RESUMEN

The genome of the highly pathogenic M1 serotype Streptococcus pyogenes isolate SF370 contains eight prophage elements. Only prophage SF370.1 could be induced by mitomycin C treatment. Prophage SF370.3 showed a 33.5-kb-long genome that closely resembled the genome organization of the cos-site temperate Siphovirus r1t infecting the dairy bacterium Lactococcus lactis. The two-phage genomes shared between 60 and 70% nucleotide sequence identity over the DNA packaging, head and tail genes. Analysis of the SF370.3 genome revealed mutations in the replisome organizer gene that may prevent the induction of the prophage. The mutated phage replication gene was closely related to a virulence marker identified in recently emerged M3 serotype S. pyogenes strains in Japan. This observation suggests that prophage genes confer selective advantage to the lysogenic host. SF370.3 encodes a hyaluronidase and a DNase that may facilitate the spreading of S. pyogenes through tissue planes of its human host. Prophage SF370.2 showed a 43-kb-long genome that closely resembled the genome organization of pac-site temperate Siphoviridae infecting the dairy bacteria S. thermophilus and L. lactis. Over part of the structural genes, the similarity between SF370.2 and S. thermophilus phage O1205 extended to the nucleotide sequence level. SF370.2 showed two probable inactivating mutations: one in the replisome organizer gene and another in the gene encoding the portal protein. Prophage SF370.2 also encodes a hyaluronidase and in addition two very likely virulence factors: prophage-encoded toxins acting as superantigens that may contribute to the immune deregulation observed during invasive streptococcal infections. The superantigens are encoded between the phage lysin and the right attachment site of the prophage genome. The genes were nearly sequence identical with a DNA segment in S. equi, suggesting horizontal gene transfer. The trend for prophage genome inactivation was even more evident for the remaining five prophage sequences that showed massive losses of prophage DNA. In these prophage remnants only 13-0.3 kb of putative prophage DNA was detected. We discuss the genomics data from S. pyogenes strain SF370 within the framework of Darwinian coevolution of prophages and lysogenic bacteria and suggest elements of genetic cooperation and elements of an arms race in this host-parasite relationship.

Asunto(s)

Bacteriófagos/genética , Evolución Molecular , Genoma Viral , Lactococcus lactis/virología , Fagos de Streptococcus/genética , Streptococcus pyogenes/virología , Lactococcus lactis/genética , Streptococcus pyogenes/genética

RESUMEN

Lactococcus lactis phage BK5-T and Streptococcus thermophilus phage Sfi21, two cos-site temperate Siphoviridae with 40-kb genomes, share an identical genome organization, sequence similarity at the amino acid level over about half of their genomes, and nucleotide sequence identity of 60% over the DNA packaging and head morphogenesis modules. Siphoviridae with similarly organized genomes and substantial protein sequence similarity were identified in several genera of low-GC-content Gram-positive bacteria. These phages demonstrated a gradient of relatedness ranging from nucleotide sequence similarity to protein sequence similarity to gene map similarity over the DNA packaging and head morphogenesis modules. Interestingly, the degree of relatedness was correlated with the evolutionary distance separating their bacterial hosts. These observations suggest elements of vertical evolution in phages. The structural genes from BK5-T shared no sequence relationships with corresponding genes/proteins from lactococcal phages belonging to distinct lactococcal phage species, including phage sk1 (phage species 936) that showed a closely related gene map. Despite a clearly distinct genome organization, lactococcal phages sk1 and c2 showed nine sequence-related proteins. Over the early gene cluster phage BK5-T shared nine regions of high nucleotide sequence similarity, covering at most two adjacent genes, with lactococcal phage r1t (phage species P335). Over the structural genes, the closest relatives of phage r1t were not lactococcal phages belonging to other phage species, but Siphoviridae from Mycobacteria (high-GC-content Gram-positive bacteria). Evidence for recent horizontal gene transfer between distinct phage species was obtained for dairy phages, but these transfers were limited to phages infecting the same bacterial host species.

Asunto(s)

Genoma Viral , Lactococcus lactis/virología , Siphoviridae/genética , Biología Computacional/métodos , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Evolución Molecular , Transferencia de Gen Horizontal , Genómica , Datos de Secuencia Molecular , Siphoviridae/clasificación , Fagos de Streptococcus/clasificación , Fagos de Streptococcus/genética

RESUMEN

Comparative phage genomics can retrace part of the evolutionary history of phage modules encoding phage-specific functions such as capsid building or establishment of the lysogenic state. The diagnosis of relatedness is not based exclusively on sequence similarity, but includes topological considerations of genome organization. The gene maps from the lambda-, psiM2-, L5-, Sfi21-, Sfi11-, phiC31-, sk1- and TM4-like phages showed a remarkable synteny of their structural genes defining a lambda supergroup within Siphoviridae (Caudovirales with long non-contractile tails). A hierarchy of relatedness within the lambda supergroup suggested elements of vertical evolution in the capsid module of Siphoviridae. Links to P22-like Podoviridae and P2-like Myoviridae were also detected. Numerous cases of horizontal gene transfer were observed, but recent transfers were limited to interbreeding phage populations. We suggest that tailed phages are the result of both vertical and horizontal evolution and are thus a good model system for web-like phylogenies.

Asunto(s)

Evolución Molecular , Genoma Viral , Siphoviridae/genética , Industria Lechera , Streptococcus/virología , Fagos de Streptococcus/genética

RESUMEN

Three prophage sequences were identified in the Lactobacillus johnsoni strain NCC533. Prophage Lj965 predicted a gene map very similar to those of pac-site Streptococcus thermophilus phages over its DNA packaging and head and tail morphogenesis modules. Sequence similarity linked the putative DNA packaging and head morphogenesis genes at the protein level. Prophage Lj965/S. thermophilus phage Sfi11/Lactococcus lactis phage TP901-1 on one hand and Lactobacillus delbrueckii phage LL-H/Lactobacillus plantarum phage phig1e/Listeria monocytogenes phage A118 on the other hand defined two sublines of structural gene clusters in pac-site Siphoviridae from low-GC Gram-positive bacteria. Bacillus subtilis phage SPP1 linked both sublines. The putative major head and tail proteins from Lj965 shared weak sequence similarity with phages from Gram-negative bacteria. A clearly independent line of structural genes in Siphoviridae from low-GC Gram-positive bacteria is defined by temperate cos-site phages including Lactobacillus gasseri phage adh, which also shared sequence similarity with phage D3 infecting a Gram-negative bacterium. A phylogenetic tree analysis demonstrated that the ClpP-like protein identified in four cos-site Siphoviridae from Lactobacillus, Lactococcus, Streptococcus, and Pseudomonas showed graded sequence relationships. The tree suggested that the ClpP-like proteins from the phages were not acquired by horizontal gene transfer from their corresponding bacterial hosts.

Asunto(s)

Genoma Viral , Lactobacillus/virología , Filogenia , Siphoviridae/genética , Secuencia de Aminoácidos , Evolución Molecular , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADN

RESUMEN

Temperate Siphoviridae from an evolutionarily related branch of low GC content gram-positive bacteria share a common genetic organization of lysogeny-related genes and the predicted proteins are linked by many sequence similarities. Their compact lysogeny modules [integrase/1-2 orfs (phage exclusion? and metalloproteinase motif proteins)/cI-like repressor/cro-like repressor/antirepressor (optional)] differ clearly from that of lambda-like and L5-like viruses, the two currently established genera of temperate Siphoviridae, while they resemble those of the P2-like genus of Myoviridae. In all known temperate Siphoviridae from low GC content gram-positive bacteria the lysogeny module is flanked by the lysis module and the DNA replication module. This modular organization is again distinct from that of the known genera of temperate Siphoviridae. On the basis of comparative sequence analysis we propose a new genus of Siphoviridae: "Sfi21-like" phages. With a larger database of phage sequences it might be possible to establish a genomics-based phage taxonomy and to retrace the evolutionary history of selected phage modules or individual phage genes. The antirepressor of Sfi21-like phages has an unusual widespread distribution since proteins with high aa similarity (40%) were found not only in phages from gram-negative bacteria, but also in insect viruses.

Asunto(s)

Bacterias Grampositivas/genética , Bacterias Grampositivas/virología , Lisogenia/genética , Siphoviridae/genética , Siphoviridae/fisiología , Secuencia de Aminoácidos , Animales , Composición de Base/genética , Secuencia de Bases , Biología Computacional , Replicación del ADN/genética , Evolución Molecular , Genoma Viral , Lactococcus/genética , Lactococcus/virología , Datos de Secuencia Molecular , Myoviridae/clasificación , Myoviridae/genética , Myoviridae/fisiología , Filogenia , Proteínas Represoras/química , Proteínas Represoras/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Siphoviridae/clasificación , Proteínas Virales/química , Proteínas Virales/genética , Activación Viral/genética

RESUMEN

The comparative analysis of five completely sequenced Streptococcus thermophilus bacteriophage genomes demonstrated that their diversification was achieved by a combination of DNA recombination events and an accumulation of point mutations. The five phages included lytic and temperate phages, both pac site and cos site, from three distinct geographical areas. The units of genetic exchange were either large, comprising the entire morphogenesis gene cluster, excluding the putative tail fiber genes, or small, consisting of one or maximally two genes or even segments of a gene. Many indels were flanked by DNA repeats. Differences in a single putative tail fiber gene correlated with the host ranges of the phages. The predicted tail fiber protein consisted of highly conserved domains containing conspicuous glycine repeats interspersed with highly variable domains. As in the T-even coliphage adhesins, the glycine-containing domains were recombinational hot spots. Downstream of a highly conserved DNA replication region, all lytic phages showed a short duplication; in three isolates the origin of replication was repeated. The lytic phages could conceivably be derived from the temperate phages by deletion and multiple rearrangement events in the lysogeny module, giving rise to occasional selfish phages that defy the superinfection control systems of the corresponding temperate phages.

Asunto(s)

Bacteriófagos/genética , Evolución Molecular , Genoma Viral , Recombinación Genética , Streptococcus/virología , Secuencia de Aminoácidos , ADN Viral/genética , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos , Alineación de Secuencia

RESUMEN

The virulent cos-site Streptococcus thermophilus bacteriophage Sfi19 has a 37,392-bp-long genome consisting of 44 open reading frames all encoded on the same DNA strand. The genome of the temperate cos-site S. thermophilus phage Sfi21 is 3.3 kb longer (40,740 bp, 53 orfs). Both genomes are very similarly organized and differed mainly by gene deletion and DNA rearrangement events in the lysogeny module; gene replacement, duplication, and deletion events in the DNA replication module, and numerous point mutations. The level of point mutations varied from <1% (lysis and DNA replication modules) to >15% (DNA packaging and head morphogenesis modules). A dotplot analysis showed nearly a straight line over the left 25 kb of their genomes. Over the right genome half, a more variable dotplot pattern was observed. The entire lysogeny module from Sfi21 comprising 12 genes was replaced by 7 orfs in Sfi19, six showed similarity with genes from temperate pac-site S. thermophilus phages. None of the genes implicated in the establishment of the lysogenic state (integrase, superinfection immunity, repressor) or remnants of it were conserved in Sfi19, while a Cro-like repressor was detected. Downstream of the highly conserved DNA replication module 11 and 13 orfs were found in Sfi19 and phiSfi21, respectively: Two orfs from Sfi21 were replaced by a different gene and a duplication of the phage origin of replication in Sfi19; a further orf was only found in Sfi21. All other orfs from this region, which included a second putative phage repressor, were closely related between both phages. Two noncoding regions of Sfi19 showed sequence similarity to pST1, a small cryptic plasmid of S. thermophilus.

Asunto(s)

Genoma Viral , Fagos de Streptococcus/genética , Streptococcus/virología , Secuencia de Aminoácidos , Sitios de Ligazón Microbiológica/genética , Secuencia de Bases , Genes Virales/genética , Variación Genética/genética , Lisogenia/genética , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta/genética , Origen de Réplica/genética , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Fagos de Streptococcus/patogenicidad , Fagos de Streptococcus/fisiología , Virulencia/genética , Ensamble de Virus/genética

RESUMEN

The temperate Streptococcus thermophilus bacteriophage Sfi21 possesses 15-nucleotide-long cohesive ends with a 3' overhang that reconstitutes a cos-site with twofold hyphenated rotational symmetry. Over the DNA packaging, head and tail morphogenesis modules, the Sfi21 sequence predicts a gene map that is strikingly similar to that of lambdoid coliphages in the absence of any sequence similarity. A nearly one to one gene correlation was found with the phage lambda genes Nu1 to H, except for gene B-to-E complex, where the Sfi21 map resembled that of coliphage HK97. The similarity between Sfi21 and HK97 was striking: both major head proteins showed an N-terminal coiled-coil structure, the mature major head proteins started at amino acid positions 105 and 104, respectively, and both major head genes were preceded by genes encoding a possible protease and portal protein. The purported Sfi21 protease is the first viral member of the ClpP protease family. The prediction of Sfi21 gene functions by reference to the gene map of intensively investigated coliphages was experimentally confirmed for the major head and tail gene. Phage Sfi21 shows nucleotide sequence similarity with Lactococcus phage BK5-T and a lactococcal prophage and amino acid sequence similarity with the Lactobacillus phage A2 and the Staphylococcus phage PVL. PVL is a missing link that connects the portal proteins from Sfi21 and HK97 with respect to sequence similarity. These observations and database searches, which demonstrate sequence similarity between proteins of phage from gram-positive bacteria, proteobacteria, and Archaea, constrain models of phage evolution.

Asunto(s)

Sitios de Ligazón Microbiológica , Fagos de Streptococcus/genética , Streptococcus/virología , Ensamble de Virus/genética , Secuencia de Aminoácidos , Archaea/virología , Sitios de Ligazón Microbiológica/genética , Secuencia de Bases , Biología Computacional , Escherichia coli/virología , Evolución Molecular , Genes Virales/genética , Genoma Viral , Datos de Secuencia Molecular , Morfogénesis , Sistemas de Lectura Abierta/genética , Filogenia , Rhodobacter/virología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Fagos de Streptococcus/fisiología , Proteínas Virales/química , Proteínas Virales/genética

RESUMEN

The structural gene cluster and the lysis module from lytic group II Streptococcus thermophilus bacteriophage phi Sfi11 was compared to the corresponding region from other Siphoviridae. The analysis revealed a hierarchy of relatedness. phi Sfi11 differed from the temperate S. thermophilus bacteriophage phi O1205 by about 10% at the nucleotide level. The majority of the changes were point mutations, mainly at the third base position. Only a single gene (orf 695) differed substantially between the two phages. Over the putative minor tail and lysis genes, phi Sfi11 and the lytic group 1 S. thermophilus phi Sfi19 shared regions with variable degrees of similarity. Orf 1291 from phi Sfi19 was replaced by four genes in phi Sfi11, two of which (orf 1000 and orf 695) showed a complicated pattern of similarity and nonsimilarity compared with phi Sfi19. The predicted orf 695 gp resembles the receptor-recognizing protein of T-even coliphages in its organization, but not its sequence. No sequence similarity was detected between phi Sfi11 and phi Sfi19 in the region covering the major head and tail genes. Comparison of the structural gene map of phi Sfi11 with that of Siphoviridae from gram-positive and -negative bacterial hosts revealed a common genomic organization. Sequence similarity was only found between phi Sfi11 and Siphoviridae from gram-positive hosts and correlated with the evolutionary distance between the bacterial hosts. Our data are compatible with the hypothesis that the structural gene operon from Siphoviridae of the low G + C group of gram-positive bacteria is derived from a common ancestor.

Asunto(s)

Genes Virales/genética , Siphoviridae/genética , Fagos de Streptococcus/genética , Evolución Biológica , Mapeo Cromosómico , Datos de Secuencia Molecular , Peso Molecular , Familia de Multigenes/genética , Sistemas de Lectura Abierta/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Streptococcus/virología , Fagos de Streptococcus/clasificación , Fagos de Streptococcus/ultraestructura , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/genética

RESUMEN

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/fisiología

RESUMEN

Comparative sequence analysis of 40% of the genomes from two prototype Streptococcus thermophilus bacteriophages (lytic group I phage phi Sfi19 and the cos site containing temperate phage phi Sfi21) suggested two processes in the evolution of their genomes. In a first evolutionarily distant phase the basic genome structure was apparently constituted by modular exchanges. Over the 17-kb-long DNA segment analyzed in the present report, we observed clusters of genes with similarity to genes from Leuconostoc oenos phage L10, Lactococcus lactis phage BK5-T, and Streptococcus pneumoniae phage Dp-1. A chimeric protein was predicted for orf 1291 which showed similarity to both phage BK5-T and phage Dp-1 proteins. The very large orf 1626 gene product showed similarity to two adjacent genes from the Lactobacillus delbrueckii phage LL-H and further phage proteins (Lactococcus lactis, Bacillus subtills). The similarities were localized to distinct parts of this apparently multifunctional protein. The putative phi Sfi19 lysin showed similarity to both lysins of phages and cellular enzymes. In a second, evolutionarily more recent, phase the S, thermophilus phage genomes apparently diversified by point mutations and small deletions/insertions. Over the investigated 17-kb DNA region phi Sfi19 differed from phi Sfi21 by 10% base pair changes, the majority of which were point mutations (mainly at the third codon position), while a third of the base pair differences were contributed by small deletions/insertions. The base pair changes were unevenly distributed. Over the Leuconostoc phage-related DNA the change rate was high, while over the Lactococcus and S. pneumoniae phage-related DNA the change rate was low. We speculate that the degree of base pair change could provide relative time scales for the modular exchange reactions observed in S. thermophilus phages.

Asunto(s)

Evolución Molecular , Genoma Viral , Mutagénesis , Fagos de Streptococcus/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Viral , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación Puntual , Análisis de Secuencia de ADN , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas Estructurales Virales/genética

RESUMEN

Mycobacterium sp. strain HL 4-NT-1, isolated from a mixed soil sample from the Stuttgart area, utilized 4-nitrotoluene as the sole source of nitrogen, carbon, and energy. Under aerobic conditions, resting cells of the Mycobacterium strain metabolized 4-nitrotoluene with concomitant release of small amounts of ammonia; under anaerobic conditions, 4-nitrotoluene was completely converted to 6-amino-m-cresol. 4-Hydroxylaminotoluene was converted to 6-amino-m-cresol by cell extracts and thus could be confirmed as the initial metabolite in the degradative pathway. This enzymatic equivalent to the acid-catalyzed Bamberger rearrangement requires neither cofactors nor oxygen. In the same crucial enzymatic step, the homologous substrate hydroxylaminobenzene was rearranged to 2-aminophenol. Abiotic oxidative dimerization of 6-amino-m-cresol, observed during growth of the Mycobacterium strain, yielded a yellow dihydrophenoxazinone. Another yellow metabolite (lambda max, 385 nm) was tentatively identified as 2-amino-5-methylmuconic semialdehyde, formed from 6-amino-m-cresol by meta ring cleavage.

Asunto(s)

Mycobacterium/metabolismo , Tolueno/análogos & derivados , Anaerobiosis , Biodegradación Ambiental , Mycobacterium/crecimiento & desarrollo , Tolueno/metabolismo

RESUMEN

A 7.6-kb DNA segment covering the putative lysogeny module of the pac-site-containing temperate Streptococcus thermophilus bacteriophage TP-J34 was sequenced. Sequence alignment with the lysogeny module from the cos-site-containing S. thermophilus bacteriophage phiSfi21 revealed areas of high sequence conservation (e.g., over the int gene), interspersed with regions of low or no sequence similarity (e.g., over the cro gene). Four of the six sharp transition zones from high to low sequence conservation were found within open reading frames coding for the CI repressor, the Anti-repressor, the Immunity protein, and a protein of unknown function. The transition points in the cI and ant genes appear to separate gene segments coding for distinct functional domains of these proteins. In addition, these two transition points were located at or near the deletion sites observed in spontaneous phage phiSfi21 deletion mutants, thus suggesting these transition points as recombinational hotspots. Furthermore, the sequence at the transition point in the cI gene resembles the attachment site of the phage, suggesting the involvement of the phage integrase in at least some of the exchange reactions. Contrary to the initial formulation of the modular theory of phage evolution the unit of the evolutionary exchange in streptococcal phages is not a group of functional genes, but can be as small as a single gene. Exchange reactions can also occur within genes, possibly between gene segments encoding distinct protein domains.

Asunto(s)

Evolución Molecular , Lisogenia/genética , Fagos de Streptococcus/genética , Streptococcus/virología , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , ADN Viral , Bases de Datos Factuales , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Proteínas Virales

RESUMEN

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 Viral

RESUMEN

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 ADN

RESUMEN

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 Virales