English | MEDLINE | ID: mdl-35544231

ABSTRACT

Bacteria are under constant assault by bacteriophages and other mobile genetic elements. As a result, bacteria have evolved a multitude of systems that protect from attack. Genes encoding bacterial defence mechanisms can be clustered into 'defence islands', providing a potentially synergistic level of protection against a wider range of assailants. However, there is a comparative paucity of information on how expression of these defence systems is controlled. Here, we functionally characterize a transcriptional regulator, BrxR, encoded within a recently described phage defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a combination of reporters and electrophoretic mobility shift assays, we discovered that BrxR acts as a repressor. We present the structure of BrxR to 2.15 Å, the first structure of this family of transcription factors, and pinpoint a likely binding site for ligands within the WYL-domain. Bioinformatic analyses demonstrated that BrxR-family homologues are widespread amongst bacteria. About half (48%) of identified BrxR homologues were co-localized with a diverse array of known phage defence systems, either alone or clustered into defence islands. BrxR is a novel regulator that reveals a common mechanism for controlling the expression of the bacterial phage defence arsenal.


Subject(s)
Bacteria , Transcription Factors , Bacteria/genetics , Bacteria/metabolism , Bacteria/virology , Bacteriophages/genetics , Genomic Islands/genetics , Plasmids , Transcription Factors/genetics , Transcription Factors/metabolism
18.
Nucleic Acids Res ; 49(19): 11257-11273, 2021 11 08.
Article in English | MEDLINE | ID: mdl-34657954

ABSTRACT

Bacteria have evolved a multitude of systems to prevent invasion by bacteriophages and other mobile genetic elements. Comparative genomics suggests that genes encoding bacterial defence mechanisms are often clustered in 'defence islands', providing a concerted level of protection against a wider range of attackers. However, there is a comparative paucity of information on functional interplay between multiple defence systems. Here, we have functionally characterised a defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a suite of thirty environmentally-isolated coliphages, we demonstrate multi-layered and robust phage protection provided by a plasmid-encoded defence island that expresses both a type I BREX system and the novel GmrSD-family type IV DNA modification-dependent restriction enzyme, BrxU. We present the structure of BrxU to 2.12 Å, the first structure of the GmrSD family of enzymes, and show that BrxU can utilise all common nucleotides and a wide selection of metals to cleave a range of modified DNAs. Additionally, BrxU undergoes a multi-step reaction cycle instigated by an unexpected ATP-dependent shift from an intertwined dimer to monomers. This direct evidence that bacterial defence islands can mediate complementary layers of phage protection enhances our understanding of the ever-expanding nature of phage-bacterial interactions.


Subject(s)
Bacterial Proteins/chemistry , Coliphages/genetics , DNA Restriction-Modification Enzymes/chemistry , Escherichia coli/genetics , Escherichia/genetics , Plasmids/chemistry , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Cloning, Molecular , Coliphages/metabolism , Crystallography, X-Ray , DNA Restriction-Modification Enzymes/genetics , DNA Restriction-Modification Enzymes/metabolism , DNA, Viral/chemistry , DNA, Viral/genetics , DNA, Viral/metabolism , Escherichia/metabolism , Escherichia/virology , Escherichia coli/metabolism , Escherichia coli/virology , Gene Expression , Genomic Islands , Genomics/methods , Models, Molecular , Plasmids/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity
19.
J Struct Biol ; 213(3): 107752, 2021 09.
Article in English | MEDLINE | ID: mdl-34116143

ABSTRACT

Bacteria use adaptive CRISPR-Cas immune mechanisms to protect from invasion by bacteriophages and other mobile genetic elements. In response, bacteriophages and mobile genetic elements have co-evolved anti-CRISPR proteins to inhibit the bacterial defense. We and others have previously shown that anti-CRISPR associated (Aca) proteins can regulate this anti-CRISPR counter-attack. Here, we report the first structure of an Aca protein, the Aca2 DNA-binding transcriptional autorepressor from Pectobacterium carotovorum bacteriophage ZF40, determined to 1.34 Å. Aca2 presents a conserved N-terminal helix-turn-helix DNA-binding domain and a previously uncharacterized C-terminal dimerization domain. Dimerization positions the Aca2 recognition helices for insertion into the major grooves of target DNA, supporting its role in regulating anti-CRISPRs. Furthermore, database comparisons identified uncharacterized Aca2 structural homologs in pathogenic bacteria, suggesting that Aca2 represents the first characterized member of a more widespread family of transcriptional regulators.


Subject(s)
Bacteriophages , CRISPR-Cas Systems , Bacteria , Bacteriophages/chemistry , Bacteriophages/genetics , CRISPR-Cas Systems/genetics , Protein Binding , Transcription Factors/genetics
20.
Viruses ; 13(3)2021 03 15.
Article in English | MEDLINE | ID: mdl-33804216

ABSTRACT

In recent years, novel lineages of invasive non-typhoidal Salmonella (iNTS) serovars Typhimurium and Enteritidis have been identified in patients with bloodstream infection in Sub-Saharan Africa. Here, we isolated and characterised 32 phages capable of infecting S. Typhimurium and S. Enteritidis, from water sources in Malawi and the UK. The phages were classified in three major phylogenetic clusters that were geographically distributed. In terms of host range, Cluster 1 phages were able to infect all bacterial hosts tested, whereas Clusters 2 and 3 had a more restricted profile. Cluster 3 contained two sub-clusters, and 3.b contained the most novel isolates. This study represents the first exploration of the potential for phages to target the lineages of Salmonella that are responsible for bloodstream infections in Sub-Saharan Africa.


Subject(s)
Bacteriophages , Salmonella Infections/therapy , Salmonella enteritidis/virology , Salmonella typhimurium/virology , Sepsis/microbiology , Humans , Malawi/epidemiology , Salmonella Infections/virology , Salmonella enteritidis/isolation & purification , Salmonella typhimurium/isolation & purification , United Kingdom/epidemiology , Water Microbiology
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