Mycoplasma arthritidis bacteriophage MAV1 prophage integration, deletions, and strain-related polymorphisms

Temperate bacteriophage MAV1 is found in certain highly virulent strains of Mycoplasma arthritidis. Integration sites, portions of the right and left prophage ends, and flanking DNA from eight prophages in seven M. arthritidis strains were characterized in this study. attb and attp sites conformed for the most part to the consensus sequence TATTTTT, although minor polymorphisms were noted. Prophages were integrated into similar sites in four strains, suggesting that these strains may have had a common ancestor. Two strains had three prophage copies each, and integration sites were identical. Two strains had two copies each. One of these shared two of the integration sites occupied in the three-copy strains, while the other shared one of these sites and harbored a second prophage in a unique site. Integration sites in the two strains with one prophage each were unique. Four MAV1 copies contained extensive substitutions within a region encoding a putative structural protein and the putative repressor protein. A 3-kb fragment was deleted from the right side of two of these copies. It is proposed that polymorphisms within MAV1 prophage integration sites and within the prophages themselves may help to identify phylogenetic relationships among virulent M. arthritidis strains.     

The vir gene of bacteriophage MAV1 confers resistance to phage infection on Mycoplasma arthritidis

Lysogenization of Mycoplasma arthritidis with the MAV1 bacteriophage increases the virulence of the mycoplasma in rats. The MAV1 vir gene is one of only two constitutively transcribed phage genes in the lysogen. We show here that Vir is a lipoprotein and is located on the outer surface of the cell membrane. To investigate whether Vir is a virulence factor, the vir gene was cloned into the transposon vector Tn4001T and inserted in the genome of the nonlysogen strain 158. The virulence of the resulting transformants was no different from that of the parent strain. Interestingly, all vir-containing transformants were resistant to infection by MAV1. Vir had no effect on MAV1 adsorption. We conclude that Vir is not a virulence factor but functions to exclude superinfecting phage, possibly by blocking the injection of phage DNA into the bacterial cytoplasm.     

Lysogenic Conversion of Pasteurella by Escherichia coli Bacteriophage P1 CM

Bacteriophage P1 CM can convert Pasteurella pestis or P. pseudotuberculosis to chloramphenicol resistance and phage restriction, but no viable phage was induced from converted Pasteurella strains.     

Immunological responses of the rat to Mycoplasma arthritidis

Arthritis was produced in rats by the intravenous injection of Mycoplasma arthritidis. Metabolic inhibiting antibody and indirect hemagglutinating antibody could not be detected in the sera of arthritic or convalescent animals. Nonmurine species of mycoplasma were capable of inducing metabolic inhibiting antibody in the rat. A hypothesis based upon the possible occurrence of heterogenetic antigens common to M. arthritidis and rat tissue was brought forward to explain these findings. Complement-fixing antibody to M. arthritidis was detected 3 to 4 days after injection and subsequently rose to high levels, depending upon the severity of arthritis and number of organisms injected. Animals that had recovered from intravenous or subcutaneous inoculation with M. arthritidis were resistant to subsequent infections by the organism. Immunity could be passively transferred by the intravenous injection of convalescent serum. Adsorption of the convalescent serum with antigen greatly reduced the complement fixation titer but did not significantly alter the protective properties of the serum. The presence of complement-fixing antibody could not be related to the development of immunity. An avirulent strain of M. arthritidis and a strain previously classified as M. hominis type 2 were capable of inducing resistance to subsequent injection by virulent M. arthritidis.     

Electron Microscopic Evidence for Linear Insertion of Bacteriophage MU-1 in Lysogenic Bacteria

Temperate bacteriophage Mu-1 was used to generate a lysogenic derivative of the F'lac episome of Escherichia coli. Intact, covalently circular molecules of F'lac and lysogenic F'lac Mu(+) deoxyribonucleic acid (DNA) were isolated and examined by electron microscopy. The mean contour lengths of F'lac and F'lac Mu(+) molecules were 37.6 +/- 0.4 mum and 53.2 +/- 0.4 mum, respectively. The mean difference, 15.6 mum, is similar to the mean contour length of 12.9 +/- 0.1 mum obtained for linear DNA molecules released by osmotic shock from mature phage Mu-1 virions. These results provide direct physical evidence that phage Mu-1 integrates by linear insertion of its genome into the DNA of lysogenic host bacteria. Chemical and physical analyses of phage Mu-1 DNA indicate that it is similar to E. coli DNA in respect of gross base composition, buoyant density, and melting temperature.     

Bacteriophage Release in a Lysogenic Strain of Agrobacterium tumefaciens

Bacteriophage release in a lysogenic strain of Agrobacterium tumefaciens V-1 is temperature-sensitive. At 25 C and 30 C, phage was released in a ratio of 1 plaque-forming unit per 100 bacteria; at 35 C, although bacterial growth was not inhibited, phage release was suppressed. Phage synthesis was induced by heat shock, 42 C for 30 min, ultraviolet irradiation, and mitomycin C. Induction by ultraviolet light was unusual-an immediate rise in phage titer followed irradiation. A large increase occurred after a 90-min latent period. The lysogenic strain was cured of the phage by incubation at 37 C, and the cured strain produced plant tumors.     

Partial purification and properties of a mitogenic factor from Mycoplasma arthritidis

M. arthritidis PG6 in a dose of 10(7) colony-forming units per ml was shown to produce a highly pronounced mitogenic effect, linked with a thermolabile factor, on the splenocytes of mice and rats. The mitogenic factor was absent in the lipid fraction and in the fraction formed by easily extractable mycoplasmal proteins. The supernatant fluid obtained after the sedimentation of M. arthritidis globular proteins with ammonium sulfate stimulated lymphocytes as actively as the intact culture of M. arthritidis. The fractionation of the supernatant fluid in columns packed with Sephadex G-200 revealed that the greatest mitogenic effect was produced by the fraction with a molecular weight of 240,000 daltons. It seems that the arthritogenic properties of M. arthritidis are linked precisely with this subcomponent.     

Exclusion of Induced Bacteriophage from Cells of a Lysogenic Bacillus megaterium Committed to Sporulation

In Escherichia coli ML 308-225, d-ribose is transported into the cell by a constitutive active transport system of high activity. The activity of this transport system is severely reduced in cells subjected to osmotic shock, and the system is not present in membrane vesicles. The mechanism by which metabolic energy is coupled to transport of ribose was investigated. Substrates which generate adenosine 5'-triphosphate primarily through oxidative phosphorylation are poor energy sources for ribose uptake in DL-54, a mutant of ML 308-225 which lacks activity for the membrane-bound Ca(2+), Mg(2+)-dependent adenosine triphosphatase required for oxidative phosphorylation. Arsenate severely inhibits ribose uptake, whereas, under the same conditions, uptake of l-proline is relatively insensitive to arsenate. Anaerobiosis does not significantly inhibit ribose uptake in ML 308-225 or DL-54 when glucose is the energy source. A significant amount of ribose uptake is resistant to uncouplers of oxidative phosphorylation such as 2,4-dinitrophenol. These results indicate that the phosphate bond energy of adenosine 5'-triphosphate, rather than an energized membrane state, couples energy to ribose transport in ML 308-225.     

Accumulation of Incomplete Particles After UV-Light Induction of Haemophilus influenzae Lysogenic for Bacteriophage HP1c1

Temperate phage HP1c1 produces large quantities of incomplete phage-like particles when grown on Haemophilus influenzae BC200, a strain apparently cured of a common defective prophage.     

The Clinically Isolated FIZ15 Bacteriophage Causes Lysogenic Conversion in Pseudomonas aeruginosa PAO1

FIZ15 bacteriophage, from a human clinical isolate of Pseudomonas aeruginosa, causes lysogenic conversion in the P. aeruginosa strain PAO1. The prophage-conferred phenotypes are: (1) increased resistance to phagocytosis by mouse peritoneal macrophages; (2) increased resistance to killing by normal human serum, and (3) increased adhesion to human buccal epithelial cells. These phenotypes are related to the prophage-induced change at the level of its own bacterial receptor, which appears to be the O-antigen. Received: 31 August 1998 / Accepted: 20 November 1998     

Targeted Curing of All Lysogenic Bacteriophage from Streptococcus pyogenes Using a Novel Counter-selection Technique

Streptococcus pyogenes is a human commensal and a bacterial pathogen responsible for a wide variety of human diseases differing in symptoms, severity, and tissue tropism. The completed genome sequences of >37 strains of S. pyogenes, representing diverse disease-causing serotypes, have been published. The greatest genetic variation among these strains is attributed to numerous integrated prophage and prophage-like elements, encoding several virulence factors. A comparison of isogenic strains, differing in prophage content, would reveal the effects of these elements on streptococcal pathogenesis. However, curing strains of prophage is often difficult and sometimes unattainable. We have applied a novel counter-selection approach to identify rare S. pyogenes mutants spontaneously cured of select prophage. To accomplish this, we first inserted a two-gene cassette containing a gene for kanamycin resistance (Kan^R) and the rpsL wild-type gene, responsible for dominant streptomycin sensitivity (Sm^S), into a targeted prophage on the chromosome of a streptomycin resistant (Sm^R) mutant of S. pyogenes strain SF370. We then applied antibiotic counter-selection for the re-establishment of the Kan^S/Sm^R phenotype to select for isolates cured of targeted prophage. This methodology allowed for the precise selection of spontaneous phage loss and restoration of the natural phage attB attachment sites for all four prophage-like elements in this S. pyogenes chromosome. Overall, 15 mutants were constructed that encompassed every permutation of phage knockout as well as a mutant strain, named CEM1ΔΦ, completely cured of all bacteriophage elements (a ~10% loss of the genome); the only reported S. pyogenes strain free of prophage-like elements. We compared CEM1ΔΦ to the WT strain by analyzing differences in secreted DNase activity, as well as lytic and lysogenic potential. These mutant strains should allow for the direct examination of bacteriophage relationships within S. pyogenes and further elucidate how the presence of prophage may affect overall streptococcal survival, pathogenicity, and evolution.     

Infection by Bacteriophage P1 and Development of Host-controlled Restriction and Modification and of Lysogenic Immunity

Shigella dysenteriae cells were infected with phage P1 or P1cl. The outcome of superinfection of these cells with phage T1.Sh or T1.Sh(P1) or P1cl was studied as a function of time after the initial infection. Cells undergoing either a lytic response or a lysogenic response to the primary infection develop the ability to specifically restrict T1.Sh between 30 and 45 min. Between 15 and 30 min, the cells seem to develop the ability to produce T1.Sh(P1) after infection by T1.Sh. However, reasons are given for believing that this apparent time difference is consistent with a simultaneous development of the two capacities (restriction and modification) within the cell. This development occurs between 30 and 45 min. Cells infected with P1cl and superinfected 45 or more min later with T1.Sh(P1) can yield both P1cl and T1. Cells infected with P1 become resistant to infection by P1cl within 5 to 10 min. It is argued that this early immunity is not necessarily different in mechanism from true lysogenic immunity.     

大肠杆菌VT2噬菌体的分离与溶源转染

本试验利用指示菌MC1061,经双层琼脂法纯化和PCR扩增vt2基因,分别从大肠杆菌O157菌株、牛粪、鸡粪和污水中分离获得5株含vt2基因的噬菌体.这些噬菌斑透明,直径为0.5-2 mm,对指示菌的感染效价均在109PFU/mL以上,抵抗氯仿和56℃C30min的作用.将噬菌体分离株SHφW1感染MC1061后,经PCR鉴定获得一株溶源菌株(MC1061/SHφW1).溶源株的LB培养滤液对Vero细胞产生了显著的病变效应,而MC1061在同等条件下培养的滤液无细胞病变,表明VT2噬菌体通过溶源将vt2毒力基因水平转移,证实了VT2噬菌体的转染与细菌毒力相关.     

大肠杆菌VT2噬菌体的分离与溶源转染

本试验利用指示菌MC1061。经双层琼脂法纯化和PCR扩增vt2基因,分别从大肠杆菌0157菌株、牛粪、鸡粪和污水中分离获得5株含vt2基因的噬菌体。这些噬菌斑透明,直径为0．5-2min,对指示菌的感染效价均在10^9PFU／mL以上,抵抗氯仿和56℃30min的作用。将噬菌体分离株SHφWl感染MC1061后。经PCR鉴定获得一株溶源菌株(MC1061／SHφW1)。溶源株的LB培养滤液对Vero细胞产生了显著的病变效应,而MC1061在同等条件下培养的滤液无细胞病变,表明VT2噬菌体通过溶源将vt2毒力基因水平转移,证实了VT2噬菌体的转染与细菌毒力相关。     

Effect of Mycoplasma arthritidis on mouse and rat lymphoid cells in experimental Mycoplasma infection]

Early stages of mycoplasma infection of mice and rats were accompanied by suppression of the populations of rosette- and plaque-forming cells. Later the character and dynamics of the immune response to M. arthritidis differed in mice and rats. In mice mycoplasma infection was accompanied by stimulation of rosette-forming cells with some suppression of the plaque-forming cells from the 7th to the 36th day of infection. In rats by the 7th day the number of plaque- and rosette-forming cells decreased in comparison with control, and the immune response was restored by the 15h day; at later periods the immune response of the infected rats exceeded the normal level considerably. The cellular and humoral immune reactions proved to depend on the mycoplasma dose.     

Persistence of Mycoplasma arthritidis in the body of mice of different strains

Mice belonging to 3 strains were shown to differ greatly in their sensitivity to M. arthritidis. This organism persisted for a year in (C57BL/6 X A/Sn)F1 mice, for 6 weeks in BALB/c mice and for 1-3 weeks in C57BL/6 mice. The sensitivity of mice to M. arthritidis infection and the persistence of the infective agent in the organs of the animals were found to depend on the state of their cell-mediated immunity.     

Identification of an isoschizomer of the HhaI DNA methyltransferase in Mycoplasma arthritidis

The genome of Mycoplasma arthritidis strain 158 has modified cytosine residues at AGCT sequences that render the DNA resistant to digestion with the AluI restriction endonuclease. The DNA methyltransferase responsible for the base modification has previously been designated MarI. From the complete genome sequence of M. arthritidis , we identify Marth_orf138 as a candidate marI gene. Marth_orf138 was cloned in Escherichia coli and its TGA codons converted to TGG. DNA isolated from E. coli cells expressing the modified Marth_orf138 gene was degraded by the AluI nuclease, indicating that Marth_orf138 does not code for MarI. However, the DNA from E. coli was found to have acquired resistance to the restriction endonuclease HhaI. Genomic DNA from M. arthritidis was also found to be resistant to HhaI (recognizes GCGC). The M. arthritidis isoschizomer of the HhaI DNA methyltransferase, coded by Marth_orf138, is designated MarII. Transformation of M. arthritidis was not significantly affected by modification of plasmid at HhaI sites, indicating that the mycoplasma lacks a restriction endonuclease that recognizes GCGC sites.     

Head Proteins from T-Even Bacteriophage: I. Molecular Weight Characterization 1

T-even bacteriophage capsid proteins were separated on 6% agarose columns by use of 6 m guanidine hydrochloride containing 5 mm dithiothreitol both to dissociate and to elute the proteins. The head capsids of T2H, T4B, T4B01, T4D, and T6r(+) contained at least three structural proteins with molecular weights of 40,000, 18,000, and 11,000 daltons, amounting to 76, 2, and 8%, respectively, of the total capsid protein. On the other hand, T2L head capsids contained only two structural proteins with molecular weights of 40,000 and 18,000 daltons (81 and 2.5%, respectively, of the total protein). A discussion of the possible role of these structural head proteins and a T-even phage head model suggesting a structural arrangement of the 40,000 dalton subunit are presented.