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1.
Nature ; 625(7994): 352-359, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-37992756

RESUMEN

It was recently shown that bacteria use, apart from CRISPR-Cas and restriction systems, a considerable diversity of phage resistance systems1-4, but it is largely unknown how phages cope with this multilayered bacterial immunity. Here we analysed groups of closely related Bacillus phages that showed differential sensitivity to bacterial defence systems, and discovered four distinct families of anti-defence proteins that inhibit the Gabija, Thoeris and Hachiman systems. We show that these proteins Gad1, Gad2, Tad2 and Had1 efficiently cancel the defensive activity when co-expressed with the respective defence system or introduced into phage genomes. Homologues of these anti-defence proteins are found in hundreds of phages that infect taxonomically diverse bacterial species. We show that the anti-Gabija protein Gad1 blocks the ability of the Gabija defence complex to cleave phage-derived DNA. Our data further reveal that the anti-Thoeris protein Tad2 is a 'sponge' that sequesters the immune signalling molecules produced by Thoeris TIR-domain proteins in response to phage infection. Our results demonstrate that phages encode an arsenal of anti-defence proteins that can disable a variety of bacterial defence mechanisms.


Asunto(s)
Fagos de Bacillus , Bacterias , Proteínas Virales , Fagos de Bacillus/clasificación , Fagos de Bacillus/genética , Fagos de Bacillus/inmunología , Fagos de Bacillus/metabolismo , Bacterias/clasificación , Bacterias/genética , Bacterias/inmunología , Bacterias/virología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Viral/genética , ADN Viral/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo
3.
Mol Cell ; 74(1): 1-2, 2019 04 04.
Artículo en Inglés | MEDLINE | ID: mdl-30951649

RESUMEN

Recently members of Bacillus phages were found to utilize a small peptide (6 aa long) to communicate with their descendants, aiding in a complex decision-making process. Proteins involved in this remarkable viral communication phenomenon were further investigated at the structural level for better understanding in this issue of Molecular Cell (Gallego del Sol et al., 2019).


Asunto(s)
Fagos de Bacillus/genética , Genoma Viral , Bases de Datos Genéticas
4.
Mol Cell ; 74(1): 59-72.e3, 2019 04 04.
Artículo en Inglés | MEDLINE | ID: mdl-30745087

RESUMEN

Bacillus phages use a communication system, termed "arbitrium," to coordinate lysis-lysogeny decisions. Arbitrium communication is mediated by the production and secretion of a hexapeptide (AimP) during lytic cycle. Once internalized, AimP reduces the expression of the negative regulator of lysogeny, AimX, by binding to the transcription factor, AimR, promoting lysogeny. We have elucidated the crystal structures of AimR from the Bacillus subtilis SPbeta phage in its apo form, bound to its DNA operator and in complex with AimP. AimR presents intrinsic plasticity, sharing structural features with the RRNPP quorum-sensing family. Remarkably, AimR binds to an unusual operator with a long spacer that interacts nonspecifically with the receptor TPR domain, while the HTH domain canonically recognizes two inverted repeats. AimP stabilizes a compact conformation of AimR that approximates the DNA-recognition helices, preventing AimR binding to the aimX promoter region. Our results establish the molecular basis of the arbitrium communication system.


Asunto(s)
Fagos de Bacillus/metabolismo , Lisogenia , Proteínas Virales/metabolismo , Fagos de Bacillus/genética , Bacillus subtilis/virología , ADN/metabolismo , Regulación Viral de la Expresión Génica , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Transducción de Señal , Relación Estructura-Actividad , Proteínas Virales/química , Proteínas Virales/genética
5.
Nucleic Acids Res ; 52(4): 2045-2065, 2024 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-38281216

RESUMEN

The genome-organizing protein p6 of Bacillus subtilis bacteriophage φ29 plays an essential role in viral development by activating the initiation of DNA replication and participating in the early-to-late transcriptional switch. These activities require the formation of a nucleoprotein complex in which the DNA adopts a right-handed superhelix wrapping around a multimeric p6 scaffold, restraining positive supercoiling and compacting the viral genome. Due to the absence of homologous structures, prior attempts to unveil p6's structural architecture failed. Here, we employed AlphaFold2 to engineer rational p6 constructs yielding crystals for three-dimensional structure determination. Our findings reveal a novel fold adopted by p6 that sheds light on its self-association mechanism and its interaction with DNA. By means of protein-DNA docking and molecular dynamic simulations, we have generated a comprehensive structural model for the nucleoprotein complex that consistently aligns with its established biochemical and thermodynamic parameters. Besides, through analytical ultracentrifugation, we have confirmed the hydrodynamic properties of the nucleocomplex, further validating in solution our proposed model. Importantly, the disclosed structure not only provides a highly accurate explanation for previously experimental data accumulated over decades, but also enhances our holistic understanding of the structural and functional attributes of protein p6 during φ29 infection.


Asunto(s)
Fagos de Bacillus , Bacillus subtilis , Fagos de Bacillus/genética , Fagos de Bacillus/química , Bacillus subtilis/virología , Replicación del ADN , ADN Viral/genética , Nucleoproteínas/metabolismo , Proteínas Virales/metabolismo
6.
Nucleic Acids Res ; 51(17): 9452-9474, 2023 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-37602373

RESUMEN

Prophages control their lifestyle to either be maintained within the host genome or enter the lytic cycle. Bacillus subtilis contains the SPß prophage whose lysogenic state depends on the MrpR (YopR) protein, a key component of the lysis-lysogeny decision system. Using a historic B. subtilis strain harboring the heat-sensitive SPß c2 mutant, we demonstrate that the lytic cycle of SPß c2 can be induced by heat due to a single nucleotide exchange in the mrpR gene, rendering the encoded MrpRG136E protein temperature-sensitive. Structural characterization revealed that MrpR is a DNA-binding protein resembling the overall fold of tyrosine recombinases. MrpR has lost its recombinase function and the G136E exchange impairs its higher-order structure and DNA binding activity. Genome-wide profiling of MrpR binding revealed its association with the previously identified SPbeta repeated element (SPBRE) in the SPß genome. MrpR functions as a master repressor of SPß that binds to this conserved element to maintain lysogeny. The heat-inducible excision of the SPß c2 mutant remains reliant on the serine recombinase SprA. A suppressor mutant analysis identified a previously unknown component of the lysis-lysogeny management system that is crucial for the induction of the lytic cycle of SPß.


Asunto(s)
Fagos de Bacillus , Bacteriófagos , Proteínas Virales , Fagos de Bacillus/genética , Bacillus subtilis/genética , Lisogenia/genética , Profagos/genética , Recombinasas/genética , Proteínas Virales/metabolismo
7.
Arch Virol ; 169(4): 81, 2024 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-38519716

RESUMEN

Bacillus subtilis is a Gram-positive bacterium that is widely used in fermentation and in the pharmaceutical industry. Phage contamination occasionally occurs in various fermentation processes and causes significant economic loss. Here, we report the isolation and characterization of a temperate B. subtilis phage, termed phi18-2, from spore powder manufactured in a fermentation plant. Transmission electron microscopy showed that phi18-2 has a symmetrical polyhedral head and a long noncontractile tail. Receptor analysis showed that phi18-2 recognizes wall teichoic acid (WTA) for infection. The phage virions have a linear double-stranded DNA genome of 64,467 bp with identical direct repeat sequences of 309 bp at each end of the genome. In lysogenic cells, the phage genome was found to be present in the cytoplasm without integration into the host cell chromosome, and possibly as a linear phage-plasmid with unmodified ends. Our data may provide some insight into the molecular basis of the unique lysogenic cycle of phage phi18-2.


Asunto(s)
Fagos de Bacillus , Bacteriófagos , Bacteriófagos/genética , Fagos de Bacillus/genética , ADN Viral/genética , Lisogenia , Genoma Viral , Plásmidos/genética , Citoplasma
8.
Arch Virol ; 169(7): 134, 2024 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-38834736

RESUMEN

Anthrax is an acute infectious zoonotic disease caused by Bacillus anthracis, a bacterium that is considered a potential biological warfare agent. Bacillus bacteriophages shape the composition and evolution of bacterial communities in nature and therefore have important roles in the ecosystem community. B. anthracis phages are not only used in etiological diagnostics but also have promising prospects in clinical therapeutics or for disinfection in anthrax outbreaks. In this study, two temperate B. anthracis phages, vB_BanS_A16R1 (A16R1) and vB_BanS_A16R4 (A16R4), were isolated and showed siphovirus-like morphological characteristics. Genome sequencing showed that the genomes of phages A16R1 and A16R4 are 36,569 bp and 40,059 bp in length, respectively. A16R1 belongs to the genus Wbetavirus, while A16R4 belongs to the genus Hubeivirus and is the first phage of that genus found to lyse B. anthracis. Because these two phages can comparatively specifically lyse B. anthracis, they could be used as alternative diagnostic tools for identification of B. anthracis infections.


Asunto(s)
Fagos de Bacillus , Bacillus anthracis , Genoma Viral , Bacillus anthracis/virología , Genoma Viral/genética , Fagos de Bacillus/aislamiento & purificación , Fagos de Bacillus/genética , Fagos de Bacillus/clasificación , Siphoviridae/genética , Siphoviridae/aislamiento & purificación , Siphoviridae/clasificación , Filogenia
9.
Appl Microbiol Biotechnol ; 108(1): 366, 2024 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-38850320

RESUMEN

This review gathers all, to the best of our current knowledge, known lysins, mainly bacteriophage-derived, that have demonstrated activity against Bacillus anthracis strains. B. anthracis is a spore-forming, toxin-producing bacteria, naturally dwelling in soil. It is best known as a potential biowarfare threat, an etiological agent of anthrax, and a severe zoonotic disease. Anthrax can be treated with antibiotics (ciprofloxacin, penicillin, doxycycline); however, their administration may take up even to 60 days, and different factors can compromise their effectiveness. Bacterial viruses, bacteriophages (phages), are natural enemies of bacteria and use their lytic enzymes, endolysins (lysins), to specifically kill bacterial cells. Harnessing the potential of lysins to combat bacterial infections holds promise for diminishing antibiotic usage and, consequently, addressing the escalating antibiotic resistance in bacteria. In this context, we list the lysins with the activity against B. anthracis, providing a summary of their lytic properties in vitro and the outcomes observed in animal models. Bacillus cereus strain ATCC 4342/RSVF1, a surrogate for B. anthracis, was also included as a target bacteria. KEY POINTS: • More than a dozen different B. anthracis lysins have been identified and studied. • They fall into three blocks regarding their amino acid sequence similarity and most of them are amidases. • Lysins could be used in treating B. anthracis infections.


Asunto(s)
Carbunco , Antibacterianos , Bacillus anthracis , Endopeptidasas , Bacillus anthracis/efectos de los fármacos , Bacillus anthracis/virología , Carbunco/tratamiento farmacológico , Carbunco/microbiología , Animales , Endopeptidasas/farmacología , Endopeptidasas/metabolismo , Endopeptidasas/genética , Antibacterianos/farmacología , Bacteriófagos/genética , Bacillus cereus/efectos de los fármacos , Bacillus cereus/virología , Humanos , Fagos de Bacillus/genética
10.
Biochemistry ; 62(20): 2902-2907, 2023 10 17.
Artículo en Inglés | MEDLINE | ID: mdl-37699513

RESUMEN

RNA thermometers are noncoding RNA structures located in the 5' untranslated regions (UTRs) of genes that regulate gene expression through temperature-dependent conformational changes. The fourU class of RNA thermometers contains a specific motif in which four consecutive uracil nucleotides are predicted to base pair with the Shine-Dalgarno (SD) sequence in a stem. We employed a bioinformatic search to discover a fourU RNA thermometer in the 5'-UTR of the blyA gene of the Bacillus subtilis phage SPßc2, a bacteriophage that infects B. subtilis 168. blyA encodes an autolysin enzyme, N-acetylmuramoyl-l-alanine amidase, which is involved in the lytic life cycle of the SPß prophage. We have biochemically validated the predicted RNA thermometer in the 5'-UTR of the blyA gene. Our study suggests that RNA thermometers may play an underappreciated yet critical role in the lytic life cycle of bacteriophages.


Asunto(s)
Fagos de Bacillus , Bacillus subtilis , Regiones no Traducidas 5' , Fagos de Bacillus/genética , Bacillus subtilis/genética , N-Acetil Muramoil-L-Alanina Amidasa/genética , Profagos/genética
11.
BMC Microbiol ; 23(1): 170, 2023 06 19.
Artículo en Inglés | MEDLINE | ID: mdl-37337195

RESUMEN

BACKGROUND: Phages play a pivotal role in the evolution of microbial populations. The interactions between phages and their hosts are complex and may vary in response to host physiology and environmental conditions. Here, we have selected the genomes of some representative Bacillus prophages and lysosomes from the NCBI database for evolutionary analysis. We explored their evolutionary relationships and analyzed the protein information encoded by hundreds of Bacillus phages. RESULTS: We obtained the following conclusions: First, Bacillus phages carried some known functional gene fragments and a large number of unknown functional gene fragments, which might have an important impact on Bacillus populations, such as the formation of spores and biofilms and the transmission of virulence factors. Secondly, the Bacillus phage genome showed diversity, with a clear genome boundary between Bacillus prophages and Bacillus lytic phages. Furthermore, genetic mutations, sequence losses, duplications, and host-switching have occurred during the evolution of the Bacillus phage, resulting in low genome similarity between the Bacillus phages. Finally, the lysis module played an important influence on the process of Bacillus phage cross-species infestation. CONCLUSIONS: This study systematically described their protein function, diversity, and genome evolution, and the results of this study provide a basis for evolutionary diversity, horizontal gene transfer and co-evolution with the host in Bacillus phages.


Asunto(s)
Fagos de Bacillus , Bacteriófagos , Fagos de Bacillus/genética , Genoma Viral , Bacteriófagos/genética , Profagos/genética , Mutación , Evolución Molecular
12.
Arch Virol ; 168(2): 54, 2023 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-36609927

RESUMEN

Mangrove is among the most carbon-rich biomes on earth, and viruses are believed to play a significant role in modulating local and global carbon cycling. However, few viruses have been isolated from mangrove sediments to date. Here, we report the isolation of a novel Bacillus phage (named phage vB_BviS-A10Y) from mangrove sediments. Phage vB_BviS-A10Y has a hexameric head with a diameter of ~ 79.22 nm and a tail with a length of ~ 548.56 nm, which are typical features of siphophages. vB_BviS-A10Y initiated host lysis at 3.5 h postinfection with a burst size of 25 plaque-forming units (PFU)/cell. The genome of phage vB_BviS-A10Y is 162,435 bp long with 225 predicted genes, and the GC content is 34.03%. A comparison of the whole genome sequence of phage vB_BviS-A10Y with those of other phages from the NCBI viral genome database showed that phage vB_BviS-A10Y has the highest similarity (73.7% identity with 33% coverage) to Bacillus phage PBC2. Interestingly, abundant auxiliary metabolic genes (AMGs) were identified in the vB_BviS-A10Y genome. The presence of a ß-1,3-glucosyltransferase gene in the phage genome supported our previous hypothesis that mangrove viruses may manipulate carbon cycling directly through their encoded carbohydrate-active enzyme (CAZyme) genes. Therefore, our study will contribute to a better understanding of the diversity and potential roles of viruses in mangrove ecosystems.


Asunto(s)
Fagos de Bacillus , Bacteriófagos , Virus , Bacteriófagos/genética , Ecosistema , Genoma Viral/genética , Virus/genética , Fagos de Bacillus/genética , Genómica , Filogenia
13.
Virus Genes ; 59(4): 624-634, 2023 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-37119398

RESUMEN

In the present study, two new Bacillus subtilis phages, BSTP4 and BSTP6, were isolated and studied further. Morphologically, BSTP4 and BSTP6 are podoviruses with complete genome of 19,145 (39.9% G + C content) and 19,367 bp (39.8% G + C content), respectively, which became among the smallest Bacillus phages. Three most prominent structural proteins were separated and identified as pre-neck appendage, major head, and head fiber proteins using LC-MS/MS. Both phages encode putative terminal proteins (TP) and contain short inverted terminal repeats (ITRs) which may be important for their replication. In addition, non-coding RNA (pRNA) and parS sites were identified which may be required for DNA packaging and their maintenance inside the host, respectively. Furthermore, the phage genome sequences show significant similarity to B. subtilis group species genome sequences. Finally, phylogenomic and phylogenetic analyses suggest that BSTP4 and BSTP6 may form a new species in the genus Salasvirus, subfamily Picovirinae of family Salasmaviridae. Considering the small numbers of ICTV-accepted B. subtilis phages and the importance of the host in the food industry and biotechnology, the current study helps to improve our understanding of the diversity of B. subtilis phages and shed light on the phage-host relationships.


Asunto(s)
Fagos de Bacillus , Podoviridae , Bacillus subtilis/genética , Filogenia , Cromatografía Liquida , Genoma Viral , Espectrometría de Masas en Tándem , Podoviridae/genética , Fagos de Bacillus/genética , Análisis de Secuencia
14.
Nature ; 544(7648): 101-104, 2017 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-28355179

RESUMEN

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide protection against viral and plasmid infection by capturing short DNA sequences from these invaders and integrating them into the CRISPR locus of the prokaryotic host. These sequences, known as spacers, are transcribed into short CRISPR RNA guides that specify the cleavage site of Cas nucleases in the genome of the invader. It is not known when spacer sequences are acquired during viral infection. Here, to investigate this, we tracked spacer acquisition in Staphylococcus aureus cells harbouring a type II CRISPR-Cas9 system after infection with the staphylococcal bacteriophage ϕ12. We found that new spacers were acquired immediately after infection preferentially from the cos site, the viral free DNA end that is first injected into the cell. Analysis of spacer acquisition after infection with mutant phages demonstrated that most spacers are acquired during DNA injection, but not during other stages of the viral cycle that produce free DNA ends, such as DNA replication or packaging. Finally, we showed that spacers acquired from early-injected genomic regions, which direct Cas9 cleavage of the viral DNA immediately after infection, provide better immunity than spacers acquired from late-injected regions. Our results reveal that CRISPR-Cas systems exploit the phage life cycle to generate a pattern of spacer acquisition that ensures a successful CRISPR immune response.


Asunto(s)
Fagos de Bacillus/genética , Fagos de Bacillus/inmunología , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , ADN Viral/genética , Staphylococcus aureus/inmunología , Staphylococcus aureus/virología , Sitios de Ligazón Microbiológica/genética , Fagos de Bacillus/crecimiento & desarrollo , Fagos de Bacillus/fisiología , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/inmunología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/inmunología , ADN Viral/inmunología , ADN Viral/metabolismo , Mutación , Staphylococcus aureus/genética , Factores de Tiempo , Transfección
15.
Int J Mol Sci ; 24(18)2023 Sep 20.
Artículo en Inglés | MEDLINE | ID: mdl-37762620

RESUMEN

Bacillus subtilis is a model organism for studying Gram-positive bacteria and serves as a cell factory in the industry for enzyme and chemical production. Additionally, it functions as a probiotic in the gastrointestinal tract, modulating the gut microbiota. Its lytic phage SPO1 is also the most studied phage among the genus Okubovrius, including Bacillus phage SPO1 and Camphawk. One of the notable features of SPO1 is the existence of a "host-takeover module", a cluster of 24 genes which occupies most of the terminal redundancy. Some of the gene products from the module have been characterized, revealing their ability to disrupt host metabolism by inhibiting DNA replication, RNA transcription, cell division, and glycolysis. However, many of the gene products which share limited similarity to known proteins remain under researched. In this study, we highlight the involvement of Gp49, a gene product from the module, in host RNA binding and heme metabolism-no observation has been reported in other phages. Gp49 folds into a structure that does not resemble any protein in the database and has a new putative RNA binding motif. The transcriptome study reveals that Gp49 primarily upregulates host heme synthesis which captures cytosolic iron to facilitate phage development.


Asunto(s)
Fagos de Bacillus , Bacteriófagos , Bacteriófagos/genética , Fagos de Bacillus/genética , Proteínas Virales/genética , División Celular , Proteínas de Unión al ARN/genética , Hemo , Bacillus subtilis/fisiología
16.
Biophys J ; 121(10): 1909-1918, 2022 05 17.
Artículo en Inglés | MEDLINE | ID: mdl-35421388

RESUMEN

The gp16 ATPase is the constituent subunit of the pentameric dsDNA (double-stranded deoxyribonucleic acid) translocation motor of the Bacillus subtilis Φ29 bacteriophage. Although recent single-molecule studies have provided tantalizing clues about the activity of this motor, the mechanism by which the gp16 subunits couple the energy obtained from the binding and hydrolysis of ATP to the mechanical work of dsDNA translocation remains unknown. To address this need, we have characterized the binding of fluorophore-labeled ATP and ADP to monomeric gp16 using a stopped-flow fluorescence assay. These experiments show that the binding of ATP/ADP occurs through a single-step mechanism with corresponding affinities of 523.8 ± 247.3 nM for ATP and a lower limit of 30 µM for ADP. When analyzed through the lens of changes in free energy of the system, this difference in binding affinities is reasonable for a cyclical process of binding, hydrolysis, and product release. In addition to answering questions about the activity of monomeric gp16, these results are also a necessary step in constructing a model for intersubunit communication within the pentameric gp16 motor.


Asunto(s)
Adenosina Trifosfatasas , Fagos de Bacillus , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Fagos de Bacillus/genética , ADN Viral/metabolismo , Hidrólisis , Cinética
17.
BMC Genomics ; 23(1): 777, 2022 Nov 28.
Artículo en Inglés | MEDLINE | ID: mdl-36443683

RESUMEN

BACKGROUND: Bacteriophages are widely considered to be highly abundant and genetically diverse, with their role in the evolution and virulence of many pathogens becoming increasingly clear. Less attention has been paid on phages preying on Bacillus, despite the potential for some of its members, such as Bacillus anthracis, to cause serious human disease. RESULTS: We have isolated five phages infecting the causative agent of anthrax, Bacillus anthracis. Using modern phylogenetic approaches we place these five new Bacillus phages, as well as 21 similar phage genomes retrieved from publicly available databases and metagenomic datasets into the Tyrovirus group, a newly proposed group named so due to the conservation of three distinct tyrosine recombinases. Genomic analysis of these large phages (~ 160-170 kb) reveals their DNA packaging mechanism and genomic features contributing to virion morphogenesis, host cell lysis and phage DNA replication processes. Analysis of the three tyrosine recombinases suggest Tyroviruses undergo a prophage lifecycle that may involve both host integration and plasmid stages. Further we show that Tyroviruses rely on divergent invasion mechanisms, with a subset requiring host S-layer for infection. CONCLUSIONS: Ultimately, we expand upon our understanding on the classification, phylogeny, and genomic organisation of a new and substantial phage group that prey on critically relevant Bacillus species. In an era characterised by a rapidly evolving landscape of phage genomics the deposition of future Tyroviruses will allow the further unravelling of the global spread and evolutionary history of these Bacillus phages.


Asunto(s)
Fagos de Bacillus , Bacillus , Humanos , Bacillus/genética , Suelo , Filogenia , Fagos de Bacillus/genética , Recombinasas , Tirosina
18.
Mol Microbiol ; 115(6): 1110-1121, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33244797

RESUMEN

In Bacillus subtilis, a sporulation-related gene, spsM, is disrupted by SPß prophage, but reconstituted during sporulation through SPß excision. The spsM reconstitution is catalyzed by a site-specific DNA recombinase, SprA, and its cognate recombination directionality factor, SprB. SprB interacts with SprA, directing the SprA-mediated recombination reaction from integration to excision; however, the details of the directionality control remains unclear. Here, we demonstrate the importance of the extreme C-terminal region (ECT) of SprA in the DNA recombination and directionality control. We created a series of SprA C-terminal deletants and examined their DNA-binding and recombination activities. Deletions in the ECT caused a loss of integration and excision activity, the magnitudes of which positively correlated with the deletion size. Gel shift study revealed that the loss of the integration activity was attributable to the failure of synaptic complex formation. The excision deficiency was caused by defective interaction with SprB. Moreover, alanine scanning analysis revealed that Phe532 is essential to interact with SprB. SprAF532A , therefore, showed almost no excision activity, while retaining the integration activity. Collectively, these results suggest that the ECT plays the crucial roles in the interaction of SprA with SprB and possibly in the directional control of the recombination.


Asunto(s)
Fagos de Bacillus/genética , Bacillus subtilis/genética , ADN Nucleotidiltransferasas/metabolismo , Integrasas/metabolismo , Recombinación Genética/genética , Bacillus subtilis/enzimología , Bacillus subtilis/metabolismo , ADN Bacteriano/genética , Proteínas de Unión al ADN/genética , Eliminación de Gen , Integrasas/genética , Lisogenia/genética
19.
Environ Microbiol ; 24(4): 2098-2118, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35293111

RESUMEN

The Bacillus phage SPß has been known for about 50 years, but only a few strains are available. We isolated four new wild-type strains of the SPbeta species. Phage vB_BsuS-Goe14 introduces its prophage into the spoVK locus, previously not observed to be used by SPß-like phages. Sequence data revealed the genome replication strategy and the genome packaging mode of SPß-like phages. We extracted 55 SPß-like prophages from public Bacillus genomes, thereby discovering three more integration loci and one additional type of integrase. The identified prophages resemble four new species clusters and three species orphans in the genus Spbetavirus. The determined core proteome of all SPß-like prophages consists of 38 proteins. The integration cassette proved to be not conserved, even though, present in all strains. It consists of distinct integrases. Analysis of SPß transcriptomes revealed three conserved genes, yopQ, yopR, and yokI, to be transcribed from a dormant prophage. While yopQ and yokI could be deleted from the prophage without activating the prophage, damaging of yopR led to a clear-plaque phenotype. Under the applied laboratory conditions, the yokI mutant showed an elevated virion release implying the YokI protein being a component of the arbitrium system.


Asunto(s)
Fagos de Bacillus , Siphoviridae , Fagos de Bacillus/genética , Fagos de Bacillus/metabolismo , Integrasas/genética , Lisogenia/genética , Profagos/genética , Integración Viral
20.
Appl Environ Microbiol ; 88(10): e0247821, 2022 05 24.
Artículo en Inglés | MEDLINE | ID: mdl-35499330

RESUMEN

The infection of a bacterium by a tailed phage starts from the adsorption process, which consists of a specific and strong interaction between viral proteins called receptor binding proteins (RBPs) and receptors located on the bacterial surface. In addition to RBPs, other tail proteins, such as evolved distal tail (evoDit) proteins and tail lysins, harboring carbohydrate binding modules (CBMs) have been shown to facilitate the phage adsorption by interacting with host polysaccharides. In this work, the proteins involved in the adsorption of Deep-Purple, a siphovirus targeting bacteria of the Bacillus cereus group, were studied. Bioinformatic analysis of Deep-Purple tail protein region revealed that it contains two proteins presenting CBM domains: Gp28, an evoDit protein, and Gp29, the potential RBP. The implication of both proteins in the adsorption of Deep-Purple particles was confirmed through cell wall decoration assays. Interestingly, whereas RBP-Gp29 exhibited the same host spectrum as Deep-Purple, evoDit-Gp28 was able to bind to many B. cereus group strains, including some that are not sensitive to the phage infection. Using immunogold microscopy, both proteins were shown to be located in the phage baseplate. Additionally, an in silico analysis of the tail regions encoded by several Siphoviridae infecting the B. cereus group was performed. It revealed that although the tail organization displayed by Deep-Purple is the most prevalent, different tail arrangements are observed, suggesting that distinct baseplate organization and adsorption mechanisms are encountered in siphoviruses targeting the B. cereus group. IMPORTANCE The B. cereus group is a complex cluster of closely related species, among which certain strains can be pathogenic (i.e., Bacillus anthracis, Bacillus cereus sensu stricto, and Bacillus cytotoxicus). Nowadays, phages are receiving increasing attention for applications in controlling and detecting such pathogens. Thus, understanding the molecular mechanisms governing the phage adsorption to its bacterial host is paramount as this step is a key determinant of the phage host spectrum. Until now, the knowledge regarding the adsorption process of tailed phage targeting the B. cereus groups was mainly restricted to the phage gamma infecting B. anthracis. With this work, we provide novel insights into the adsorption of Deep-Purple, a siphovirus infecting the B. cereus group. We showed that this phage recognizes polysaccharides and relies on two different viral proteins for its successful adsorption.


Asunto(s)
Fagos de Bacillus , Siphoviridae , Adsorción , Fagos de Bacillus/genética , Bacillus cereus , Siphoviridae/genética , Proteínas Virales
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