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1.
Nat Commun ; 11(1): 4403, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32879312

RESUMO

Bacteriophage genomes rapidly evolve via mutation and horizontal gene transfer to counter evolving bacterial host defenses; such arms race dynamics should lead to divergence between phages from similar, geographically isolated ecosystems. However, near-identical phage genomes can reoccur over large geographical distances and several years apart, conversely suggesting many are stably maintained. Here, we show that phages with near-identical core genomes in distant, discrete aquatic ecosystems maintain diversity by possession of numerous flexible gene modules, where homologous genes present in the pan-genome interchange to create new phage variants. By repeatedly reconstructing the core and flexible regions of phage genomes from different metagenomes, we show a pool of homologous gene variants co-exist for each module in each location, however, the dominant variant shuffles independently in each module. These results suggest that in a natural community, recombination is the largest contributor to phage diversity, allowing a variety of host recognition receptors and genes to counter bacterial defenses to co-exist for each phage.


Assuntos
Bacteriófagos/genética , Camada de Gelo/virologia , Metagenoma , Cianobactérias/virologia , Ecossistema , Transferência Genética Horizontal , Genes Virais , Genoma Viral , Interações entre Hospedeiro e Microrganismos/genética , Camada de Gelo/microbiologia , Metagenômica , Filogenia
2.
Methods Mol Biol ; 2203: 147-165, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833211

RESUMO

We have developed a reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) in which a full-length cDNA corresponding to the IBV genome is inserted into the vaccinia virus genome under the control of a T7 promoter sequence. Vaccinia virus as a vector for the full-length IBV cDNA has the advantage that modifications can be introduced into the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. Here, we describe the use of transient dominant selection as a method for introducing modifications into the IBV cDNA that has been successfully used for the substitution of specific nucleotides, deletion of genomic regions, and the exchange of complete genes. Infectious recombinant IBVs are generated in situ following the transfection of vaccinia virus DNA, containing the modified IBV cDNA, into cells infected with a recombinant fowlpox virus expressing T7 DNA-dependent RNA polymerase.


Assuntos
Vírus da Bronquite Infecciosa/genética , Transfecção/métodos , Vírus Vaccinia/genética , Animais , Bacteriófagos/genética , Chlorocebus aethiops , DNA Polimerase Dirigida por DNA/metabolismo , Vírus da Varíola das Aves Domésticas/genética , Recombinação Homóloga , Microrganismos Geneticamente Modificados , Vírus Vaccinia/isolamento & purificação , Células Vero
4.
Virology ; 548: 160-167, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838937

RESUMO

Filamentous Inoviridae phages integrate into the chromosome of plant pathogens Xanthomonas as prophages, but their diversity and integrative mechanism are not completely understood. A proviral Cf2 sequence of 6454 bases from Xanthomonas citri genome was revived as infectious virions able to lysogenize its host. Unlike other Xanthomonas phages (Cf1c, φLf, Xf109, XacF1), Cf2 phage has RstA/RstB replication protein, and its attP has XerD binding arm and dif central region but lacks XerC binding arm. XerC+/Xf109 and XerD+/Cf2 attPs are in the opposite direction in phage genomes. Moreover, XerCD binding and XerD catalysis for strand exchange are necessary for site-specific integration of XerD+/Cf2 and XerC+/Xf109 attPs. Taken together, these results provide a new insight into the mechanism of XerCD-mediated recombination at XerD + attP.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/fisiologia , Inovirus/fisiologia , Integrases/metabolismo , Xanthomonas/enzimologia , Xanthomonas/virologia , Sítios de Ligação Microbiológicos , Proteínas de Bactérias/genética , Bacteriófagos/genética , Genoma Bacteriano , Inovirus/genética , Integrases/genética , Lisogenia , Integração Viral , Xanthomonas/genética
5.
Science ; 369(6501): 333-337, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32675376

RESUMO

CRISPR-Cas systems are found widely in prokaryotes, where they provide adaptive immunity against virus infection and plasmid transformation. We describe a minimal functional CRISPR-Cas system, comprising a single ~70-kilodalton protein, CasΦ, and a CRISPR array, encoded exclusively in the genomes of huge bacteriophages. CasΦ uses a single active site for both CRISPR RNA (crRNA) processing and crRNA-guided DNA cutting to target foreign nucleic acids. This hypercompact system is active in vitro and in human and plant cells with expanded target recognition capabilities relative to other CRISPR-Cas proteins. Useful for genome editing and DNA detection but with a molecular weight half that of Cas9 and Cas12a genome-editing enzymes, CasΦ offers advantages for cellular delivery that expand the genome editing toolbox.


Assuntos
Bacteriófagos/genética , Sistemas CRISPR-Cas , Edição de Genes , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
6.
BMC Bioinformatics ; 21(Suppl 12): 305, 2020 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-32703190

RESUMO

BACKGROUND: Horizontal gene transfer, i.e. the acquisition of genetic material from nonparent organism, is considered an important force driving species evolution. Many cases of horizontal gene transfer from prokaryotes to eukaryotes have been registered, but no transfer mechanism has been deciphered so far, although viruses were proposed as possible vectors in several studies. In agreement with this idea, in our previous study we discovered that in two eukaryotic proteins bacteriophage recombination site (AttP) was adjacent to the regions originating via horizontal gene transfer. In one of those cases AttP site was present inside the introns of cysteine-rich repeats. In the present study we aimed to apply computational tools for finding multiple horizontal gene transfer events in large genome databases. For that purpose we used a sequence of cysteine-rich repeats to identify genes potentially acquired through horizontal transfer. RESULTS: HMMER remote similarity search significantly detected 382 proteins containing cysteine-rich repeats. All of them, except 8 sequences, belong to eukaryotes. In 124 proteins the presence of conserved structural domains was predicted. In spite of the fact that cysteine-rich repeats are found almost exclusively in eukaryotic proteins, many predicted domains are most common for prokaryotes or bacteriophages. Ninety-eight proteins out of 124 contain typical prokaryotic domains. In those cases proteins were considered as potentially originating via horizontal transfer. In addition, HHblits search revealed that two domains of the same fungal protein, Glycoside hydrolase and Peptidase M15, have high similarity with proteins of two different prokaryotic species, hinting at independent horizontal gene transfer events. CONCLUSIONS: Cysteine-rich repeats in eukaryotic proteins are usually accompanied by conserved domains typical for prokaryotes or bacteriophages. These proteins, containing both cysteine-rich repeats, and characteristic prokaryotic domains, might represent multiple independent horizontal gene transfer events from prokaryotes to eukaryotes. We believe that the presence of bacteriophage recombination site inside cysteine-rich repeat coding sequence may facilitate horizontal genes transfer. Thus computational approach, described in the present study, can help finding multiple sequences originated from horizontal transfer in eukaryotic genomes.


Assuntos
Bacteriófagos/genética , Transferência Genética Horizontal/genética , Genes Virais , Recombinação Genética/genética , Proteínas Virais/química , Sequência de Aminoácidos , Sequência de Bases , Sequência Conservada , Domínios Proteicos , Proteínas Virais/classificação
7.
Artigo em Inglês | MEDLINE | ID: mdl-32645350

RESUMO

In this issue of Cell Host & Microbe, two papers by Osuna et al. describe the characterization of AcrIIA1, an anti-CRISPR protein distributed widely among Listeria phages. AcrIIA1 functions as an anti-CRISPR and as a dynamic repressor of acr loci, suggesting it may play an important role in lysogeny.


Assuntos
Bacteriófagos/genética , Listeria , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Lisogenia
8.
Nat Commun ; 11(1): 3784, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32728052

RESUMO

The CRISPR-Cas are adaptive bacterial and archaeal immunity systems that have been harnessed for the development of powerful genome editing and engineering tools. In the incessant host-parasite arms race, viruses evolved multiple anti-defense mechanisms including diverse anti-CRISPR proteins (Acrs) that specifically inhibit CRISPR-Cas and therefore have enormous potential for application as modulators of genome editing tools. Most Acrs are small and highly variable proteins which makes their bioinformatic prediction a formidable task. We present a machine-learning approach for comprehensive Acr prediction. The model shows high predictive power when tested against an unseen test set and was employed to predict 2,500 candidate Acr families. Experimental validation of top candidates revealed two unknown Acrs (AcrIC9, IC10) and three other top candidates were coincidentally identified and found to possess anti-CRISPR activity. These results substantially expand the repertoire of predicted Acrs and provide a resource for experimental Acr discovery.


Assuntos
Bacteriófagos/genética , Proteína 9 Associada à CRISPR/antagonistas & inibidores , Aprendizado de Máquina , Análise de Sequência de Proteína/métodos , Proteínas Virais/genética , Archaea/genética , Archaea/virologia , Bactérias/genética , Bactérias/virologia , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Biologia Computacional/métodos , Conjuntos de Dados como Assunto , Edição de Genes/métodos , Interações Hospedeiro-Parasita/genética , Homologia de Sequência de Aminoácidos
9.
Nat Commun ; 11(1): 2730, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483187

RESUMO

Bacteria have evolved sophisticated adaptive immune systems, called CRISPR-Cas, that provide sequence-specific protection against phage infection. In turn, phages have evolved a broad spectrum of anti-CRISPRs that suppress these immune systems. Here we report structures of anti-CRISPR protein IF9 (AcrIF9) in complex with the type I-F CRISPR RNA-guided surveillance complex (Csy). In addition to sterically blocking the hybridization of complementary dsDNA to the CRISPR RNA, our results show that AcrIF9 binding also promotes non-sequence-specific engagement with dsDNA, potentially sequestering the complex from target DNA. These findings highlight the versatility of anti-CRISPR mechanisms utilized by phages to suppress CRISPR-mediated immune systems.


Assuntos
Bactérias/metabolismo , Bacteriófagos/metabolismo , Sistemas CRISPR-Cas , DNA/metabolismo , RNA Guia/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Bactérias/genética , Bactérias/virologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófagos/genética , Microscopia Crioeletrônica , DNA/química , DNA/genética , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Proteus penneri/genética , Proteus penneri/metabolismo , Proteus penneri/virologia , RNA Guia/química , RNA Guia/genética , Homologia de Sequência de Aminoácidos , Proteínas Virais/química , Proteínas Virais/genética
10.
Arch Virol ; 165(9): 2111-2114, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32556600

RESUMO

A novel myovirus, vB_PagM_AAM22 (AAM22), was isolated in Lithuania using Pantoea agglomerans as the host for phage propagation. The 49,744-bp genome of AAM22 has a G + C content of 48.4% and contains 96 probable protein-encoding genes and no genes for tRNA. In total, 34 ORFs were given a putative functional annotation, including genes associated with virion morphogenesis, DNA metabolism, and phage-host interactions. Based on comparative phylogenetic analysis, AAM22 cannot be assigned to any genus currently recognized by the ICTV and is a potential candidate to form a new genus within the family Myoviridae.


Assuntos
Bacteriófagos/isolamento & purificação , Genoma Viral , Myoviridae/isolamento & purificação , Pantoea/virologia , Bacteriófagos/classificação , Bacteriófagos/genética , Composição de Bases , Sequência de Bases , DNA Viral/genética , Myoviridae/classificação , Myoviridae/genética , Fases de Leitura Aberta , Filogenia
11.
PLoS Negl Trop Dis ; 14(6): e0008387, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32574158

RESUMO

Environmental enteric dysfunction (EED) is characterized by diffuse villous atrophy of the small bowel. EED is strongly associated with stunting, a major public health problem linked to increased childhood morbidity and mortality. EED and subsequent stunting of linear growth are surmised to have microbial origins. To interrogate this relationship, we defined the comprehensive virome (eukaryotic virus and bacteriophage) and bacterial microbiome of a longitudinal cohort of rural Malawian children with extensive metadata and intestinal permeability testing at each time point. We found thirty bacterial taxa differentially associated with linear growth. We detected many eukaryotic viruses. Neither the total number of eukaryotic families nor a specific viral family was statistically associated with improved linear growth. We identified 3 differentially abundant bacteriophage among growth velocities. Interestingly, there was a positive correlation between bacteria and bacteriophage richness in children with subsequent adequate/moderate growth which children with subsequent poor growth lacked. This suggests that a disruption in the equilibrium between bacteria and bacteriophage communities might be associated with subsequent poor growth. Future studies of EED and stunting should include the evaluation of viral communities in addition to bacterial microbiota to understand the complete microbial ecology of these poorly understood entities.


Assuntos
Bactérias/classificação , Bacteriófagos/classificação , Microbioma Gastrointestinal , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/virologia , Transtornos do Crescimento/microbiologia , Transtornos do Crescimento/virologia , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/virologia , Bacteriófagos/genética , Bacteriófagos/crescimento & desenvolvimento , Bacteriófagos/isolamento & purificação , Pré-Escolar , Feminino , Gastroenteropatias/microbiologia , Gastroenteropatias/virologia , Humanos , Lactente , Intestino Delgado/microbiologia , Intestino Delgado/virologia , Malaui , Masculino , Viabilidade Microbiana , Permeabilidade , RNA Ribossômico 16S
12.
Arch Virol ; 165(9): 1995-2002, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32588241

RESUMO

Genomic evolution among bacteriophages infecting Caulobacter crescentus is inevitable. However, the conservation of the proteins associated with intact phage particles has not been investigated. In this study, we compared the structural proteins associated with two genomically diverse but morphologically similar C. crescentus-infecting bacteriophages, phiCbK and CcrSC. We were able to detect more than 20 proteins that are part of the bacteriophage particle in both phages, and we were able to identify a small number of proteins that were found in only one of the two phage particles. All but one of the genes coding for these structural proteins were located in a region of the genome that had been designated a structural region, confirming the idea that the genes in these phage genomes are clustered according to their function. During the purification process, we also discovered that phiCbk has a replication complex that can be recovered from the cell lysate, and this complex allowed us to identify many of the phage proteins involved in phage genome replication.


Assuntos
Bacteriófagos/isolamento & purificação , Caulobacter crescentus/virologia , Siphoviridae/isolamento & purificação , Proteínas Virais/genética , Bacteriófagos/classificação , Bacteriófagos/genética , Bacteriófagos/crescimento & desenvolvimento , Genoma Viral , Genômica , Siphoviridae/classificação , Siphoviridae/genética , Siphoviridae/crescimento & desenvolvimento
13.
PLoS One ; 15(6): e0233961, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479512

RESUMO

Hundreds of target specific peptides are routinely discovered by peptide display platforms. However, due to the high cost of peptide synthesis only a limited number of peptides are chemically made for further analysis. Here we describe an accurate and cost effective method to bin peptides on-phage based on binding region(s), without any requirement for peptide or protein synthesis. This approach, which integrates phage and yeast display platforms, requires display of target and its alanine variants on yeast. Flow cytometry was used to detect binding of peptides on-phage to the target on yeast. Once hits were identified, they were synthesized to confirm their binding region(s) by HDX (Hydrogen deuterium exchange) and crystallography. Moreover, we have successfully shown that this approach can be implemented as part of a panning process to deplete non-functional peptides. This technique can be applied to any target that can be successfully displayed on yeast; it narrows down the number of peptides requiring synthesis; and its utilization during selection results in enrichment of peptide population against defined binding regions on the target.


Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Biblioteca de Peptídeos , Alanina/genética , Alanina/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Técnicas de Visualização da Superfície Celular/economia , Análise Custo-Benefício , Citometria de Fluxo/economia , Citometria de Fluxo/métodos , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/metabolismo , Mutação , Ligação Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
14.
PLoS One ; 15(6): e0234636, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555720

RESUMO

The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics.


Assuntos
Actinobacteria/virologia , Bacteriófagos/genética , Variação Genética , Genoma Viral , Bacteriófagos/classificação , Bacteriófagos/isolamento & purificação , Composição de Bases , DNA Viral/genética , Genes Virais , Genômica , Filogenia , Proteínas Virais de Fusão/genética
15.
PLoS One ; 15(6): e0234438, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32525945

RESUMO

Shiga toxin-producing Escherichia coli (STECs) contamination of produce, as a result of contact with ruminant fecal material, has been associated with serious foodborne illness. Bacteriophages (phages) that infect STECs have primarily been reported to be of cattle origin. However, they likely exist in other environments or in animals that share habitats with cattle, such as goats. To explore the presence and diversity of phages specific to STEC O157 and the top six non-O157 STECs in goat-associated environments, environmental samples consisting of feces (goat and cattle) and soil samples were collected monthly for six months from an organic produce farm. A variety of phages belonging to the Myoviridae, Siphoviridae, and Podoviridae families were isolated from all goat fecal and half of the soil samples. The most commonly isolated phages belonged to Myoviridae and were lytic against STEC O103. The isolated phages had different host ranges, but collectively, showed lytic activity against O157 and the top six non-O157 STEC strains excluding O121. Two non-O157 STECs (O174: H21 and O-antigen-negative: H18) were isolated from soil and cattle feces, respectively. Although prior studies have reported that goats shed STEC into the environment, the findings of the current study suggest that goat feces may also contain lytic STEC-specific phages. The phages of goat origin have the capacity to infect STECs implicated in causing foodborne outbreaks, making them potential candidates for biocontrol pending additional characterization steps. Further work is needed to determine if the addition of goats to the farm environment could potentially reduce the presence of STECs.


Assuntos
Bacteriófagos/isolamento & purificação , Fezes/virologia , Cabras/microbiologia , Escherichia coli Shiga Toxigênica/virologia , Criação de Animais Domésticos/métodos , Animais , Bacteriófagos/genética , California , Bovinos/microbiologia , DNA Viral/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Fazendas , Alimentos Orgânicos/microbiologia , Doenças Transmitidas por Alimentos/microbiologia , Doenças Transmitidas por Alimentos/prevenção & controle , Microbiologia do Solo
16.
Nat Commun ; 11(1): 3034, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32541663

RESUMO

Alphaproteobacteria, which are the most abundant microorganisms of temperate oceans, produce phage-like particles called gene transfer agents (GTAs) that mediate lateral gene exchange. However, the mechanism by which GTAs deliver DNA into cells is unknown. Here we present the structure of the GTA of Rhodobacter capsulatus (RcGTA) and describe the conformational changes required for its DNA ejection. The structure of RcGTA resembles that of a tailed phage, but it has an oblate head shortened in the direction of the tail axis, which limits its packaging capacity to less than 4,500 base pairs of linear double-stranded DNA. The tail channel of RcGTA contains a trimer of proteins that possess features of both tape measure proteins of long-tailed phages from the family Siphoviridae and tail needle proteins of short-tailed phages from the family Podoviridae. The opening of a constriction within the RcGTA baseplate enables the ejection of DNA into bacterial periplasm.


Assuntos
Bacteriófagos/fisiologia , Técnicas de Transferência de Genes , Rhodobacter capsulatus/genética , Rhodobacter capsulatus/virologia , Siphoviridae/fisiologia , Bacteriófagos/genética , Bacteriófagos/ultraestrutura , Microscopia Crioeletrônica , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Transferência Genética Horizontal , Siphoviridae/genética , Siphoviridae/ultraestrutura
17.
PLoS One ; 15(6): e0234159, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32525961

RESUMO

Bacteriophages (phages) play a key role in shaping microbial communities, including those of the human body. Phages are abundant members of the urogenital tract, most often persisting through the lysogenic life cycle as prophages integrated within the genomes of their bacterial hosts. While numerous studies of the urogenital microbiota have focused on the most abundant bacterial member of this niche-Lactobacillus species-very little is known about Lactobacillus phages. Focusing on Lactobacillus jensenii strains from the urinary tract, we identified numerous prophages related to the previously characterized Lv-1 phage from a vaginal L. jensenii strain. Furthermore, we identified a new L. jensenii phage, Lu-1. Evidence suggests that both phages are abundant within the urogenital tract. CRISPR spacer sequences matching to Lv-1 and Lu-1 prophages were identified. While first detected in urinary isolates, the Lu-1 phage was also discovered in L. jensenii isolates from vaginal and perineal swabs, and both phages were found in metagenomic data sets. The prevalence of these phages in the isolates suggests that both phages are active members of the urogenital microbiota.


Assuntos
Bacteriófagos/isolamento & purificação , Lactobacillus/virologia , Períneo/microbiologia , Vagina/microbiologia , Bacteriófagos/genética , Biologia Computacional , Feminino , Humanos , Microbiota
18.
Arch Virol ; 165(8): 1929-1932, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32514690

RESUMO

Phages, viruses targeting bacteria, have potential therapeutic applications in the control of infections with antibiotic-resistant bacteria. In this study, an Enterobacter cloacae phage, Ec_L1, was isolated from sewage sludge samples collected from a hospital. The genome of phage Ec_L1 consists of 51,894 bp with 48.24% G+C content. Nineteen of the 85 putative proteins encoded by this phage have known functions, and no rRNA or tRNA genes were found. Comparative analysis of genome sequences suggested that phage Ec_L1 should be considered a member of the subfamily Tunavirinae, which includes T1-like phages. According to the International Committee on Taxonomy of Viruses (ICTV), phage Ec_L1 is the type member of the new genus "Eclunavirus", whose name was derived from Ec_L1.


Assuntos
Bacteriófagos/genética , Enterobacter cloacae/virologia , Genoma Viral/genética , Composição de Bases/genética , Fases de Leitura Aberta/genética , Análise de Sequência/métodos
19.
Nat Commun ; 11(1): 2816, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499527

RESUMO

The intense arms race between bacteria and phages has led to the development of diverse antiphage defense systems in bacteria. Unlike well-known restriction-modification and CRISPR-Cas systems, recently discovered systems are poorly characterized. One such system is the Thoeris defense system, which consists of two genes, thsA and thsB. Here, we report structural and functional analyses of ThsA and ThsB. ThsA exhibits robust NAD+ cleavage activity and a two-domain architecture containing sirtuin-like and SLOG-like domains. Mutation analysis suggests that NAD+ cleavage is linked to the antiphage function of Thoeris. ThsB exhibits a structural resemblance to TIR domain proteins such as nucleotide hydrolases and Toll-like receptors, but no enzymatic activity is detected in our in vitro assays. These results further our understanding of the molecular mechanism underlying the Thoeris defense system, highlighting a unique strategy for bacterial antiphage resistance via NAD+ degradation.


Assuntos
Bacteriófagos/genética , Escherichia coli/virologia , NAD/metabolismo , Bacillus cereus/metabolismo , Sistemas CRISPR-Cas , Clonagem Molecular , Cristalografia por Raios X , Análise Mutacional de DNA , Escherichia coli/metabolismo , Hidrolases/metabolismo , Cinética , Mutação , Domínios Proteicos , Estrutura Secundária de Proteína , Receptores Toll-Like/metabolismo
20.
Int J Food Microbiol ; 329: 108686, 2020 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-32516659

RESUMO

Clostridium tyrobutyricum has been identified as a major species associated with the late blowing defect (LBD) of semi-hard and hard cheeses, due to undesirable butyric acid fermentation. To find new strategies to control this spoilage bacterium, we investigated the delivery of a bacteriophage endolysin by a cheese starter culture. The nisin producer Lactococcus lactis subsp. lactis INIA 415 was engineered to produce the CTP1L endolysin, encoded by the virulent bacteriophage ΦCTP1 of C. tyrobutyricum and with a demonstrated lytic activity in vitro, to the cheese matrix. The presence of the nisRK two-component regulatory system in the host strain allowed constitutive expression of the endolysin under the control of the nisA promoter (PnisA), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media. Engineered lysins with a second cell wall binding domain were also tested and shown to have improved lytic activity. Transformation of L. lactis subsp. lactis INIA 415 with endolysin delivery plasmids had a detrimental effect on its ability to produce nisin in milk, but did not affect its acidifying capacity. Transformed L. lactis subsp. lactis INIA 415 were evaluated as starters in cheeses contaminated with spores of C. tyrobutyricum. Evolution of microbiological parameters, pH and dry matter of cheeses were studied, and Clostridium metabolism and LBD in cheeses were monitored by sensory and instrumental analyses during ripening. Cheese made with the parental strain L. lactis subsp. lactis INIA 415 delayed LBD by one month, attributable to the activity of the nisin, but it was not sufficient to arrest the growth of C. tyrobutyricum during ripening completely. The use of the endolysin-producing strains in cheese manufacture as single cultures also delayed the appearance of LBD by one month, attributable to the activity of the endolysin produced in situ during ripening, because nisin activity in these cheeses was very low at day 1 and undetectable from 15 days onwards. Endolysin was more effective than nisin in inhibiting Clostridium growth, since cheeses made with the CTP1L or the chimeric derivative producers only as starters showed lower LBD symptoms, higher lactic acid levels and lower concentrations of propionic and butyric acids (associated with off-flavours) than cheese made with the parental strain. Investigation of different promoters to maximise endolysin production may help to implement CTP1L as a tool to control C. tyrobutyricum by L. lactis cheese starter and reduce LBD even further.


Assuntos
Bacteriófagos , Queijo/microbiologia , Clostridium tyrobutyricum/efeitos dos fármacos , Endopeptidases/genética , Endopeptidases/farmacologia , Microbiologia de Alimentos/métodos , Lactococcus lactis/genética , Bacteriófagos/enzimologia , Bacteriófagos/genética , Lactococcus lactis/enzimologia , Nisina/farmacologia , Organismos Geneticamente Modificados
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