The role of the HCR system in the repair of lethal lesions ofBacillus subtilis phages and their transfecting DNA damaged by radiation and alkylating agents

The role of the HCR system in the repair of prelethal lesions induced by UV-light, γ-rays and alkylating agents was studied in theBacillus subtilis SPP1 phage, its thermosensitive mutants (N3, N73 endts ₁) and corresponding infectious DNA. The survival of phages and their transfecting DNA after treatment with UV light is substantially higher inhcr ⁺ cells than inhcr cells, the differences being more striking in intact phages than in their transfecting DNA’s. Repair inhibitors reduce the survival inhcr ⁺ cells: caffeine lowers the survival of UV-irradiated phage SPP1 in exponentially growinghcr ⁺ cells but has no effect on its survival in competenthcr ⁺ cells; acriflavin and ethidium bromide decrease the survival of UV-irradiated SPP1 phage in both exponentially growing and competenthcr ⁺ cells to the level of survival observed inhcr cells; moreover, ethidium bromide lowers the number of infective centres inhcr ⁺ cells of UV-irradiated DNA of the SPP1 phage. Repair inhibitors do not lower the survival of UV-irradiated phages or their DNA inhcr cells. The repair mechanism under study repairs effectively also lesions induced by polyfunctional alkylating agents in transfecting DNA’s ofB. subtilis phages but is not functional with lesions induced by these agents in free phages and lesions caused in phages and their DNA by ethyl methanesulphonate or γ-rays.     

The occurrence and taxonomic value of PBS X-like defective phages in the genus Bacillus

72 strains of 24 Bacillus species were induced with mitomycin C. The lysates were examined for the presence of defective phages resembling PBS X in morphology. All strains tested of B. amyloliquefaciens. B, licheniformis, B. pumilus and B. subtilis contained such phages. Five morphological types of defective, PBS X-like phage could be distinguished, differing in their tail lengths and in the number of cross-striations on the tail. The quaternary structure of the tail, the molecular weight of the main tail protein and the antigenic properties of the phages were identical. The killing ranges of the defective phages have been determined and their possible use in taxonomy discussed.     

Molecular characterization of new group A streptococcal bacteriophages containing the gene for streptococcal erythrogenic toxin A (speA)

Summary Bacteriophage T12 is the prototype phage carrying the streptococcal erythrogenic toxin A (speA) gene. To examine more closely the phages involved in lysogenic conversion, we examined 300 group A streptococcal strains, and identified and isolated two new phages that carry the speA gene. The molecular sizes of these phage genomes were between 32 and 40 kb, similar to that of phage T12 (35 kb). However, as ascertained by restriction analysis, the physical maps of the new phage genomes were different from phage T12 and from each other. Hybridization analysis also showed that all of these phages were only partially related to one another and the speA gene was always located close to the phage attachment site. Additionally, colony hybridization showed that whereas phage T12 or one of its close relatives is the most common phage associated with the group A streptococci, phage 49 has a much stronger association with the speA gene. A defective phage was also found following pulsed field gel electrophoresis of total phage DNA. This phage appears to be a resident of strain T25₃c and is found only following induction of a T25₃c lysogen. Restriction enzyme analysis of the isolated defective phage DNA suggests that it is the source of the submolar amounts of DNA previously found in association with phage T12 digestion patterns. Additionally, the defective phage may serve as the site of integration of the speA gene-carrying phages described above.     

Classification of virulent bacteriophages of Streptococcus salivarius subsp. thermophilus isolated from yoghurt and Swiss-type cheese

Summary Twelve isometric-headed bacteriophages virulent against Streptococcus salivarius subsp. thermophilus were differentiated into three subgroups by analysis of the phage genomes and the structural proteins. Subgroup I is composed of two phages (P6 and P8) with a genome size of 41.2 and 44.2 kb pairs, respectively, complete DNA homology, and identical protein composition (main proteins of sizes 39.8, 24.0, 14.8 kilodaltons in sodium dodecyl sulphate-polyacrylamide gel electrophoresis). One phage (a10/J9) with low DNA homology to the other phages was classified into subgroup II. Subgroup III consists of nine phages with a genome size of 33.8 to 36.7 kb pairs and two major structural proteins (30.9 and 24.0 kilodaltons, or 30.9 and 26.3 kilodaltons). In general, phages with different host spectra revealed different restriction enzyme patterns, and DNA homologies of various degrees were detected among all phages tested.     

Thirteen Virulent and Temperate Bacteriophages of Lactobacillus bulgaricus and Lactobacillus lactis Belong to a Single DNA Homology Group

Thirteen virulent phages and two temperate phages of two closely related bacterial species (Lactobacillus lactis and L. bulgaricus) were compared for their protein composition, their antigenic properties, their restriction endonuclease patterns, and their DNA homology. The immunoblotting studies and the DNA-DNA hybridizations showed that the phages could be differentiated into two groups. One group contained 2 temperate phages of L. bulgaricus and 11 virulent phages of L. lactis. Inside each group, at least two common proteins of identical sizes could be detected for each phage. These proteins were able to cross-react in immunoblotting experiments with an antiserum raised against one phage of the same group. Temperate phage DNAs showed partial homology with DNAs from some virulent phages. These homologies seem to be located on the region coding for the structural proteins since recombinant plasmids coding for one of the major phage proteins of one phage were able to hybridize with the DNAs from phages of the same group. These results suggest that temperate and virulent phages may be related to one another.     

Molecular Phylogeny of φ29-Like Phages and Their Evolutionary Relatedness to Other Protein-Primed Replicating Phages and Other Phages Hosted by Gram-Positive Bacteria

The φ29-like phage genus of Podoviridae family contains phages B103, BS32, GA-1, M2, Nf, φ15, φ29, and PZA that all infect Bacillus subtilis. They have very similar morphology and their genomes consist of linear double-stranded DNA of approximately 20 kb. The nucleotide sequences of individual genomes or their parts determined thus far show that these phages evolved from a common ancestor. A terminal protein (TP) that is covalently bound to the DNA 5′-end primes DNA replication of these phages. The same mechanism of DNA replication is used by the Cp-1 related phages (also members of the Podoviridae family) and by the phage PRD1 (member of the Tectoviridae family). Based on the complete or partial genomic sequence data of these phages it was possible to analyze the evolutionary relationship within the φ29-like phage genus as well as to other protein-primed replicating phages. Noncoding regions containing origins of replication were used in the analysis, as well as amino acid sequences of DNA polymerases, and with the φ29-like phages also amino acid sequences of the terminal proteins and of the gene 17 protein product, an accessory component of bacteriophage DNA replicating machinery. Included in the analysis are also results of a comparison of these phage DNAs with the prophages present in the Bacillus subtilis genome. Based on this complex analysis we define and describe in more detail the evolutionary branches of φ29-like phages, one branch consisting of phages BS32, φ15, φ29, and PZA, the second branch composed of phages B103, M2, and Nf, and the third branch having phage GA-1 as its sole member. In addition, amino acid sequences of holins, proteins involved in phage lysis were used to extend the evolutionary study to other phages infecting Gram-positive bacteria. The analysis based on the amino acid sequences of holins showed several weak points in present bacteriophage classification. Received: 14 April 1998 / Accepted: 31 July 1998     

A mutant bacteriophage evolved to infect resistant bacteria gained a broader host range

Bacteriophages (phages) are the most abundant entities in nature, yet little is known about their capacity to acquire new hosts and invade new niches. By exploiting the Gram‐positive soil bacterium Bacillus subtilis (B. subtilis) and its lytic phage SPO1 as a model, we followed the coevolution of bacteria and phages. After infection, phage‐resistant bacteria were readily isolated. These bacteria were defective in production of glycosylated wall teichoic acid (WTA) polymers that served as SPO1 receptor. Subsequently, a SPO1 mutant phage that could infect the resistant bacteria evolved. The emerging phage contained mutations in two genes, encoding the baseplate and fibers required for host attachment. Remarkably, the mutant phage gained the capacity to infect non‐host Bacillus species that are not infected by the wild‐type phage. We provide evidence that the evolved phage lost its dependency on the species‐specific glycosylation pattern of WTA polymers. Instead, the mutant phage gained the capacity to directly adhere to the WTA backbone, conserved among different species, thereby crossing the species barrier.     

BREX is a novel phage resistance system widespread in microbial genomes

The perpetual arms race between bacteria and phage has resulted in the evolution of efficient resistance systems that protect bacteria from phage infection. Such systems, which include the CRISPR‐Cas and restriction‐modification systems, have proven to be invaluable in the biotechnology and dairy industries. Here, we report on a six‐gene cassette in Bacillus cereus which, when integrated into the Bacillus subtilis genome, confers resistance to a broad range of phages, including both virulent and temperate ones. This cassette includes a putative Lon‐like protease, an alkaline phosphatase domain protein, a putative RNA‐binding protein, a DNA methylase, an ATPase‐domain protein, and a protein of unknown function. We denote this novel defense system BREX (Bacteriophage Exclusion) and show that it allows phage adsorption but blocks phage DNA replication. Furthermore, our results suggest that methylation on non‐palindromic TAGGAG motifs in the bacterial genome guides self/non‐self discrimination and is essential for the defensive function of the BREX system. However, unlike restriction‐modification systems, phage DNA does not appear to be cleaved or degraded by BREX, suggesting a novel mechanism of defense. Pan genomic analysis revealed that BREX and BREX‐like systems, including the distantly related Pgl system described in Streptomyces coelicolor, are widely distributed in ~10% of all sequenced microbial genomes and can be divided into six coherent subtypes in which the gene composition and order is conserved. Finally, we detected a phage family that evades the BREX defense, implying that anti‐BREX mechanisms may have evolved in some phages as part of their arms race with bacteria.     

Structural Similarity and Genetic Homology between Lactobacillus casei Bacteriophages Isolated in Japan and in Finland

Three Lactobacillus casei bacteriophages, LC-Nu, PL-1, and ?FSW, were compared. Phage LC-Nu, which has not been previously characterized, originated from a local cheese plant in Finland. Phages PL-1 and ?FSW (isolated in Japan) represent the most thoroughly studied L.casei phages so far. All three phages had similar morphotypes, but still had different patterns of structural proteins, as analyzed by SDS-PAGE. The phages differed also in types of genome organization: LC-Nu and PL-1 had cohesive ends in their DNAs, and the DNA of ?FSW was circularly permuted. The initiation site and orientation of packaging of ?FSW DNA were identified. The homologies between the phage genomes were analyzed by Southern hybridization. About one-third of each phage gem me was highly homologous with other phages (homology over 85%), and two-thirds were slightly homologous (homology between 65% and 76%). DNAs from five industrial L. casei strains were also tested for homology with phage LC-Nu DNA. Phage LC-Nu related sequences were present in all the L. casei strains tested.     

Lysogeny in Lactobacillus delbrueckii strains and characterization of two new temperate prolate-headed bacteriophages

Aims: Frequency of lysogeny in Lactobacillus delbrueckii strains (from commercial and natural starters) and preliminary characterization of temperate bacteriophages isolated from them. Methods and Results: Induction of strains (a total of 16) was made using mitomycin C (MC) (0·5 μg ml⁻¹). For 37% of the MC-treated supernatants, it was possible to detect phage particles or presence of killing activity, but only two active bacteriophages were isolated. The two temperate phages isolated were prolate-headed phages which belonged to group c of Lact. delbrueckii bacteriophages classification. Different DNA restriction patterns were obtained for each phage, while the structural protein profiles and packaging sites were identical. Distinctive one-step growth curves were exhibited by each phage. An influence of calcium ions was observed for their lysis in broth but not on the adsorption levels. Conclusions: Our study showed that lysogeny is also present in Lact. delbrueckii strains, including commercial strains. Significance and Impact of the Study: Commercial strains could be lysogenic and this fact has a great practical importance since they could contribute to the dissemination of active-phage particles in industrial environments.     

Inversion and deletion mutants in Bacillus subtilis bacteriophage SPP1 as a consequence of cloning

Summary Properties of an inversion and a deletion mutant of B. subtilis phage SPP1 which arose during cloning are described. The results are related to the biology of this bacteriophage.In preceding communications from our laboratories (Heilmann and Reeve 1982, Behrens et al. 1983) we reported the properties of genetically engineered SPP1 bacteriophages, which could be used as cloning vehicles in B. subtilis. These phages contain a unique restriction site within a dispensable region of their genomes. In the course of cloning experiments using these phage vectors, we have occasionally observed the appearance of not only the original vector and desired hybrid phages, but also of SPP1 phages which had undergone extensive genomic rearrangements. Properties of two such phages, SPP1 inv1, which was found to contain a large inversion and of SPP1 delV, a deletion mutant, which defines an additional dispensable region of the SPP1 genome, are described in this communication.     

Lysogenization by bacteriophage lambda: II. - Identification of genes involved in the multiplicity dependent processes

We previously showed that, under conditions of rapid exponential growth, lysogenization of E. coli cells by phage λ requires that the cell is infected by at least 2 phages able to replicate their DNA, or 3 or 4 phages unable to replicate their DNA [ref. 4]. Since genes dealing with prophage integration appear not to be involved in these multiplicity dependent processes, a determination was made as to whether more than one copy of the genes involved in repressor synthesis or its activation are needed for lysogenization. The complementation patterns which we obtained indicate multiplicity effects involving gene cII (and, perhaps, cIII) in lysogenization by both phage able or unable to replicate. In the former case, we propose that cII protein (and, perhaps, cIII) both induces repressor synthesis and inhibits phage DNA replication. In lysogenization by phage unable to replicate, the data suggest that the expression of early phage genes and repressor synthesis in the course of lysogenization are mutually exclusive processes which do not take place on the same phage chromosome.     

一株枯草芽孢杆菌原噬菌体Bsu-yong1的基因组分析

【目的】枯草芽孢杆菌（Bacillus subtilis）是在自然界中广泛存在的革兰氏阳性菌，其抗逆性极强，能抑制大多数有害菌的繁殖，是常用的产酶菌，用其生产的蛋白酶、淀粉酶占全球工业酶产量的50%。原噬菌体（prophage）整合在宿主基因组中，可为宿主提供基因和生物学功能，非常具有研究价值。以往，有关B. subtilis原噬菌体的报道主要集中于缺陷型原噬菌体（defective prophage），本研究对一株非缺陷型活性原噬菌体（active prophage）的基因组进行解析，以扩充对非缺陷型原噬菌体的认知。【方法】使用丝裂霉素C从枯草芽孢杆菌中诱导一株噬菌体，命名为Bacillus phage Bsu-yong1（简称Bsu-yong1）。对Bsu-yong1进行负染、透射电镜（transmission electron microscopy，TEM）观察，用Illumina MiSeq测定其基因组序列、综合运用生物信息学工具对其进行基因功能注释和系统进化分析。【结果】Bsu-yong1与PBSX类缺陷型原噬菌体在形态上相似，但Bsu-yong1具有完整的噬菌体基因组，这与缺陷型原噬菌体不同，后者在包装过程中不能正确包裹自身的基因组，而是随机包裹一段宿主染色体。Bsu-yong1基因组全长为43 590 bp，G+C含量为41%，含有62个开放阅读框（open reading frame，ORF），呈模块化分布。Bsu-yong1拥有基因编码T7SS效应器LXG多态性毒素（T7SS effector LXG polymorphic toxin）、ImmA/IrrE蛋白和SMI1/KNR4蛋白。前二者为细菌毒素（toxin），后者为抗毒素（antitoxin），toxin-antitoxin是细菌免疫系统重要成员，参与菌间竞争与环境适应。此前，尚未有编码LXG polymorphic toxin的基因在噬菌体中被发现和报道。在基于全基因组比对构建的蛋白谱进化树（proteomic tree）中，Bsu-yong1与噬菌体sv105、rho14、vB_BteM-A9Y聚集形成一个独立的进化支（clade），基因组比对显示它们基因组的复制与调控模块具有高度保守性，它们共享29个核心基因（core gene），均具有PBSX样形态特征。Bsu-yong1与其他噬菌体的进化距离较远。将Bsu-yong1与所有噬菌体进行比对，得到的成对序列比较（pairwise sequence comparison，PASC）最大值为46.72%，小于属边界值（70%）。【结论】vB_Bsu-yong1在有尾纲中代表一个新的未知的属；建议构建一个新的科（family），该科由Bsu-yong1与噬菌体sv105、rho14、vB_BteM-A9Y组成。vB_Bsu-yong携带免疫相关基因，它可能有利于宿主在菌间竞争中获胜和适应环境。本研究丰富了噬菌体基因数据库，拓展了对芽孢杆菌活性原噬菌体的认知。     

Characterization of phage isolates from a phage-carrying culture of Leuconostoc oenos 58N

Summary During large-scale cultivation of Leuconostoc oenos strain 58N, growth inhibition was detected and attributed to the presence of the virulent phage P581. To determine if this phage originated from a temperate phage, L. oenos 58N was exposed to mitomycin C, and this treatment led indeed to release of phages (P58II). Further examination of the lytic potential of phages P581 and P58II revealed that these two phages were able to lyse the same strains of L. oenos with the exception of the original host strain, which was only sensitive to P581. Results of DNA/DNA hybridization experiments failed to show homology between the DNA of phage P58II and the chromosomal DNA of L. oenos 58N. A phage-free culture of L. oenos 58N could be obtained after repeated subculture. These results indicate that the original L. oenos 58N was in a special type of phage-carrier state. Phages P58I and P58II were compared on the basis of morphology, lytic spectra, restriction enzyme analysis, DNA homology, genome size and protein structure and proved to be identical. It is assumed that P58I arose from the phage-carrier culture of L. oenos 58N and became virulent by some mutational event.Offprint requests to: E. K. Arendt     

Novel organization of the site-specific integration and excision recombination functions of the Staphylococcus aureus serotype F virulence-converting phages φ13 and φ42

Functions required for site-specific integration and excision of the Staphylococcus aureus serotype F virulence-converting phages φ13 and φ42 were localized and characterized. Like other temperate phages, integration of φ13 and φ42 sequences was found to require the product of an int gene located close to the phage attP site. Both int genes are almost identical, express proteins possessing characteristic features of the Int (integrase) family of recombinases, but share very little homology with previously described int genes, including those of the serotype B S. aureus phages L54a and φ11. Nevertheless, all four S. aureus phages share an almost identical short sequence located immediately 5′ to these distinct int genes, suggesting a common mechanism of int gene regulation. Upstream from these common sequences, the sequences of φ13 and φ42 are quite distinct from each other, and from the corresponding regions of φ11 and L54a which encode the Xis proteins that are required with Int to mediate site-specific excision of the latter phages. Surprisingly, φ13 and φ42 sequences encompassing the attP sites and int genes, but lacking either an adjacent or more distant phage excision protein gene, were sufficient to mediate site-specific excision of integrated phage DNA sequences.     

Pervasive prophage recombination occurs during evolution of spore-forming Bacilli

Phages are the main source of within-species bacterial diversity and drivers of horizontal gene transfer, but we know little about the mechanisms that drive genetic diversity of these mobile genetic elements (MGEs). Recently, we showed that a sporulation selection regime promotes evolutionary changes within SPβ prophage of Bacillus subtilis, leading to direct antagonistic interactions within the population. Herein, we reveal that under a sporulation selection regime, SPβ recombines with low copy number phi3Ts phage DNA present within the B. subtilis population. Recombination results in a new prophage occupying a different integration site, as well as the spontaneous release of virulent phage hybrids. Analysis of Bacillus sp. strains suggests that SPβ and phi3T belong to a distinct cluster of unusually large phages inserted into sporulation-related genes that are equipped with a spore-related genetic arsenal. Comparison of Bacillus sp. genomes indicates that similar diversification of SPβ-like phages takes place in nature. Our work is a stepping stone toward empirical studies on phage evolution, and understanding the eco-evolutionary relationships between bacteria and their phages. By capturing the first steps of new phage evolution, we reveal striking relationship between survival strategy of bacteria and evolution of their phages.Subject terms: Bacterial genetics, Evolution     

DNA relatedness among bacteriophages of the morphological group C3

Six bacteriophages with an elongated head and a short, noncontractile tail were compared by DNA-DNA hybridization, seroneutralization kinetics, mol% G+C and molecular weight of DNA, and host range. Three phage species could be identified. Phage species 1 containedEnterobacter sakazakii phage C2,Erwinia herbicola phages E3 and E16P, andSalmonella newport phage 7–11. These phages had a rather wide host range (4 to 13 bacterial species). DNA relatedness among species 1 phages was above 75% relative binding ratio (S1 nuclease method, 60°C) when labeled DNA from phage C2 was used, and above 41% when labeled DNA from phage E3 was used. Molecular weight of DNA was about 58×10⁶ (C2) to 67 ×10⁶ (E3). The mol% G+C of DNA was 43–45. Anti-C2 serum that neutralizes all phages of species 1 does not neutralize phages of the other two species. Species 2 contains only coliphage Esc-7-11, whose host range was only oneEscherichia coli strain out of 188 strains of Enterobacteriaceae studied; it was unrelated to the other two species by seroneutralization and DNA hybridization. DNA from phage Esc-7-11 had a base composition of 43 mol% G+C and a molecular weight of about 45×10⁶. Species 3 contains onlyProteus mirabilis phage 13/3a. Its host range was limited to swarmingProteus species. Species 3 was unrelated to the other two species by seroneutralization and DNA hybridization. DNA from phage 13/3a had a base composition of 35 mol% G+C and molecular weight of about 53×10⁶. It is proposed that phage species be defined as phage nucleic acid hybridization groups.     

An evaluation of the rate constants for the adsorption of the defective phage PBS Z to Bacillus subtilis

The adsorption of the defective phage PBS Z of Bacillus subtilis has previously been assumed to proceed in two steps, a reversible adsorption of extended phages followed by contraction of the adsorbed particles (Steensma, 1981a). This model, also used for other phages, explained the biphasic character of the adsorption curve, but a discrepancy was found between the calculated and observed concentrations of adsorbed, extended phages. Computer simulations indicated that this might be caused by inhomogeneity of the phage preparations with respect to their adsorption properties and that in that case other models would also fit the experimental data. Discrimination between the models was not possible on the basis of the available information on PBS Z and it was therefore concluded that the values reported previously for the rate constants (Steensma, 1981a) should be used with caution.     

Genomic analysis of Bacillus subtilis lytic bacteriophage ϕNIT1 capable of obstructing natto fermentation carrying genes for the capsule-lytic soluble enzymes poly-γ-glutamate hydrolase and levanase

Bacillus subtilis strains including the fermented soybean (natto) starter produce capsular polymers consisting of poly-γ-glutamate and levan. Capsular polymers may protect the cells from phage infection. However, bacteriophage ?NIT1 carries a γ-PGA hydrolase gene (pghP) that help it to counteract the host cell’s protection strategy. ?NIT had a linear double stranded DNA genome of 155,631-bp with a terminal redundancy of 5,103-bp, containing a gene encoding an active levan hydrolase. These capsule-lytic enzyme genes were located in the possible foreign gene cluster regions between central core and terminal redundant regions, and were expressed at the late phase of the phage lytic cycle. All tested natto origin Spounavirinae phages carried both genes for capsule degrading enzymes similar to ?NIT1. A comparative genomic analysis revealed the diversity among ?NIT1 and Bacillus phages carrying pghP-like and levan-hydrolase genes, and provides novel understanding on the acquisition mechanism of these enzymatic genes.     

Bacteriophage of Haemophilus influenzae III. Morphology, DNA Homology, and Immunity Properties of HP1c1, S2, and the Defective Bacteriophage from Strain Rd

The phages HP1c1 and S2 and a defective phage of Haemophilus influenzae have been compared. The morphology of the phages and the mol wt of their DNAs are similar, although the defective phage appears to have a different tail plate region. Electron microscope observation indicates that the defective phage does not attach to the cell surface, and its DNA appears to lack cohesive ends. The homology of the DNAs of the phages has been measured by hydridization. DNA from the defective phage shows little or no homology with the other phage DNAs. HP1c1 and S2 DNAs show a high level of homology. Each of these phages can form plaques on lawns of the lysogen of the other phage but at reduced plating efficiencies, suggesting that the two phages have related but not identical immunity systems.