Properties of the streptomycete temperate bacteriophage FP43.

FP43 is a temperate bacteriophage for Streptomyces griseofuscus that forms plaques on many Streptomyces species. FP43 virions contain 56 kb of double-strand DNA that is circularly permuted and terminally redundant, and contains 65% G + C. A physical map of the FP43 genome was constructed, and the origin for headful packaging (pac) was localized to an 8.8-kb region of the genome (hft) that mediates high-frequency transduction by FP43 of plasmid pRHB101. The phage attachment site (attP), a replication origin (rep), a region that inhibits plaque formation (pin), and a 3-kb deletion (rpt) that caused a 100-fold reduction in plasmid transduction were mapped.     

Cloning a replication function from the streptomycete bacteriophage FP43.

Streptomyces griseofuscus cells carrying a 4.4-kb SphI DNA fragment from bacteriophage FP43 inhibited plaque formation (Pin) by FP43, and the Pin function was localized to a 0.96-kb SacII fragment. The same 4.4-kb SphI fragment was able to replicate freely in several streptomycetes, including S. griseofuscus, and the replication (Rep) function was localized to a 1.2-kb SphI-FspI fragment. Plasmids with FP43 Rep function are unstable and are present at about 20-50 copies per chromosome. Plasmids with FP43 Rep function are compatible with SCP2* plasmids.     

Isolation and characteristics of the bacteriophage from the vaccine strain Tohama phase I

Some properties of bacteriophage phi T isolated from the vaccine strain Bordetella pertussis Tohama phase I and propagated in Bordetella parapertussis 504 cells are presented. Phage phi T belongs to the IV group in accordance with Tikhonenko classification. The diameter of head and length of noncontractile tail sheath are 49.5 +/- 0.5 and 145 +/- 7 nm, respectively. Diameter of the tail sheath is 3.2 +/- 0.6 nm. Molecular mass of the phage DNA is 37 +/- 3 kb. Population of phi T phage is polymorphous and consists of particles the genomes or which vary from each other by the "insert" located 6.8 +/- 0.6 kb from the end of molecule. The blot hybridization has demonstrated that the bacteriophage genome is not inserted into the chromosome of the lysogenic strain. Autonomous location of the phage genome in the host cell is suggested. The temperature and hydrogen ions concentration effects on bacteriophage phi T stability were studied. The conditions for phage suspension storage are described.     

Isolation and characterization of the Streptomyces cattleya temperate phage TG1

A temperate actinophage, TG1, was isolated from soil by growth on Streptomyces cattleya and has been shown to be potentially useful for the cloning of DNA in this organism and other streptomycetes. It forms stable lysogens by integration at a unique site on the chromosome. The phage genome consists of 41 kb of double-stranded DNA with cohesive ends. It has unique sites for ClaI, NdeI, PstI, SmaI, and XbaI. The PstI site has been shown to be in a dispensable region of the phage genome. Deletions (2 kb in length) were obtained which retain this site and should be useful for the cloning of DNA.     

Isolation and characterization of bacteriophage BCJA1, a novel temperate bacteriophage active against the alkaliphilic bacterium, Bacillus clarkii

The isolation and characterization of a novel bacteriophage active against the obligately alkaliphilic bacterium Bacillus clarkii is described. The bacteriophage, designated BCJA1, is a member of the Siphoviridae family with a B1 morphology. It possesses an isometric head, which measures 65 nm between opposite apices, and a noncontractile tail of 195 nm length. It had a buoyant density of 1.518 g/ml and an estimated particle mass of 37 × 10⁷ daltons. BCJA1 was stable over the pH range of 6–11. A one-step growth experiment conducted at pH 10 demonstrated a latent period of about 40 min and a burst size of approximately 40. The purified bacteriophage appeared to consist of 10 proteins with the major head and tail proteins likely to be of molecular weight 36 500 and 28 000, respectively. The genome size was estimated to be between 32.1 and 34.8 kb. The percent G + C content of purified bacteriophage DNA was 45.6. The wildtype bacteriophage is temperate but a clear plaque mutant was isolated. Received: May 25, 1997 / Accepted: August 5, 1997     

Repair of double-strand breaks by incorporation of a molecule of homologous DNA

An in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to monitor repair of double-strand breaks in the T7 genome. The efficiency of double-strand break repair was markedly increased by DNA molecules ('donor' DNA) consisting of a 2.1 kb DNA fragment, generated by PCR, that had ends extending approximately 1 kb on either side of the break site. Repair proceeded with greater than 10% efficiency even when T7 DNA replication was inhibited. When the donor DNA molecules were labelled with 32P, repaired genomes incorporated label only near the site of the double-strand break. When repair was carried out with unlabelled donor DNA and [32P]-dCTP provided as precursor for DNA synthesis the small amount of incorporated label was distributed randomly throughout the entire T7 genome. Repair was performed using donor DNA that had adjacent BamHI and PstI sites. When the BamHI site was methylated and the PstI site was left unmethylated, the repaired genomes were sensitive to PstI but not to BamHI endonuclease, showing that the methyl groups at the BamHI recognition site had not been replaced by new DNA synthesis during repair of the double-strand break. These observations are most consistent with a model for double-strand break repair in which the break is widened to a small gap, which is subsequently repaired by physical incorporation of a patch of donor DNA into the gap.     

Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization

Recombinant lambda bacteriophage clone H3 containing a human DNA segment of 14.9 kb present in one or two copies per haploid genome was isolated. In situ hybridization to human metaphase chromosomes of the ³H-labeled cloned DNA resulted in highly significant labeling (53% of cells) of band p36 of chromosome 1, such that 22% of all chromosomal grains were located on this region. Hybridization was dependent upon the presence of dextran sulfate in the hybridization mixture and was not affected by repetitive DNA competitor. These results demonstrate localization of a single copy sequence on human metaphase chromosomes.     

Isolation and characterization of a new ruminal bacteriophage lytic toStreptococcus bovis

A new bacteriophage, designated F4, was isolated from the ruminal fluid of a calf. The host range of F4 phage was limited to five strains ofStreptococcus bovis out of ten tested on which clear plaques 0.6–1.2 mm in diameter were found. Bacteriophage F4 had an elongated head 75 nm long and 33 nm wide with a noncontractile flexible tail 100 nm in length on average. This phage is defective in the generation of plaques at low multiplicities of infection. Its genome consists of double-stranded linear DNA of 60.38 kb lacking cohesive ends. The F4 DNA was analyzed with 13 restriction enzymes. The restriction enzymes that did not cleave it wereBamHI,EcoRI,PvuI, andSmaI. The circular restriction map was constructed with four restriction endonucleases (XbaI,EcoI,SalI, andBglI).     

Structure of the FBJ murine osteosarcoma virus genome: molecular cloning of its associated helper virus and the cellular homolog of the v-fos gene from mouse and human cells.

The 8.2-kilobase (kb) unintegrated circular DNA form of the FBJ murine leukemia virus (FBJ-MLV) was linearized by cleavage at the single HindIII site, molecularly cloned into bacteriophage Charon 30, and subsequently subcloned into pBR322 (pFBJ-MLV-1). Both FBJ-MLV virion RNA and pFBJ-MLV-1 DNA were used to investigate the arrangement of helper virus sequences in the FBJ murine osteosarcoma virus genome (FBJ-MSV) by heteroduplex formation with cloned FBJ-MSV proviral DNA. The results showed that the FBJ-MSV genome contained 0.8 kb of helper virus sequence at its 5' terminus and 0.98 kb at its 3' terminus. Approximately 6.8 kb of helper virus sequence had been deleted, and 1.7 kb of unrelated sequence was inserted into the FBJ-MSV genome. This substituted region contains v-fos, the transforming gene of FBJ-MSV. Using a probe specific for v-fos, we have cloned homologous sequences (c-fos) from mouse and human chromosomal DNA. Heteroduplex analysis of FBJ-MSV DNA with these recombinant clones showed that both the c-fos(mouse) and the c-fos(human) sequences hybridized to the entire 1.7-kb v-fos region. However, five regions of homology of 0.27, 0.26, 0.14, 0.5, and 0.5 kb were separated by four regions of nonhomology of 0.76, 0.55, 0.1, and 0.1 kb from 5' to 3' with respect to the FBJ-MSV genome. The size of these sequences showed striking similarity in both c-fos(mouse) and c-fos(human).     

明永冰川融水中一株裂解性低温噬菌体的分离及特征

【目的】高山冰川是一类独特的生态系统,本研究探索从明永冰川地区分离和培养低温菌噬菌体,并对其特征进行研究。【方法】利用已分离的低温菌为宿主,采用"双层平板法"从明永冰川融水中分离纯化低温菌噬菌体;对噬菌体及其宿主进行电镜形态观察,并进行噬菌体基因组限制性酶切片段长度多态性分析、衣壳蛋白组成分析及噬菌体生理特征研究。【结果】从明永冰川融水中分离获得一株裂解性低温噬菌体,命名为MYSP03(Mingyong Flavobacterium Siphoviridae Bacteriophage),其宿主菌MYB03鉴定为Flavobacterium菌株。噬菌体MYSP03为长尾型,无囊膜,头部具典型的正多面体立体对称结构,直径约72 nm;尾管长约240 nm,直径约10 nm;4℃时具侵染活性,在4℃-20℃范围内均可产生边缘清晰、透明的噬菌斑,最适感染温度约10℃,pH耐受范围较广,最适感染pH约9.4,对氯仿不敏感,基因组为双链DNA,大小约66 kb。     

Temperate bacteriophage ZF40 of Erwinia carotovora: phage particle structure and DNA restriction analysis

Structural organization of the temperate bacteriophage ZF40 of Erwinia carotovora was studied. Phage ZF40 proved to be a typical member of the Myoviridae family (morphotype A1). Phage particles consist of an isometric head 58.3 nm in diameter and a contractile 86.3-nm-long tail with a complex basal plate and short tail fibers (31.5 nm). Phage tail sheath, a truncated cone in shape, is characterized by specific packaging of structural subunits. The ZF40 phage genome is 45.8 kb in size, as determined by restriction analysis, and contains DNA cohesive ends. The ZF40 phage of Erwinia carotovora is assumed to be a new species of bacteriophages specific for enterobacteria.     

DNA analysis of temperate bacteriophage Aa/23 isolated from Actinobacillus actinomycetemcomitans

The DNA of the temperate bacteriophage Aaφ23 isolated from the oral bacterium Actinobacillus actinomycetemcomitans was examined structurally both in the phage head and in the prophage. The DNA in phage particles comprises 44?kb linear molecules with a terminal redundancy of 1.6?kb, which represent circular permutations. Thus, DNA is packaged into phage heads by the headful mechanism. The Aaφ23 prophage is integrated into the host chromosome.     

Complete genomic nucleotide sequence of the temperate bacteriophage Aa Phi 23 of Actinobacillus actinomycetemcomitans

The entire double-stranded DNA genome of the Actinobacillus actinomycetemcomitans bacteriophage Aa Phi 23 was sequenced. Linear DNA contained in the phage particles is circularly permuted and terminally redundant. Therefore, the physical map of the phage genome is circular. Its size is 43,033 bp with an overall molar G+C content of 42.5 mol%. Sixty-six potential open reading frames (ORFs) were identified, including an ORF resulting from a translational frameshift. A putative function could be assigned to 23 of them. Twenty-three other ORFs share homologies only with hypothetical proteins present in several bacteria or bacteriophages, and 20 ORFs seem to be specific for phage Aa Phi 23. The organization of the phage genome and several genetic functions share extensive similarities to that of the lambdoid phages. However, Aa Phi 23 encodes a DNA adenine methylase, and the DNA packaging strategy is more closely related to the P22 system. The attachment sites of Aa Phi 23 (attP) and several A. actinomycetemcomitans hosts (attB) are 49 bp long.     

DNA inversion mediated by the r-determinant of plasmid NR1: evidence for the intramolecular replicative transposition of a 23 kb IS1-flanked transposon?

Summary The r-determinant (r-det) of the R plasmid NR1-Basel is a 23 kb, IS1-flanked transposon, called Tn2671, which has been shown to transpose to the genome of bacteriophage P7. Among the derivatives of phage P7::r-det we found one which carried two copies of the r-det as inverted repeats and which also contained the P7 genome segment between them in inverted orientation. Its generation is best explained by assuming that the entire 23 kb Tn2671 transposon has undergone intramolecular replicative transposition.     

Bacteriophage influence Campylobacter jejuni types populating broiler chickens

The characteristics that allow one Campylobacter jejuni genotype to succeed over another under the influence of bacteriophage predation have been examined in experimental broiler chickens following the observation that this succession appeared to occur in naturally colonized broiler chicken flocks. Examination of three C. jejuni strains from a single flock indicated that horizontal transfer of at least 112 kb of genomic DNA from strain F2C10 (bacteriophage sensitive) to strain F2E1 (bacteriophage insensitive) had created strain F2E3. Transfer of this DNA was associated with acquisition of sensitivity to 6 of 25 lytic bacteriophage isolated from the same flock. All strains tested were capable of colonizing broiler chickens but cocolonization revealed that the bacteriophage sensitive strains F2E3 and F2C10 had a competitive advantage over the bacteriophage insensitive strain F2E1. With the addition of lytic bacteriophage the situation was completely reversed, with F2E1 dominating. The inability to replicate bacteriophage is associated with a significant fitness cost that renders the insensitive strain competitive only in the presence of bacteriophage. We demonstrate that interstrain recombination in vivo can generate genome diversity in C. jejuni and that bacteriophage predation is a strong selective pressure that influences the relative success of emergent strains in broiler chickens.     

Molecular characterization of the genome of Bacillus subtilis (natto) bacteriophage PM1, a phage associated with disruption of food production

“Natto”, regarded as a traditional food, is made by fermenting boiled soybeans with Bacillus subtilis (natto), which is a natto-producing strain related to B. subtilis. Natto production is disrupted by bacteriophage infection of B. subtilis (natto); thus, it is necessary to control bacteriophage infection. A bacteriophage of B. subtilis (natto), PM1, was isolated during interrupted natto production in a factory. As PM1 was shown to have a long non-contractile tail in a morphological study, it was believed to belong to the family Siphoviridae. The genome of PM1 was shown to be a linear double-stranded DNA of approximately 50 kb. Based on the results of studies using restriction endonucleases, PM1 DNA was found to be circularly permuted, similar to bacteriophage DNA without definite ends (e.g. bacteriophage T4). The nucleotide sequence of a 1.1 kb segment of PM1 was determined and used to design a PCR assay. A 0.5 kb product was amplified from eight of ten bacteriophage isolates that infect B. subtilis (natto), and the nucleotide sequences of the PCR-amplified products were identical to those of PM1, suggesting that PM1-related bacteriophages are the most prevalent infectious agents associated with the disruption of natto production. The PCR method might be useful to detect PM1-related bacteriophages and will help to control bacteriophage infection.     

A physical map of the Salmonella typhimurium LT2 genome made by using XbaI analysis.

XbaI digestion and pulsed-field gel electrophoresis of the genome of Salmonella typhimurium LT2 yields 24 fragments: 23 fragments (total size, 4,807 kb) are from the chromosome, and one fragment (90 kb) is from the virulence plasmid pSLT. Some of the 23 fragments from the chromosome were located on the linkage map by the use of cloned genes as probes and by analysis of strains which gain an XbaI site from the insertion of Tn10. Twenty-one of the fragments were arranged as a circular physical map by the use of linking probes from a set of 41 lysogens in which Mud-P22 was stably inserted at different sites of the chromosome; fragment W (6.6 kb) and fragment X (6.4 kb) were not located on the physical map. XbaI digestion of strains with Tn10 insertions allowed the physical locations of specific genes along the chromosome to be determined on the basis of analysis of new-fragment sizes. There is good agreement between the order of genes on the linkage map, which is based primarily on P22 joint transduction and F-mediated conjugation, and the physical map, but there are frequently differences in the length of the interval from the two methods. These analyses allowed the measurement of the amount of DNA packaged in phage P22 heads by Mud-P22 lysogens following induction; this varies from ca. 100 kb (2 min) to 240 kb (5 min) in different parts of the chromosome.     

The Pasteurella multocida toxin is encoded within a lysogenic bacteriophage

Toxigenic strains of Pasteurella multocida produce a 146 kDa toxin (PMT) that acts as a potent mitogen. Sequence analysis of the structural gene for PMT, toxA, previously suggested it was horizontally acquired, because it had a low G + C content relative to the P. multocida genome. To address this, the sequence of DNA flanking toxA was determined. The sequence analysis showed the presence of homologues to bacteriophage tail protein genes and a bacteriophage antirepressor, suggesting that the toxin gene resides within a prophage. In addition to phage genes, the toxA flanking DNA contained a homologue of a restriction/modification system that was shown to be functional. The presence of a bacteriophage was demonstrated in spent medium from toxigenic P. multocida isolates. Its production was increased by mitomycin C addition, a treatment that is known to induce the lytic cycle of many temperate bacteriophages. The genomes of bacteriophages from three different toxigenic P. multocida strains had similar but not identical restriction profiles, and were approximately 45-50 kb in length. The prophages from two of these had integrated at the same site in the chromosome, in a tRNA gene. Southern blot analysis confirmed that these bacteriophages contained the toxA gene.