Genetic analysis of the erf region of the bacteriophage P22 chromosome

Derivatives of P22 with deletions of DNA sequences around and including the erf gene were obtained by crossing phages with plasmids containing fragments of the P22 chromosome. In some cases, the parent phages carried a large insertion in sequences not borne by the plasmids. In these cases, deletion of DNA from the phage chromosome to restore terminal repetition (a selectable trait) could be accomplished by recombination between phage and plasmid DNA in chosen sequences flanking the insertion on both sides and borne by the plasmid. In other cases, the parent phages had deletions of a selectable gene, which could be acquired from the plasmid parents only by acquisition of an overlapping deletion. Deletion-bearing P22 strains were tested for growth and homologous genetic recombination in wild-type, recA-, and rec(B or C)- hosts. This analysis indicated the existence of a gene, mapping to the left of erf, that is helpful (but not completely essential) for growth of P22 in a wild-type host. Because P22 lacking this gene grows as well as wild-type P22 on a recBC- host, it has been designated abc (anti-recBC). The abc gene does not appear to be essential for homologous genetic recombination in any host. A plasmid bearing a 1900 base pair fragment of P22 DNA, that expresses erf and abc under the control of the E. coli lac promoter, was constructed. It supports growth and recombination in a recA- host by a phage that lacks all of the genes known to lie between 24 and 9.     

Insertion of host DNA into PVL-encoding phages of the Staphylococcus aureus lineage ST80 by intra-chromosomal recombination

Temperate bacteriophages play a critical role in the pathogenicity of the human pathogen Staphylococcus aureus by mediating positive lysogenic conversion for different virulence factors such as Panton-Valentine leukocidin (PVL) or by interrupting chromosomal virulence genes. PVL-encoding phages are integrated in the S. aureus genome within a conserved ORF which is surrounded by a cluster of tandemly repeated genes. Here we demonstrate that in S. aureus clonal complex ST80 strains PVL-phage induction led to the acquisition of host DNA into the phage genome probably due to a homologous recombination event between direct repeats of the two paralogous genes adjacent to the phage integration site. Phage excision was accompanied by an additional chromosomal deletion in this region. This so far unrecognized mechanism of DNA uptake into the phage genome may play an important role in the co-evolution of phages and bacteria.     

Replication and plasmid-bacteriophage recombination I. Marker rescue analysis

Recombinant DNA plasmids of pBR322 and T7 DNA were constructed and tested for the presence of various T7 genes. A plasmid containing part of T7 gene 5 (DNA polymerase) was used to transform various strains of Escherichia coli, including a dna B mutant. [³H]Thymidine incorporation and a new method of copy number analysis showed that plasmid DNA was not degraded after infection by T7 as is the E. coli host DNA. Marker rescue between recombinant T7 plasmids and mutant infecting bacteriophage was quantitated by determining the percentage of wild-type progeny phage produced after infection. Replication of the plasmid DNA and infecting phage DNA was controlled independently by a dna B mutation in the host and by gene 5 mutations in the phage, respectively. Marker rescue frequencies decreased slightly, if either plasmid or phage replication was blocked. However, marker rescue dropped below detectable levels, if neither the plasmid nor the phage could replicate. These results show clearly that replication plays a role in plasmid-phage recombination, and possible roles for replication in this process are discussed.     

An orthologue of the cor gene is involved in the exclusion of temperate lambdoid phages. Evidence that Cor inactivates FhuA receptor functions

A new set of lambdoid phages (mEp) classified into different immunity groups was previously described. Phages mEp213, mEp237, and mEp410 were unable to grow in mEp167 lysogenic cells, presumably due to an exclusion mechanism expressed constitutively by the mEp167 repressed prophage. In this work, to analyze the exclusion phenomenon, we constructed a genomic library from mEp167 phage in a pPROEX derivative plasmid. A DNA fragment containing an open reading frame for a 77 amino acid polypeptide was selected by its ability to confer resistance to heteroimmune phage infection. This ORF shows high amino acid sequence identity with putative Cor proteins of phages HK022, phi80 and N15. Cells expressing the mEp167 cor gene from a plasmid (Cor(+) phenotype) excluded 13 of 20 phages from different infection immunity groups. This exclusion was observed in both tonB(-) and tonB(+) cells. Lambdoid mEp phages that were excluded in these cells were unable to infect cells defective in the outer membrane FhuA receptor (fhuA(-)). Thus, Cor-mediated exclusion was only observed in fhuA(+) cells. Phage production after DNA transfection or the spontaneous induction of mEp prophage in Cor(+) cells was not blocked. In addition, ferrichrome uptake, which is mediated by FhuA, was inhibited in Cor(+) cells. Our results show that not only phage infection via FhuA but also a FhuA transport activity (ferrichrome uptake) are inhibited by Cor, presumably by inactivation of FhuA.     

Comparative genomics of lactococcal phages: insight from the complete genome sequence of Lactococcus lactis phage BK5-T

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.     

A beta-related corynebacteriophage which lacks a tox allele but can acquire it by recombination with converting phage.

Corynebacteriophage 782, a phage highly related to the beta family of corynebacteriophages but lacking a tox allele, was isolated from a nontoxinogenic clinical isolate of Corynebacterium diphtheriae. Phage 782 exhibits beta immunity but has a wider host range than beta, forming plaques on strains of C. ulcerans and C. pseudotuberculosis as well as on C. diphtheriae. Phage 782 and beta differed in their DNA mass and in their restriction endonuclease digest patterns, but were similar in possessing cos (cohesive) and attP (phage attachment) sites. Moreover, all the BamHI fragments of 782 and beta except one hybridized with a DNA probe of the other. The exception in both cases was the attP-containing fragment, which in beta also carries the tox gene. Recombinants between phage 782 and pi phage, a tox+ beta-related phage, were isolated which contained ca. 70% of phage 782 DNA but carried the attP-tox-bearing fragment of pi and were thus now converting phages. The recombinants had lost the wide-host-range phenotype of 782 and had the narrower host range of pi. The significance of the tox-less, beta-related phages to the natural history of diphtheria is discussed.     

T4 phages against Escherichia coli diarrhea: Potential and problems

A combination of in vitro and in vivo experiments with comparative phage genomics was used for the rational design of a phage cocktail against E. coli diarrhea. Orally applied T4 coliphages representing three different subgroups (T4-, RB49- and JS98-like phages) had no negative impact on the murine gut microbiota. T4 phages were found with high titers in the cecum and colon and lower titers in the small intestine, but were not detected in the blood, liver or spleen. No adverse effects were observed after one-month exposure to phage nor were serum anti-T4 antibodies detected. T4 phages belonging to the same subgroup showed closely related genomes that differed by 12 (phage JS10 vs. JS98 reference) to 17 (phage JSE vs. RB49 reference) insertion/deletions mostly representing single small ORFs. Bioinformatic analysis did not reveal undesired genes in the T4 genomes. Sequence variability was seen over the tail fibre genes, but the variability did not correlate with phage host range. The investigated T4 phages were not only species- but also strain-specific, necessitating the use of phage cocktails consisting of 10 and 16 T4 phage isolates to cover half to two thirds of E. coli strains representing the five main pathotypes isolated from diarrhea patients.     

Detection of phages infecting Bacteroides fragilis HSP40 using a specific DNA probe

Nine bacteriophage isolates of Bacteroides fragilis, obtained from urban sewage and pig faeces samples using four different host strains (HSP40, RYC4023, RYC2056 and RYC3318), were compared on the basis of morphology, host range, DNA restriction patterns, DNA hybridisation and protein composition. All the phages are siphovirus and, as judged from cleavage by restriction endonucleases, their genome is composed of double-stranded DNA of similar size ( approximately 51-kb). Host range analysis differentiated two types of phages: (1) phages that clearly infect B. fragilis strains HSP40 (B40-2, B23-1, B23-2, B23-3 and B23-4, of which B40-8 is the phage type); and (2) the group of bacteriophages that were not infectious for HSP40 (B56-1, B56-2 and B18-1). Similarity in DNA restriction patterns and protein characteristics was found in the HSP40 infectious phages. Anti-B40-8 serum recognised only the proteins of the phages of this type. Although all phages showed similar major protein sizes, minor specific bands were detected. Bacteriophages B56-1, B56-2 and B18-1 showed heterogeneity in their DNA restriction profiles although some degree of DNA-DNA homology between all genomes was observed. Southern blot analysis with phage B40-8 DNA based probes identified a 1.5-kb DNA region homologous for all HSP40 phage isolates, but absent in the genome of the other phage isolates that did not infect this bacterial strain. The homologous region was used as a specific probe to specifically detect B. fragilis HSP40 phages.     

Genotypic variations of Shiga toxin-converting phages from enterohaemorrhagic Escherichia coli O157: H7 isolates

Pulsed-field gel electrophoresis (PFGE) analysis revealed that enterohaemorrhagic Escherichia coli (EHEC) O157:H7 strains had considerable variations in their genomes. This study investigated whether or not the molecular profile of Shiga toxin (Stx) 1- and Stx2-converting phages isolated from EHEC O157:H7 strains, derived from various sources in the USA and Japan, corresponded to the variations of host strains' genotypes as determined by PFGE. A total of 51 Stx-converting phages including 12 Stx1-converting phages and 37 Stx2-converting phages was isolated from seven USA isolates and 20 Japanese isolates. The average Dice coefficient values showed 44% similarity between phage DNAs in Stx2-converting phages digested with SmaI and 55% in Stx1-converting phages digested with HindIII, indicating considerable variation among phage DNA. In particular, restriction fragment length polymorphism (RFLP) patterns of Stx2-converting phage DNA varied according to the PFGE type of their host strain, which suggests that the phage genomes have altered their genotypic characteristics with those of host genomes. However, there are several exceptions: the RFLP patterns of some Stx2-converting phages were quite similar irrespective of the different genotypes of the host strains, indicating that horizontal transfer of Stx2-converting phage may also occur under some circumstances.     

Comparative study of vaginal Lactobacillus phages isolated from women in the United States and Turkey: prevalence, morphology, host range, and DNA homology

Lactobacilli play an important role in maintaining vaginal health. However, during bacterial vaginosis lactobacilli decrease for unknown reasons. Our preliminary study showed that phages could infect vaginal lactobacilli. Therefore, the aim of this study was to analyze the distribution, virulence, and types of vaginal Lactobacillus phages isolated from women of two countries: the United States and Turkey. A total of 209 vaginal lactobacilli were isolated from reproductive-aged women in the United States (n = 107) and Turkey (n = 102). By analysis of 16S rRNA gene sequence and by comparison of protein profiles, most lactobacilli were identified as L. crispatus, L. gasseri, and L. jensenii. After mitomycin C induction, 28% of American lactobacilli and 36% of Turkish lactobacilli released phages. A total of 67 phages were isolated and further characterized by their host range, electron microscopy, and DNA homology. All 67 phages were infective against lactobacilli from both collections. The host ranges of most phages were broad, including multiple Lactobacillus species. Even though the phages were all temperate, they were able to cause lytic infection in various strains. The electron micrographs of these phages showed a hexagon-shaped head and a long tail with or without a contractile tail sheath. Based on their morphology, these phages belonged to Bradley's phage groups A and B, and could be further classified into four morphotypes. All four types were found among American phages, but only three were found among Turkish isolates. DNA hybridization with labeled probes of the four types of phages revealed that additional genetic types existed within each morphotype among these phages. The phage genomic sizes ranged between 34 and 55 kb. Many of the lysogenic Lactobacillus strains released phages spontaneously at a high frequency of 10(-3) to 10(-4) PFU/cell. In conclusion, lysogeny in vaginal lactobacilli is widely spread. Some lysogenic lactobacilli spontaneously release phages with a broad host range, which can be lytic against other vaginal lactobacilli regardless of their geographic origin.     

Characterization of a Shiga Toxin-Encoding Temperate Bacteriophage of Shigella sonnei

A Shiga toxin (Stx)-encoding temperate bacteriophage of Shigella sonnei strain CB7888 was investigated for its morphology, DNA similarity, host range, and lysogenization in Shigella and Escherichia coli strains. Phage 7888 formed plaques on a broad spectrum of Shigella strains belonging to different species and serotypes, including Stx-producing Shigella dysenteriae type 1. With E. coli, only strains with rough lipopolysaccharide were sensitive to this phage. The phage integrated into the genome of nontoxigenic S. sonnei and laboratory E. coli K-12 strains, which became Stx positive upon lysogenization. Moreover, phage 7888 is capable of transducing chromosomal genes in E. coli K-12. The relationships of phage 7888 with the E. coli Stx1-producing phage H-19B and the E. coli Stx2-producing phage 933W were investigated by DNA cross-hybridization of phage genomes and by nucleotide sequencing of an 8,053-bp DNA region of the phage 7888 genome flanking the stx genes. By these methods, a high similarity was found between phages 7888 and 933W. Much less similarity was found between phages H-19B and 7888. As in the other Stx phages, a regulatory region involved in Q-dependent expression is found upstream of stxA and stxB (stx gene) in phage 7888. The morphology of phage 7888 was similar to that of phage 933W, which shows a hexagonal head and a short tail. Our findings demonstrate that stx genes are naturally transferable and are expressed in strains of S. sonnei, which points to the continuous evolution of human-pathogenic Shigella by horizontal gene transfer.     

Analysis of some phenotypic traits of feces-borne temperate lambdoid bacteriophages from different immunity groups: a high incidence of cor+, FhuA-dependent phages

A group of previously isolated heterogeneous mEp lambdoid phages (43) from 19 different immunity groups for phage infection was further characterized to gain insight into some phenotypic traits and to assess their relationship with phage lambda. Interestingly, the FhuA host receptor was required by the majority of mEp phages (37 out of 43; approximately 85%). The cor gene, which has been reported to be involved in FhuA-dependent exclusion of lambdoid phages, was also found in most of the FhuA-dependent phages. Accordingly, no cor amplification by PCR was obtained among the six FhuA-independent mEp lambdoid phages. In contrast, it was found that around 25% of the population (10 out of 43 phages) required the specific and essential lambda N antitermination function, and the lambda site-specific DNA recombination function was observed only in two members (4.6%). Thus, a larger proportion of phages require the FhuA receptor for infection, and this is frequently correlated with the cor gene.     

铜绿假单胞菌噬菌体的分离鉴定及其控制生物被膜的应用研究

目的 分离鉴定铜绿假单胞菌烈性噬菌体,研究噬菌体控制宿主菌形成牛物被膜的效率.方法 以铜绿假单胞菌临床菌株为指示菌,从不同环境样品中分离噬菌体;采用限制性内切酶图谱分析和宿主范围测定方法,对分离的噬菌体进行分类;利用透射电子显微镜对分离噬菌体进行形态学研究;以TJC729为指示菌,开展噬菌体控制牛物被膜形成的应用研究.结果 分别以14株铜绿假单胞菌临床分离菌株为指示菌,共分离得到13株烈性噬菌体,命名为C1～C13.利用限制性内切酶EcoR Ⅰ分析结果表明,13株噬菌体基因组均为双链DNA,并可被分成8组;宿主谱测定结果显示,c1和C13、C6和C7、C9和C11分别具有相同的宿主范围,其余7株噬菌体的宿主范围各不相同.随机挑选噬菌体C1进行形态学研究,发现噬菌体C1头部具有二十面体结构,尾部较长且无收缩性尾鞘,属于长尾噬菌体科.生物被膜控制实验结果显示,混合噬菌体能够较好地抑制TJC729生物被膜的形成.进一步实验结果显示,噬菌体C1、C10和C12分别与牛物被膜混合培养24 h后,牛物被膜的量分别下降到初始量的32.7%、57.6%、32.8%.结论 分离了13株铜绿假单胞菌烈性噬菌体,它们能够显著抑制宿主菌生物被膜的形成,并对生物被膜造成一定程度的破坏,为控制铜绿假单胞菌引起的感染提供了一个新方法.

Abstract：

Objective To isolate and classigy the bacteriophages specific to Pseudomonas aetuginosa and to investigate biofilm control efficaey of the isolated virulent phages.Methods With P. aeruginosa clinical strains as indicators.bacteriophages were isolated by screening difierent environmental samples.Classification of the isolated phages was done with the methods of restriction fragment analysis of phage genome and host range analysis.Transmission electron microscopy(TEM)was used in phage morphology study.In biogilm control tests,TJC729 was used as the jndicator strain to study the biofilm control efficacy of the isolated phages.Results Total 13 lytic phages specific to P.aeruginosa strains were isolated and named as C1-C13.According to the result of restriction fragment analysis.all 13 phages were double-stranded DNA viruses and could be divided into eight groups.Host range experiments were conducted with 5 laboratory strains and 12 clinical strains of P. aeruginosa.The same infection profiles were observed among phage C1 and C13,C6 and C7,and C9 and C11,respectively.While the remaining 7 phages each had different unique infection profile.Phage C1 was selected randomly to study its morphology.The obtained images showed that phage C1 had an icosahedral head with a non-contractile tail,belonging to the Siphoviridae family.Compared with the single phage,phage cocktail had the best effect on biofilm control.Further experiment results showed that phage C1.C10 and C12 can destroy biofilm after treatment of the biofilm for 24 h.The biofilm amounts were deceased to 32.7%,57.6%and 32.8%of the initial values,respectively.Conclusion Thirteen virulent phages specific to P. aeruginosa had been isolated.The phages could significantly inhibit the biofilm formation and had a certain degree of damage on the biofilm.The results suggested an alternative method for the treatments of P.aeruginosa infections.     

P087, a lactococcal phage with a morphogenesis module similar to an Enterococcus faecalis prophage

The virulent lactococcal phage P087 was isolated from a dairy environment in 1978. This phage was then recognized as the reference member for one of the ten phage groups currently known to infect Lactococcus lactis strains. The double-stranded DNA genome of this Siphoviridae phage is composed of 60,074 bp and is circularly permuted. Five tRNA and 88 orfs were found within an uncommon genome architecture. Eleven structural proteins were also identified through SDS-PAGE and LC-MS/MS analyses. Of note, 11 translated orfs from the structural module of phage P087 have identities to gene products found in a prophage located in the genome of Enterococcus faecalis V583. The alignment of both genomic sequences suggests that DNA exchanges could occur between these two phages which are infecting low G+C bacteria found in similar ecological niches.     

Comparative genomics of the late gene cluster from Lactobacillus phages

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.     

Optimisation and standardisation of a method for detecting and enumerating bacteriophages infecting Bacteroides fragilis

A method for the detection and enumeration of bacteriophages infecting Bacteroides fragilis has been standardised. The recommended host strain is RYC2056 (ATCC 700786) because of the relatively high counts (10(4)-10(5) PFU/100ml) that it recovers in sewage from very different geographical areas. The addition of 0.25% bile to the culture and assay media and the manipulation of the host strain under strict anaerobic conditions resulted in a significant increase (more than 100%) in the number of phages detected. No other changes in the media and culture conditions resulted in changes in the phage counts detected. However, these increases do not justify changing the culture conditions and media described, taking into consideration that bile renders the media cloudy making it difficult to follow the host growth and that most laboratories do not have the facilities to work under strict anaerobic conditions. Nalidixic acid (100 microg/ml) and kanamycin (100 microg/ml) in the assay medium significantly reduce the background flora from polluted samples without affecting the phage counts. Freezing cultures just before the end of the log-phage growth at (-70+/-10) degrees C with BSA-sucrose as cryoprotector, storing of 1-2 ml in glass vials at (-70+/-10) degrees C and using them directly to inoculate fresh broth allows the obtention of cultures ready for phage enumeration in about 2.5 h. All these developments have been incorporated into a procedure that makes the method for detecting phages infecting B. fragilis as workable as the standardised methods available for the detection of coliphages.     

Effect of calcium ions on the infection of Bacillus subtilis by bacteriophage SF 6.

Infection of Bacillus subtilis 168Wt by SF 6 resulted in a rapid reduction in the number of phages. This could be counteracted by the addition of calcium, barium or strontium ions. At the optimum concentration of 7.5 x 10(-2) M, the number of p.f.u. remained constant until lysis began. Although cultures of another host. B. subtilis 31 try- his-, at the end of the logarithmic growth phase produced a substance which inactivated free phages, this was not the major cause of the reduction in the numbers of p.f.u. during infection experiments at low Ca2+ concentrations. The diminution of the number of p.f.u. was therefore attributed to the fact that at least one of the steps of the lytic cycle was calcium dependent. Adsorption of SF 6 was equally effective in media containing high or low concentrations of calcium ions. Infection experiments with phages whose DNA had been labelled radioactively revealed that, at high concentrations of calcium ions, the label remained associated with the host cells until lysis commenced. At low concentrations, however, a dissociation between phage DNA and the host was found, although adsorption took place at a normal rate. From these experiments we concluded that a high concentration of calcium ions was required for the penetration of phage DNA. Similar experiments with phages whose protein coat had been labelled showed the same results, indicating that desorption of the inactivated phages occurred. Both electron microscopy and column chromatogarphy with hydroxyapatite showed that a considerable fraction of the inactivated phages had ejected their DNA into the medium. A hypothesis explaining these results is presented.     

A modular view of the bacteriophage genomic space: identification of host and lifestyle marker modules

Bacteriophage genomes can be regarded as an ensemble of modules which are accessible to the whole phage population via recombination. The time spent by prophages in the bacterial host provides them with the opportunity to exchange modules with other prophages or infecting phages. Here we analyze the modular structure of a set of 457 phages and 760 prophages extracted from completely sequenced bacterial genomes using the ACLAME database and its associated tools. We identified 91 modules of proteins with similar phylogenetic profiles. Of these, 25 and 6 are associated with temperate and virulent phages, respectively; 57 are restricted to a host or small group of hosts; and 55 could be annotated with a phage function. We use the transposable phages as a study case and show how the inclusion of prophages allows us to unveil new types of genome organization (i.e. novel module combinations) and obtain insight into the host range for this particular group, highlighting the utility of prophage prediction to better characterize phage diversity.     

Biology of the temperate Streptococcus thermophilus bacteriophage TP-J34 and physical characterization of the phage genome

The temperate Streptococcus thermophilus bacteriophage TP-J34 was identified in the lysogenic host strain J34. The majority of phage particles produced upon induction was defective and noninfectious, consisting of DNA-filled heads lacking tails. A physical map (45.6 kb) was established. Analysis of minor restriction bands of the DNA isolated from phage particles as well as the analysis of the protein pattern indicated that phage TP-J34 is a pac-type phage. This was confirmed by immunoelectron microscopy using antisera raised against virulent cos- and pac-type S. thermophilus phages. The lysogenic host J34 but not its noninducible derivate J34-12 contained phage DNA in the nonintegrated state and exhibited autolysis at elevated temperatures. Prophage-carrying strains grew homogeneously while 16 of 20 prophage-cured derivatives aggregated and sedimented rapidly. When phage TP-J34 was propagated lytically on a prophage-cured host strain, a 2.7-kb site-specific deletion occurred in the phage genome. This deletion was also identified in the prophage DNAs of relysogenized strains.