RESUMO
Bacteriophage replication requires specific host-recognition. Some siphophages harbour a large complex, the baseplate, at the tip of their non-contractile tail. This baseplate holds receptor binding proteins (RBPs) that can recognize the host cell-wall polysaccharide (CWPS) and specifically attach the phage to its host. While most phages possess a dedicated RBP, the phage J-1 that infects Lactobacillus casei seemed to lack one. It has been shown that the phage J-1 distal tail protein (Dit) plays a role in host recognition and that its sequence comprises two inserted modules compared with 'classical' Dits. The first insertion is similar to carbohydrate-binding modules (CBMs), whereas the second insertion remains undocumented. Here, we determined the structure of the second insertion and found it also similar to several CBMs. Expressed insertion CBM2, but not CBM1, binds to L. casei cells and neutralize phage attachment to the bacterial cell wall and the isolated and purified CWPS of L. casei BL23 prevents CBM2 attachment to the host. Electron microscopy single particle reconstruction of the J-1 virion baseplate revealed that CBM2 is projected at the periphery of Dit to optimally bind the CWPS receptor. Taken together, these results identify J-1 evolved Dit as the phage RBP.
Assuntos
Proteínas da Cauda Viral/metabolismo , Proteínas da Cauda Viral/ultraestrutura , Bacteriófagos/metabolismo , Carboidratos , Especificidade de Hospedeiro , Ácido Láctico , Lactobacillus , Lacticaseibacillus casei/metabolismo , Lactococcus lactis/metabolismo , Microscopia Eletrônica , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Proteínas da Cauda Viral/genética , VírionRESUMO
Lactic acid bacteria (LAB) have many applications in food and industrial fermentations. Prophage induction and generation of new virulent phages is a risk for the dairy industry. We identified three complete prophages (PLE1, PLE2, and PLE3) in the genome of the well-studied probiotic strain Lactobacillus casei BL23. All of them have mosaic architectures with homologous sequences to Streptococcus, Lactococcus, Lactobacillus, and Listeria phages or strains. Using a combination of quantitative real-time PCR, genomics, and proteomics, we showed that PLE2 and PLE3 can be induced-but with different kinetics-in the presence of mitomycin C, although PLE1 remains as a prophage. A structural analysis of the distal tail (Dit) and tail associated lysin (Tal) baseplate proteins of these prophages and other L. casei/paracasei phages and prophages provides evidence that carbohydrate-binding modules (CBM) located within these "evolved" proteins may replace receptor binding proteins (RBPs) present in other well-studied LAB phages. The detailed study of prophage induction in this prototype strain in combination with characterization of the proteins involved in host recognition will facilitate the design of new strategies for avoiding phage propagation in the dairy industry.
Assuntos
Lacticaseibacillus casei/genética , Lacticaseibacillus casei/virologia , Prófagos/genética , Prófagos/fisiologia , Ativação Viral , Microbiologia de Alimentos , Mitomicina/metabolismo , Inibidores da Síntese de Ácido Nucleico/metabolismo , Proteínas da Cauda Viral/genéticaRESUMO
To understand the interaction between lipopolysaccharide (LPS) and proteins in molecular detail, a molecular genetic approach has been employed, using phage as a model system. The phage epsilon(34) is a Salmonella phage whose tailspike protein (TSP) uses the host LPS as its initial host cell receptor. Previous studies indicated that there was a similarity between the well-studied tail protein of Salmonella phage P22 and the epsilon(34). This study reports the identification of the gene for the epsilon(34) TSP as well as its initial characterization. In addition, some aspects of the structure of the epsilon(34) TSP have been deduced.
Assuntos
Fagos de Salmonella/genética , Proteínas da Cauda Viral/genética , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , Salmonella enterica/genética , Salmonella enterica/virologiaRESUMO
To study the interaction between lipopolysaccharide and protein, a comparative approach was employed using seven Salmonella enterica serovar Typhimurium typing phages as the protein model systems. This interaction has been studied in detail in the Salmonella enterica serovar Typhimurium phage P22 system and involves only the viral tailspike protein. Similarity between these phages and phage P22 was monitored in this Report by assaying restriction endonuclease digestions, capsid size, reactivity to the P22 tailspike protein monoclonal antibody, mAb92, which reacts with the N-terminus of the P22 tail protein and the ability to produce a PCR fragment using primers made to the ends of the P22 tailspike gene. The data indicate that tailspike similarity exists between most of these phages and a scheme reclassifying them is presented and that the N-terminus of the P22 tailspike protein may be a motif for many phage systems and may serve as a aid in the taxonomy of phages. The data suggest a classification scheme in which the N-terminus of some tailspike proteins (head-binding region in some tail proteins) may play a critical element role in the classification of Salmonella viruses.
Assuntos
Sequência Conservada , Fagos de Salmonella/genética , Proteínas da Cauda Viral/genética , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Bacteriófago P22/genética , Bacteriófago P22/imunologia , Western Blotting , Impressões Digitais de DNA , DNA Viral/análise , Glicosídeo Hidrolases , Reação em Cadeia da Polimerase , Fagos de Salmonella/classificação , Fagos de Salmonella/imunologia , Salmonella typhi/virologia , Proteínas da Cauda Viral/imunologiaRESUMO
This brief report describes the isolation and initial characterization of revertants to the most severe temperature sensitive folding mutant known. The revertants or suppressors may describe amino acid interactions that occur during the folding of the P22 tailspike polypeptide chain. Results indicate that several different types of suppressors may have been obtained.
Assuntos
/genética , Genes Supressores/genética , Glicosídeo Hidrolases/genética , Mutação , Dobramento de Proteína , Proteínas da Cauda Viral/genéticaRESUMO
This review describes the use of a simple genetic system that has provided important insight into the process of folding and, of its flipside, that of protein aggregation. These studies make use of the tail protein of the bacterial virus P22 which infects Salmonella typhimurium. This folding system serves as a model for a number protein structural elements and may also provide important insights into disease-related protein folding defects at a time when an increasing number of diseases are being shown to be due to protein folding alterations.
Assuntos
Bacteriófago P22/genética , Dobramento de Proteína , Proteínas da Cauda Viral/genética , Aminoácidos/genética , Aminoácidos/metabolismo , Bacteriófago P22/fisiologia , DNA Viral/genética , Humanos , Hidrólise , Técnicas In Vitro , Mutação , Conformação Proteica , Salmonella typhimurium/virologiaRESUMO
This review describes the use of a simple genetic system that has provided important insight into the process of folding and, of its flipside, that of protein aggregation. These studies make use of the tail protein of the bacterial virus P22 which infects Salmonella typhimurium. This folding system serves as a model for a number protein structural elements and may also provide important insights into disease-related protein folding defects at a time when an increasing number of diseases are being shown to be due to protein folding alterations
Assuntos
Humanos , /genética , Técnicas In Vitro , Dobramento de Proteína , Proteínas da Cauda Viral/genética , Aminoácidos/genética , Aminoácidos/metabolismo , /fisiologia , DNA Viral/genética , Hidrólise , Mutação , Conformação Proteica , Salmonella typhimurium/virologiaRESUMO
This brief report describes the isolation and initial characterization of revertants to the most severe temperature sensitive folding mutant known. The revertants or suppressors may describe amino acid interactions that occur during the folding of the P22 tailspike polypeptide chain. Results indicate that several different types of suppressors may have been obtained.