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Much like social networks are used to connect with friends or relatives, bacteria communicate with relatives through quorum sensing. Viruses, though, were thought to be asocial-until now. Erez et al. (2017) reveal that viruses are also sharing information with relatives.
Assuntos
Bactérias , Percepção de QuorumRESUMO
Bacterial viruses (bacteriophages, phages) of the gut have increasingly become a focus in microbiome studies, with an understanding that they are likely key players in health and disease. However, characterization of the virome remains largely based on bioinformatic approaches, with the impact of these viromes inferred based on a century of knowledge from aerobic phage work. Studying the phages infecting anaerobes is difficult, as they are often technically demanding to isolate and propagate. In this review, we primarily discuss the phages infecting three well-studied anaerobes in the gut: Bifidobacterium, Clostridia and Bacteroides, with a particular focus on the challenges in isolating and characterizing these phages. We contrast the lessons learned from these to other anaerobic work on phages infecting facultative anaerobes of the gut: Enterococcus and Lactobacillus. Phages from the gut do appear to adhere to the lessons learned from aerobic work, but the additional challenges of working on them has required ingenious new approaches to enable their study. This, in turn, has uncovered remarkable biology likely underpinning phage-host relationships in many stable environments.
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Bactérias/virologia , Bacteriófagos/isolamento & purificação , Viroma , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bacteriófagos/classificação , Bacteriófagos/genética , Bacteriófagos/fisiologia , Microbioma Gastrointestinal , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/virologia , HumanosRESUMO
Gene transfer agents (GTAs) are phage-like particles that can package and transfer a random piece of the producing cell's genome, but are unable to transfer all the genes required for their own production. As such, GTAs represent an evolutionary conundrum: are they selfish genetic elements propagating through an unknown mechanism, defective viruses, or viral structures "repurposed" by cells for gene exchange, as their name implies? In Rhodobacter capsulatus, production of the R. capsulatus GTA (RcGTA) particles is associated with a cluster of genes resembling a small prophage. Utilizing transcriptomic, genetic and biochemical approaches, we report that the RcGTA "genome" consists of at least 24 genes distributed across five distinct loci. We demonstrate that, of these additional loci, two are involved in cell recognition and binding and one in the production and maturation of RcGTA particles. The five RcGTA "genome" loci are widespread within Rhodobacterales, but not all loci have the same evolutionary histories. Specifically, two of the loci have been subject to frequent, probably virus-mediated, gene transfer events. We argue that it is unlikely that RcGTA is a selfish genetic element. Instead, our findings are compatible with the scenario that RcGTA is a virus-derived element maintained by the producing organism due to a selective advantage of within-population gene exchange. The modularity of the RcGTA "genome" is presumably a result of selection on the host organism to retain GTA functionality.
Assuntos
Evolução Biológica , Transferência Genética Horizontal , Rhodobacter capsulatus/genética , Proteínas de Bactérias/genética , Bacteriófagos/genética , Regulação Bacteriana da Expressão Gênica , Transferência Genética Horizontal/genética , Prófagos/genéticaRESUMO
A recent demonstration of synergy between a temperate phage and the antibiotic ciprofloxacin suggested a scalable approach to exploiting temperate phages in therapy, termed temperate phage-antibiotic synergy, which specifically interacted with the lysis-lysogeny decision. To determine whether this would hold true across antibiotics, we challenged Escherichia coli with the phage HK97 and a set of 13 antibiotics spanning seven classes. As expected, given the conserved induction pathway, we observed synergy with classes of drugs known to induce an SOS response: a sulfa drug, other quinolones, and mitomycin C. While some ß-lactams exhibited synergy, this appeared to be traditional phage-antibiotic synergy, with no effect on the lysis-lysogeny decision. Curiously, we observed a potent synergy with antibiotics not known to induce the SOS response: protein synthesis inhibitors gentamicin, kanamycin, tetracycline, and azithromycin. The synergy results in an eightfold reduction in the effective minimum inhibitory concentration of gentamicin, complete eradication of the bacteria, and, when administered at sub-optimal doses, drastically decreases the frequency of lysogens emerging from the combined challenge. However, lysogens exhibit no increased sensitivity to the antibiotic; synergy was maintained in the absence of RecA; and the antibiotic reduced the initial frequency of lysogeny rather than selecting against formed lysogens. Our results confirm that SOS-inducing antibiotics broadly result in temperate-phage-specific synergy, but that other antibiotics can interact with temperate phages specifically and result in synergy. This is the first report of a means of chemically blocking entry into lysogeny, providing a new means for manipulating the key lysis-lysogeny decision.IMPORTANCEThe lysis-lysogeny decision is made by most bacterial viruses (bacteriophages, phages), determining whether to kill their host or go dormant within it. With over half of the bacteria containing phages waiting to wake, this is one of the most important behaviors in all of biology. These phages are also considered unusable for therapy because of this behavior. In this paper, we show that many antibiotics bias this behavior to "wake" the dormant phages, forcing them to kill their host, but some also prevent dormancy in the first place. These will be important tools to study this critical decision point and may enable the therapeutic use of these phages.
Assuntos
Antibacterianos , Escherichia coli , Lisogenia , Antibacterianos/farmacologia , Escherichia coli/virologia , Escherichia coli/efeitos dos fármacos , Resposta SOS em Genética/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Colífagos/fisiologia , Colífagos/efeitos dos fármacos , Sinergismo Farmacológico , Bacteriófagos/fisiologia , Bacteriófagos/efeitos dos fármacos , Mitomicina/farmacologiaRESUMO
Rhodobacter capsulatus produces a gene transfer agent (GTA) called RcGTA. RcGTA is a phage-like particle that packages R. capsulatus DNA and transfers it to other R. capsulatus cells. We quantified the relative frequency of packaging for each gene in the genome by hybridization of DNA from RcGTA particles to an R. capsulatus microarray. All genes were found within the RcGTA particles. However, the genes encoding the RcGTA particle were under-packaged compared with other regions. Gene transfer bioassays confirmed that the transfer of genes within the RcGTA structural cluster is reduced relative to those of other genes. Single-cell expression analysis, by flow cytometry analysis of cells containing RcGTA-reporter gene fusion constructs, demonstrated that RcGTA gene expression is not uniform within a culture. This phenomenon was accentuated when the constructs were placed in a strain lacking a putative lysis gene involved in RcGTA release; a small subpopulation was found to be responsible for â¼ 95% of RcGTA activity. We propose a mechanism whereby high levels of RcGTA gene transcription in the most active RcGTA-producing cells cause a reduction in their packaging frequency. This subpopulation's role in producing and releasing the RcGTA particles explains the lack of observed cell lysis in cultures.
Assuntos
Proteínas de Bactérias/biossíntese , Empacotamento do DNA , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica , Transferência Genética Horizontal , Rhodobacter capsulatus/genética , Rhodobacter capsulatus/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Genes Reporter , Dados de Sequência MolecularRESUMO
Urinary tract infections (UTIs) are a problem worldwide, affecting almost half a billion people each year. Increasing antibiotic resistance and limited therapeutic options have led to the exploration of alternative therapies for UTIs, including bacteriophage (phage) therapy. This systematic review aims at evaluating the efficacy of phage therapy in treating UTIs. We employed a comprehensive search strategy for any language, any animal, and any publication date. A total of 55 in vivo and clinical studies were included. Of the studies, 22% were published in a non-English language, 32.7% were before the year 1996, and the rest were after 2005. The results of this review suggest that phage therapy for UTIs can be effective; more than 72% of the included articles reported microbiological and clinical improvements. On the other hand, only 5 randomized controlled trials have been completed, and case reports and case series information were frequently incomplete for analysis. Overall, this comprehensive systematic review identifies preliminary evidence supporting the potential of phage therapy as a safe and viable option for the treatment of UTIs.
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In the original publication [...].
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There is renewed interest in bacterial viruses (phages) as alternatives to antibiotics. All phage treatments to date have used virulent phages rather than temperate ones, as these can integrate into the genome of the bacterial host and lie dormant. However, temperate phages are abundant and easier to isolate. To make use of these entities, we leverage stressors known to awaken these dormant, integrated phages. Co-administration of the temperate phage HK97 with sub-inhibitory concentrations of the antibiotic ciprofloxacin results in bacterial eradication (≥8 log reduction) in vitro. This synergy is mechanistically distinct from phage-antibiotic-synergy described for virulent phages. Instead, the antibiotic specifically selects against bacteria in which the phage has integrated. As the interaction between temperate phages and stressors such as ciprofloxacin are known to be widespread, this approach may be broadly applicable and enable the use of temperate phages to combat bacterial infections.
Assuntos
Antibacterianos/uso terapêutico , Bactérias/efeitos dos fármacos , Bacteriófagos/genética , Lisogenia/genética , Antibacterianos/farmacologia , HumanosRESUMO
Many bacteria carry bacteriophages (bacterial viruses) integrated in their genomes in the form of prophages, which replicate passively alongside their bacterial host. Environmental conditions can lead to prophage induction; the switching from prophage replication to lytic replication, that results in new bacteriophage progeny and the lysis of the bacterial host. Despite their abundance in the gut, little is known about what could be inducing these prophages. We show that several medications, at concentrations predicted in the gut, lead to prophage induction of bacterial isolates from the human gut. We tested five medication classes (non-steroidal anti-inflammatory, chemotherapy, mild analgesic, cardiac, and antibiotic) for antimicrobial activity against eight prophage-carrying human gut bacterial representative isolates in vitro. Seven out of eight bacteria showed signs of growth inhibition in response to at least one medication. All medications led to growth inhibition of at least one bacterial isolate. Prophage induction was confirmed in half of the treatments showing antimicrobial activity. Unlike antibiotics, host-targeted medications led to a species-specific induction of Clostridium beijerinckii, Bacteroides caccae, and to a lesser extent Bacteroides eggerthii. These results show how common medication consumption can lead to phage-mediated effects, which in turn would alter the human gut microbiome through increased prophage induction.
Assuntos
Bactérias/crescimento & desenvolvimento , Bactérias/virologia , Bacteriófagos/crescimento & desenvolvimento , Lisogenia/efeitos dos fármacos , Preparações Farmacêuticas/administração & dosagem , Ativação Viral/efeitos dos fármacos , Bactérias/efeitos dos fármacos , Bactérias/genética , Bacteriófagos/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , HumanosRESUMO
Gut microbiota have myriad roles in host physiology, development, and immunity. Though confined to the intestinal lumen by the epithelia, microbes influence distal systems via poorly characterized mechanisms. Recent work has considered the role of extracellular vesicles in interspecies communication, but whether they are involved in systemic microbe-host interaction is unclear. Here, we show that distinctive nanoparticles can be isolated from mouse blood within 2.5 h of consuming Lacticaseibacillus rhamnosus JB-1. In contrast to blood nanoparticles from saline-fed mice, they reproduced lipoteichoic acid-mediated immune functions of the original bacteria, including activation of TLR2 and increased IL-10 expression by dendritic cells. Like the fed bacteria, they also reduced IL-8 induced by TNF in an intestinal epithelial cell line. Though enriched for host neuronal proteins, these isolated nanoparticles also contained proteins and viral (phage) DNA of fed bacterial origin. Our data strongly suggest that oral consumption of live bacteria rapidly leads to circulation of their membrane vesicles and phages and demonstrate a nanoparticulate pathway whereby beneficial bacteria and probiotics may systemically affect their hosts.
Assuntos
Bacteriófagos/isolamento & purificação , Sangue/microbiologia , Sangue/virologia , Células Dendríticas/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Lacticaseibacillus rhamnosus/metabolismo , Probióticos/farmacologia , Animais , Bacteriófagos/genética , Células Dendríticas/imunologia , Vesículas Extracelulares/química , Interleucina-8/genética , Interleucina-8/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Lacticaseibacillus rhamnosus/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C/genéticaRESUMO
Modern bacteriophage encapsulation methods based on polymers such as alginate have been developed recently for their use in phage therapy for veterinary purposes. In birds, it has been proven that using this delivery system allows the release of the bacteriophage in the small intestine, the site of infection by Salmonella spp. This work designed an approach for phage therapy using encapsulation by ionotropic gelation of the lytic bacteriophage S1 for Salmonella enterica in 2% w/v alginate beads using 2% w/v calcium chloride as crosslinking agent. This formulation resulted in beads with an average size of 3.73 ± 0.04 mm and an encapsulation efficiency of 70%. In vitro, the beads protected the bacteriophages from pH 3 and released them at higher pH. To confirm that this would protect the bacteriophages from gastrointestinal pH changes, we tested the phage infectivity in vivo assay. Using a model chicken (Gallus gallus domesticus) infected with Salmonella Enteritidis, we confirmed that after 3 h of the beads delivery, infective phages were present in the chicken's duodenal and caecal sections. This study demonstrates that our phage formulation is an effective system for release and delivery of bacteriophage S1 against Salmonella Enteritidis with potential use in the poultry sector.
Assuntos
Terapia por Fagos/métodos , Fagos de Salmonella/metabolismo , Alginatos/química , Animais , Bacteriófagos , Ceco/metabolismo , Encapsulamento de Células/métodos , Galinhas/microbiologia , Trato Gastrointestinal/metabolismo , Microesferas , Aves Domésticas/virologia , Fagos de Salmonella/genética , Salmonella enterica/metabolismo , Salmonella enterica/virologiaRESUMO
If, as we all know, only the strong survive, why do bacterial viruses (phages) encode weak suppressors of a bacterial immune system? In this issue of Cell Host & Microbe, Chevallereau et al. (2019) expertly demonstrate how, in the context of competition with other phages, weakness can be a strength.
Assuntos
Bacteriófagos/genética , Bactérias , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Comportamento CooperativoRESUMO
Pseudomonas pseudoalcaligenes KF707 is naturally resistant to the toxic metalloid tellurite, but the mechanisms of resistance are not known. In this study we report the isolation of a KF707 mutant (T5) with hyperresistance to tellurite. In order to characterize the bacterial response and the pathways leading to tolerance, we utilized Phenotype MicroArray technology (Biolog) and a metabolomic technique based on nuclear magnetic resonance spectroscopy. The physiological states of KF707 wild-type and T5 cells exposed to tellurite were also compared in terms of viability and reduced thiol content. Our analyses showed an extensive change in metabolism upon the addition of tellurite to KF707 cultures as well as different responses when the wild-type and T5 strains were compared. Even in the absence of tellurite, T5 cells displayed a "poised" physiological status, primed for tellurite exposure and characterized by altered intracellular levels of glutathione, branched-chain amino acids, and betaine, along with increased resistance to other toxic metals and metabolic inhibitors. We conclude that hyperresistance to tellurite in P. pseudoalcaligenes KF707 is correlated with the induction of the oxidative stress response, resistance to membrane perturbation, and reconfiguration of cellular metabolism.
Assuntos
Farmacorresistência Bacteriana , Metabolômica , Pseudomonas pseudoalcaligenes/efeitos dos fármacos , Pseudomonas pseudoalcaligenes/metabolismo , Telúrio/toxicidade , Citoplasma/química , Espectroscopia de Ressonância Magnética , Viabilidade Microbiana , Pseudomonas pseudoalcaligenes/química , Compostos de Sulfidrila/análiseRESUMO
CRISPR-Cas systems are bacterial anti-viral systems, and bacterial viruses (bacteriophages, phages) can carry anti-CRISPR (Acr) proteins to evade that immunity. Acrs can also fine-tune the activity of CRISPR-based genome-editing tools. While Acrs are prevalent in phages capable of lying dormant in a CRISPR-carrying host, their orthologs have been observed only infrequently in virulent phages. Here we identify AcrIIA6, an Acr encoded in 33% of virulent Streptococcus thermophilus phage genomes. The X-ray structure of AcrIIA6 displays some features unique to this Acr family. We compare the activity of AcrIIA6 to those of other Acrs, including AcrIIA5 (also from S. thermophilus phages), and characterize their effectiveness against a range of CRISPR-Cas systems. Finally, we demonstrate that both Acr families from S. thermophilus phages inhibit Cas9-mediated genome editing of human cells.
Assuntos
Proteína 9 Associada à CRISPR/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Bacteriófagos/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiologia , Edição de Genes , Humanos , Virulência/genética , Virulência/fisiologiaRESUMO
The CRISPR-Cas system owes its utility as a genome-editing tool to its origin as a prokaryotic immune system. The first demonstration of its activity against bacterial viruses (phages) is also the first record of phages evading that immunity 1 . This evasion can be due to point mutations 1 , large-scale deletions 2 , DNA modifications 3 , or phage-encoded proteins that interfere with the CRISPR-Cas system, known as anti-CRISPRs (Acrs) 4 . The latter are of biotechnological interest, as Acrs can serve as off switches for CRISPR-based genome editing 5 . Every Acr characterized to date originated from temperate phages, genomic islands, or prophages 4-8 , and shared properties with the first Acr discovered. Here, with a phage-oriented approach, we have identified an unrelated Acr in a virulent phage of Streptococcus thermophilus. In challenging a S. thermophilus strain CRISPR-immunized against a set of virulent phages, we found one that evaded the CRISPR-encoded immunity >40,000× more often than the others. Through systematic cloning of its genes, we identified an Acr solely responsible for the abolished immunity. We extended our findings by demonstrating activity in another S. thermophilus strain, against unrelated phages, and in another bacterial genus immunized using the heterologous SpCas9 system favoured for genome editing. This Acr completely abolishes SpCas9-mediated immunity in our assays.
Assuntos
Sistemas CRISPR-Cas/efeitos dos fármacos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/efeitos dos fármacos , Fagos de Streptococcus/genética , Fagos de Streptococcus/metabolismo , Streptococcus pyogenes/efeitos dos fármacos , Streptococcus pyogenes/virologia , Proteínas Virais/genética , Proteínas Virais/farmacologia , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/virologia , Edição de Genes , Ilhas Genômicas/genética , Imunidade , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Fenótipo , Mutação Puntual , Prófagos , Streptococcus pyogenes/imunologia , Streptococcus thermophilus/genética , Streptococcus thermophilus/virologia , Transformação Bacteriana , Proteínas Virais/imunologiaRESUMO
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems have been adapted into a powerful genome-editing tool. The basis for the flexibility of the tool lies in the adaptive nature of CRISPR-Cas as a bacterial immune system. Here, we describe a protocol to experimentally demonstrate the adaptive nature of this bacterial immune system by challenging the model organism for the study of CRISPR adaptation, Streptococcus thermophilus, with phages in order to detect natural CRISPR immunization. A bacterial culture is challenged with lytic phages, the surviving cells are screened by PCR for expansion of their CRISPR array and the newly acquired specificities are mapped to the genome of the phage. Furthermore, we offer three variants of the assay to (i) promote adaptation by challenging the system using defective viruses, (ii) challenge the system using plasmids to generate plasmid-resistant strains and (iii) bias the system to obtain natural immunity against a specifically targeted DNA sequence. The core protocol and its variants serve as a means to explore CRISPR adaptation, discover new CRISPR-Cas systems and generate bacterial strains that are resistant to phages or refractory to undesired genes or plasmids. In addition, the core protocol has served in teaching laboratories at the undergraduate level, demonstrating both its robust nature and educational value. Carrying out the core protocol takes 4 h of hands-on time over 7 d. Unlike sequence-based methods for detecting natural CRISPR adaptation, this phage-challenge-based approach results in the isolation of CRISPR-immune bacteria for downstream characterization and use.
Assuntos
Adaptação Fisiológica , Pesquisa , Streptococcus thermophilus/genética , Streptococcus thermophilus/fisiologia , Ensino , Bacteriófagos/fisiologia , Sistemas CRISPR-Cas , Streptococcus thermophilus/imunologia , Streptococcus thermophilus/virologiaRESUMO
UNLABELLED: The adaptive immune system of prokaryotes, called CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes), results in specific cleavage of invading nucleic acid sequences recognized by the cell's "memory" of past encounters. Here, we exploited the properties of native CRISPR-Cas systems to program the natural "memorization" process, efficiently generating immunity not only to a bacteriophage or plasmid but to any specifically chosen DNA sequence. IMPORTANCE: CRISPR-Cas systems have entered the public consciousness as genome editing tools due to their readily programmable nature. In industrial settings, natural CRISPR-Cas immunity is already exploited to generate strains resistant to potentially disruptive viruses. However, the natural process by which bacteria acquire new target specificities (adaptation) is difficult to study and manipulate. The target against which immunity is conferred is selected stochastically. By biasing the immunization process, we offer a means to generate customized immunity, as well as provide a new tool to study adaptation.
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Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células Procarióticas , Bacteriófagos , PlasmídeosRESUMO
Key components of CRISPR-Cas systems have been adapted into a powerful genome-editing tool that has caught the headlines and the attention of the public. Canonically, a customized RNA serves to guide an endonuclease (e.g. Cas9) to its DNA target, resulting in precise genomic lesions that can be repaired in a personalized fashion by cellular machinery. Here, we turn to the microbes that are the source of this system to explore many of its other notable applications. These include mining the CRISPR 'memory' arrays for functional genomic data, generation of customized virus-resistant or plasmid-refractory bacterial cells, editing of previously intractable viral genomes, and exploiting the unique properties of a catalytically inactive Cas9, dCas9, to serve as a highly customizable anti-nucleic acid 'antibody'.
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Bactérias/virologia , Bacteriófagos/imunologia , Sistemas CRISPR-Cas , Bactérias/genética , Bactérias/imunologia , Bacteriófagos/genética , Genoma Bacteriano , Genoma ViralRESUMO
Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated cas genes serve as a prokaryotic 'adaptive' immune system, protecting against foreign DNA elements such as bacteriophages. CRISPR-Cas systems function by incorporating short DNA 'spacers', homologous to invading DNA sequences, into a CRISPR array (adaptation). The array is then transcribed and matured into RNA molecules (maturation) that target homologous DNA for cleavage (interference). It is unclear how these three stages could occur quickly enough in a naive phage-infected cell to interfere with phage replication before this cell would be irrevocably damaged by the infection. Here we demonstrate that cells can acquire spacers from defective phages at a rate directly proportional to the quantity of replication-deficient phages to which the cells are exposed. This process is reminiscent of immunization in humans by vaccination with inactivated viruses.
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Imunidade Adaptativa/imunologia , Bacteriófagos/imunologia , Sistemas CRISPR-Cas/imunologia , Streptococcus thermophilus/imunologia , Imunidade Adaptativa/genética , Bacteriófagos/genética , Sistemas CRISPR-Cas/genética , Dano ao DNA/genética , DNA Bacteriano/genética , DNA Intergênico/genética , Mutação/genética , Streptococcus thermophilus/genéticaRESUMO
The α-proteobacterium Rhodobacter capsulatus is a model organism for the study of bacterial photosynthesis and the bacteriophage-like gene transfer agent. Characterization of phages that infect Rhodobacter is extremely rare, and scarce for the α-proteobacteria in general. Here, we describe the discovery of the only functional Mu-like transposing phage to have been identified in the α-proteobacteria, RcapMu, resident in the genome-sequenced R. capsulatus SB1003 strain. RcapMu packages ~42kb of total DNA, including <3kb of host DNA with no conserved motifs, indicative of replicative transposition with little insertion site preference. The phage genome contains 58 ORFs with comparable organization to known transposable phages. Shotgun proteomics of purified RcapMu particles detected all proteins with predicted structural functions as well as seven hypothetical proteins. Overall, comparison of RcapMu to enterobacteria phage Mu and other Mu-like phages revealed only regional homology to these phages, providing further evidence for the promiscuous, modular nature of bacteriophage evolution.