RESUMEN
Of 62 Streptococcus thermophilus bacteriophages isolated from various ecological settings, half contain a lysin gene interrupted by a group IA2 intron. Phage mRNA splicing was demonstrated. Five phages possess a variant form of the intron resulting from three distinct deletion events located in the intron-harbored open reading frame (orf 253). The predicted orf 253 gene sequence showed a significantly lower GC content than the surrounding intron and lysin gene sequences, and the predicted protein shared a motif with endonucleases found in phages from both gram-positive and gram-negative bacteria. A comparison of the phage lysin genes revealed a clear division between intron-containing and intron-free alleles, leading to the establishment of a 14-bp consensus sequence associated with intron possession. The conserved intron was not found elsewhere in the phage or S. thermophilus bacterial genomes. Folding of the intron RNA revealed secondary structure elements shared with other phage introns: first, a 38-bp insertion between regions P3 and P4 that can be folded into two stem-loop structures (shared with introns from Bacillus phage SPO1 and relatives); second, a conserved P7.2 region (shared with all phage introns); third, the location of the stop codon from orf 253 in the P8 stem (shared with coliphage T4 and Bacillus phage SPO1 introns); fourth, orf 253, which has sequence similarity with the H-N-H motif of putative endonuclease genes found in introns from Lactococcus, Lactobacillus, and Bacillus phages.
Asunto(s)
Empalme Alternativo , Enzimas/genética , Genes Virales , Intrones , Fagos de Streptococcus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , ADN Viral , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta , ARN Viral/química , Eliminación de Secuencia , Homología de Secuencia de Ácido Nucleico , Streptococcus/virologíaRESUMEN
Bacteriophages attacking Streptococcus thermophilus, a lactic acid bacterium used in milk fermentation, are a threat to the dairy industry. These small isometric-headed phages possess double-stranded DNA genomes of 31 to 45 kb. Yoghurt-derived phages exhibit a limited degree of variability, as defined by restriction pattern and host range, while a large diversity of phage types have been isolated from cheese factories. Despite this diversity all S. thermophilus phages, virulent and temperate, belong to a single DNA homology group. Several mechanisms appear to create genetic variability in this phage group. Site-specific deletions, one type possibly mediated by a viral recombinase/integrase, which transformed a temperate into a virulent phage, were observed. Recombination as a result of superinfection of a lysogenic host has been reported. Comparative DNA sequencing identified up to 10% sequence diversity due to point mutations. Genome sequencing of the prototype temperate phage phi Sfi21 revealed many predicted proteins which showed homology with phages from Lactococcus lactis suggesting horizontal gene transfer. Homology with phages from evolutionary unrelated bacteria like E. coli (e.g. lambdoid phage 434 and P1) and Mycobacterium phi L5 was also found. Due to their industrial importance, the existence of large phage collections, and the whole phage genome sequencing projects which are currently underway, the S. thermophilus phages may present an interesting experimental system to study bacteriophage evolution.
Asunto(s)
Bacteriófagos/genética , Bacteriófagos/patogenicidad , Evolución Molecular , Streptococcus/virología , Ecología , Virulencia/fisiologíaRESUMEN
The temperate bacteriophage phiSfi21 integrates its DNA into the chromosome of Streptococcus thermophilus strains via site-specific recombination. Nucleotide sequencing of the attachment sites identified a 40-bp identity region which surprisingly overlaps both the 18-terminal bp of the phage integrase gene and the 11-terminal bp of a host tRNAArg gene. A 2.4-kb phage DNA segment, covering attP, the phage integrase, and a likely immunity gene contained all the genetic information for faithful integration of a nonreplicative plasmid into the attB site. A deletion within the int gene led to the loss of integration proficiency. A number of spontaneous deletions were observed in plasmids containing the 2.4-kb phage DNA segment. The deletion sites were localized to the tRNA side of the identity region and to phage or vector DNA with 3- to 6-bp-long repeats from the border region. A similar type of deletion was previously observed in a spontaneous phage mutant.
Asunto(s)
Bacteriófagos/fisiología , Recombinación Genética , Streptococcus/virología , Integración Viral , Secuencia de Bases , Eliminación de Gen , Vectores Genéticos , Datos de Secuencia Molecular , Plásmidos , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
A highly conserved DNA region extending over 5 kb was observed in Streptococcus thermophilus bacteriophages. Comparative sequencing of one temperate and 26 virulent phages demonstrated in the most extreme case an 18% aa difference for a predicted protein, while the majority of the phages showed fewer, if any aa changes. The relative degree of aa conservation was not homogeneous over the DNA segment investigated. Sequence analysis of the conserved segment revealed genes possibly involved in DNA transactions. Three predicted proteins (orf 233, 443, and 382 gene product (gp)) showed nucleoside triphosphate binding motifs. Orf 443 gp showed in addition a DEAH box motif, characteristically found in a subgroup of helicases, and a variant zinc finger motif known from a phage T7 helicase/primase. Tree analysis classified orf 443 gp as a distant member of the helicase superfamily. Orf 382 gp showed similarity to putative plasmid DNA primases. Downstream of orf 382 a noncoding repeat region was identified that showed similarity to a putative minus origin from a cryptic S. thermophilus plasmid. Four predicted proteins showed not only high degrees of aa identity (34 to 63%) with proteins from Lactococcus lactis phages, but their genes showed a similar topological organization. We interpret this as evidence for a horizontal gene transfer event between phages of the two bacterial genera in the distant past.
Asunto(s)
Bacteriófagos/genética , Replicación del ADN , ADN Viral , Lactococcus lactis/virología , Streptococcus/virología , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia Conservada , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADN , Replicación ViralRESUMEN
A mozzarella cheese factory using an undefined, milk-derived Streptococcus thermophilus starter system was monitored longitudinally for 2 years to determine whether the diversity of the resident bacteriophage population arose from environmental sources or from genetic changes in the resident phage in the factory. The two hypotheses led to different predictions about the genetic diversity of the phages. With respect to host range, 12 distinct phage types were observed. With two exceptions, phages belonging to different lytic groups showed clearly distinct restriction patterns and multiple isolates of phages showing the same host range exhibited identical or highly related restriction patterns. Sequencing studies in a conserved region of the phage genome revealed no point mutations in multiple isolates of the same phage type, while up to 12% nucleotide sequence diversity was observed between the different phage types. This diversity is as large as that between the most different sequences from phages in our collection. These observations make unlikely a model that postulates a single phage invasion event and diversification of the phage during its residence in the factory. In the second stage of our factory study, a defined starter system was introduced that could not propagate the resident factory phage population. Within a week, three new phage types were observed in the factory while the resident phage population was decreased but not eliminated. Raw milk was the most likely source of these new phages, as phages with identical host ranges and restriction patterns were isolated from raw milk delivered to the factory during the intervention trial. Apparently, all of the genetic diversity observed in the S. thermophilus phages isolated during our survey was already created in their natural environment. A better understanding of the raw-milk ecology of S. thermophilus phages is thus essential for successful practical phage control.
Asunto(s)
Queso/microbiología , Fagos de Streptococcus/genética , Streptococcus/virología , Animales , Secuencia de Bases , Ecología , Microbiología Ambiental , Microbiología de Alimentos , Variación Genética , Estudios Longitudinales , Leche/virología , Datos de Secuencia Molecular , Mutagénesis , Hibridación de Ácido Nucleico , Mutación Puntual , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Phage phi Sfi21, the only temperate Streptococcus thermophilus phage from our phage collection, showed extensive DNA homology with virulent phages from lytic group I. Southern blot hybridizations demonstrated that the phi Sfi21-specific DNA was clustered in an approximately 6.6-kb-long region, the putative lysogeny module. Sequence analysis and database research identified an integrase within this module; orf 203 with homology to an anonymous orf 258 from the temperate lactococcal phage BK5-T; orf 127 and orf 122 with weak homology to the N- and C-terminal parts, respectively, of the cl-like repressor from lactococcal phages Tuc2009 and BK5-T; orf 75 with homology to a repressor protein from lambdoid phage 434 and an anti-repressor ant with homology to phage P1. The molecular arrangement of the predicted orfs in phage phi Sfi21 was very similar to that of the lactococcal phage BK5-T. The transition from phi Sfi21-specific DNA into DNA shared with virulent phages was abrupt and flanked at one side by notable DNA repeats. Sequence analysis identified a holin protein to the left of the lysogeny module. A site-specific deletion of 2.4 kb, which reproducibly transformed phi Sfi21 into a lytic phage, was localized in the lysogeny module. It was flanked at both sides by conspicuous DNA repeats. One repeat region reflected the DNA around the attP site, while the other reflected the putative genetic switch region between repressor and anti-repressor genes. S. thermophilus host Sfi1 transformed with a plasmid containing int and orf 203 showed resistance to superinfection by heterologous phages, but not by the homologous phi Sfi21. Part of the int gene could be deleted without loss of this activity, while a deletion in orf 203 resulted in loss of the phage resistance. We speculate on the possibility of a bipartite immunity system for the control of lysogeny in phi Sfi21.
Asunto(s)
ADN Viral , Proteínas de Unión al ADN , Lisogenia , Fagos de Streptococcus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Integrasas/clasificación , Integrasas/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas Represoras/genética , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Streptococcus/virología , Proteínas Virales/genética , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Site-specific spontaneous deletions were observed with high frequency in three regions of the genome of the temperate Streptococcus thermophilus phage phi SFi21. Deletion sizes were 750 bp (type 1), 2.7 kb (type 2), and 1 kb (type 3). Combinations of types 1 and 3 and 2 and 3 were observed. The mutants grew lytically although with reduced burst sizes. Type 2 mutants lost the capacity to lysogenize host cells. Upon serial passage, the deletion mutants overgrew the wild-type phage. No direct or inverted DNA repeats were associated with type 1 or 2 deletion sites. Several independent phage isolates showed deletions at identical nucleotide positions, suggesting a site-specific recombination system. Sequencing of an Xbal restriction fragment covering the type 1 deletion predicted a single long open reading frame (ORF) showing a high degree of amino acid similarity with two proteins from bacteriophage P1 implicated in its immunity control (KiIA, Ant). Type 1 deletion leads to a loss of the conserved C-terminal part of this ORF.
Asunto(s)
Eliminación de Secuencia , Fagos de Streptococcus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Cartilla de ADN , ADN Viral , Genoma Viral , Lisogenia , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Streptococcus/virología , Fagos de Streptococcus/crecimiento & desarrollo , Fagos de Streptococcus/aislamiento & purificaciónRESUMEN
The temperate Streptococcus thermophilus bacteriophage phi SFi21 showed an 38-kb-long double-stranded DNA genome with cohesive ends. A single integration site was used in lysogens established in three different S. thermophilus strains. The attP and attB sites were localized on the restriction map of phage DNA and by hybridization on pulsed field separated bacterial DNA. All laboratory-established lysogens showed in addition to integrated prophage DNA unintegrated monomer phage DNA with unligated cos sites. The genetic relatedness of phi SFi21 DNA with DNA from lytic phages was studied in dot blot and Southern blot hybridization by using individual restriction fragments of phiSFi21 DNA as probes. Lytic group I phages hybridized with fragments of the central and the right part of the phiSFi21 genome but failed to hybridize with a fragment joining both parts. Lytic group II phages showed hybridization with the right half of the phiSFi21 genome. In lytic group IV phages, biologically a heterogeneous group, many different combinations of cross hybridization were detected in accordance with the hypothesis of the modular evolution of phage genomes.
Asunto(s)
ADN Viral/genética , Lisogenia/genética , Fagos de Streptococcus/genética , Sitios de Ligazón Microbiológica/genética , Southern Blotting , Sondas de ADN , Genoma Viral , Mitomicina/farmacología , Hibridación de Ácido Nucleico , Mapeo Restrictivo , Fagos de Streptococcus/crecimiento & desarrollo , Activación Viral/efectos de los fármacosRESUMEN
In the last 30 years, 81 Streptococcus thermophilus bacteriophage isolates were collected from industrial yogurt (n = 40) and cheese (n = 41) fermentation. Forty-six distinct restriction patterns of phage DNA (11 in yogurt and 35 in cheese) were observed. The phages were investigated for host range, serological properties, and DNA homology to study whether these three independent techniques can be used to classify the phages into taxonomic groups. Yogurt factory-derived phages were classified into the same two subgroups by serology, host range analysis, and hybridization with subgroup-specific DNA sequences. Cheese factory-derived phages, however, could not be classified: the 35 cheese phage isolates with distinct restriction patterns showed 34 different host ranges. All but one cheese phage isolate showed serological cross-reactivity with yogurt phages. A phage DNA fragment that hybridized with all phage DNA samples was cloned, establishing the genetic relatedness of all S. thermophilus phages from our collection. With the sequence information from an unusually conserved S. thermophilus phage DNA element (H. Brüssow, A. Probst, M. Frémont, and J. Sidoti, Virology 200:854-857, 1994), a PCR-based phage detection method was developed for cheese whey from a factory that produced mozzarella cheese with complex undefined starter mixes. PCR allowed the detection of phages in cheese whey (detection limit, 10(3) PFU/ml) which could not be detected by dot blot hybridization techniques (detection limit, 10(7) PFU/ml).
Asunto(s)
Queso/virología , Leche/virología , Fagos de Streptococcus/clasificación , Fagos de Streptococcus/aislamiento & purificación , Animales , Secuencia de Bases , Clonación Molecular , Sondas de ADN , ADN Viral/análisis , ADN Viral/genética , Fermentación , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Sensibilidad y Especificidad , Fagos de Streptococcus/ultraestructura , YogurRESUMEN
Microbiological developments during industrial meat fermentations (salami), made with and without commercial starter cultures, were followed at two factories in Germany and Italy. In the German product microbial growth was evident only for the first 48 h, followed by a gradual decline in numbers of most micro-organisms. The pH fell from 5.8 to 4.8 in the 28 d required for production. In Italy a similar situation was seen, except that a second period of bacterial growth began around 15 d, coincident with the appearance of intentional surface mould growth which reversed the pH fall, the final pH being 6.2. The German starter culture was a mixture of Lactobacillus plantarum and Staphylococcus carnosus, whereas in Italy only Staph. carnosus was used. The strain of Lact. plantarum used did not grow in the German product whereas the Staph. carnosus grew well in both products to form a substantial proportion of the final microflora.
Asunto(s)
Fermentación , Microbiología de Alimentos , Lactobacillus/crecimiento & desarrollo , Productos de la Carne/microbiología , Staphylococcus/crecimiento & desarrollo , Bacteriófagos/aislamiento & purificación , Alemania , Concentración de Iones de Hidrógeno , ItaliaRESUMEN
Bovine rotavirus (BRV) V1005 is serologically distinct from rotavirus serotypes 1, 2, 3, 4, 5, 6, 8 and 9. BRV V1005 showed cross-reactions with BRV B223, the American prototype of serotype 10 rotavirus, and with BRV E4049, a British serotype 10 isolate. BRV V1005 was, however, not neutralized by four monoclonal antibodies directed against VP7 of BRV B223. Two-way cross-reactions were observed between BRV V1005 and a reassortant rotavirus containing the VP4 from BRV UK. In addition the major tryptic cleavage product of VP4, VP5*, from BRV V1005 is indistinguishable by peptide mapping and its isoelectric point from the homologous protein of BRV UK, but is clearly different from VP5* of BRV NCDV. The peptide map of VP7 from BRV V1005 differed from that obtained for VP7 of BRV UK.
Asunto(s)
Bovinos/microbiología , Rotavirus/inmunología , Proteínas de la Matriz Viral/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Células Cultivadas , Reacciones Cruzadas , Epítopos , Cobayas , Focalización Isoeléctrica , Pruebas de Neutralización , Mapeo Peptídico , Conejos , Rotavirus/clasificaciónRESUMEN
Reassortants between serotype 3 SA11 and serotype 6 NCDV rotaviruses were used to determine the relative amounts of serum-neutralizing antibody to VP4 and VP7 of serotype 3 SA11 rotavirus in children after natural rotavirus exposure. Sera from Ecuadorian children of a population-based study and sera from children of a hospital-based study in Germany (excluding diarrhea patients) demonstrated high titers of VP7-specific but only low titers of VP4-specific antibodies. In contrast, paired sera from German children hospitalized with a symptomatic primary rotavirus gastroenteritis demonstrated a titer increase to VP4 more frequently than to VP7 protein by neutralization test and immunoblotting. For these rotavirus patients, we provided, previously, direct evidence for the development of cross-neutralizing antibodies.
Asunto(s)
Anticuerpos Antivirales/inmunología , Antígenos Virales , Proteínas de la Cápside , Infecciones por Rotavirus/inmunología , Rotavirus/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Cápside/inmunología , Preescolar , Ecuador , Alemania Occidental , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina A/aislamiento & purificación , Inmunoglobulina G/inmunología , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina M/inmunología , Inmunoglobulina M/aislamiento & purificación , Lactante , Pruebas de Neutralización , Rotavirus/clasificación , SerotipificaciónRESUMEN
Two types of empty capsid particles that differed with respect to the presence of the two outer shell proteins were isolated from MA-104 cells infected with bovine rotavirus V1005. Three previously uncharacterized polypeptides, I, II, and III, migrating between VP2 and VP6, were detected in empty capsids but not in single- and double-shelled rotavirus particles. Peptide mapping revealed that all three proteins were related to VP2. Polypeptides I, II, and III could be generated by in vitro trypsin digestion of empty capsids not exposed to trypsin in the infection medium. Labeled polypeptides appeared in empty capsids before they were detected in intracellular single- or double-shelled rotavirus particles. Empty capsids were also observed in MA-104 cells infected with bovine rotaviruses UK and NCDV, simian rotavirus SA11, and human rotavirus KU. VP7-containing empty capsid is the minimal subunit vaccine for cows; we failed to induce a substantial neutralizing antibody increase with VP7 purified under denaturating or nondenaturating conditions or with synthetic peptides corresponding to two regions of VP7.
Asunto(s)
Antígenos Virales/aislamiento & purificación , Cápside/aislamiento & purificación , Rotavirus/análisis , Vacunas Virales , Animales , Cápside/biosíntesis , Cápside/inmunología , Bovinos , Línea Celular , Centrifugación Zonal , Electroforesis en Gel de Poliacrilamida , Humanos , Macaca mulatta , Sustancias Macromoleculares , Metionina/metabolismo , Rotavirus/inmunología , Serina Endopeptidasas , Tripsina , Uridina/metabolismoRESUMEN
Single serotype vaccination of mature cows with nine different strains of bovine, simian and human rotaviruses induced heterotypic milk and serum neutralizing antibodies against two bovine and four human rotavirus serotypes. Immunization with single-shelled simian rotavirus SA11 increased milk and serum neutralization titres fivefold over those of control cows, without inducing antibodies to outer shell polypeptides of rotavirus. Vaccination with double-shelled SA11 virions also elicited cross-reacting antibodies to the outer shell proteins VP3 and/or VP7 which neutralized rotavirus seven times more efficiently than antisera to single-shelled SA11 virus. A related rotavirus similar to simian rotavirus SA11, but from a different host, might thus be an attractive vaccine for immunization of pregnant cows to confer passive immunity to calves.