Lysogenic conversion in Klebsiella pneumoniae: system which requires active immunity regulation for expression of the conversion phenomenon.

We have previously described Klebsiella pneumoniae MirM7b, which, although stably lysogenic for the inducible and nondefective phages FR2 and AP3, is not immune to superinfection by these same viruses. MirA12b, a strain which is lysogenic for FR2 and AP3 and immune to superinfection, has been derived from MirM7b. The sensitivity of this strain and that of the nonimmune parent to several bacteriophages have been compared in this work. It has been found that, whereas MirM7b is sensitive to coliphages P1, T3, T7, and phiI, MirA12b is fully resistant to all of them. It is shown that phages FR2 and AP3 convert Klebsiella strains to resistance to coliphage P1 and coliphages T3, T7, and phiI, respectively, and cause loss of surface antigens in lysogenic cells. To determine such a conversion, both FR2 and AP3 require expression of immunity to superinfection. This explains the differences that exist between MirM7b and MirA12b in both phage sensitivity and surface antigens. Hypotheses are presented to explain the peculiar need for an active superinfection repressor to express lysogenic conversion.     

Control of cell septation by lateral wall extension in a pH-conditional morphology mutant of Klebsiella pneumoniae.

The pH-conditional morphology mutant of Klebsiella pneumoniae strain MirM7 grows as cocci at pH 7 and as rods at pH 5.8. The mutant has a high-level mecillinam resistance (50% lethal dose greater than 200 micrograms/ml) in both forms. When broth cultures of the rod-shaped mutant were grown with 0.7 microgram of mecillinam per ml, cells assumed a round shape and continued to divided at a higher rate than the untreated control. A MirM7 rod-shaped revertant (MirA12), when treated with the same antibiotic concentration, changed to coccal shape and stopped dividing. The penicillin-binding proteins (PBPs) of strains MirA12 and MirM7 were analyzed. K. pneumoniae had six major PBPs quite similar to those of Escherichia coli. No differences were seen in the PBPs of MirM7 cocci and rods and MirA12 cells. In particular, PBP2 was found to be present and similar in MirM7 rods and cocci and MirA12 cells. We suggest that that in gram-negative rods, a control mechanism exists which prevents further septation in the absence of lateral cell wall elongation. The unique behavior of MirM7 is due to the fact that the control mechanism is not active in this strain. This model allows us to explain the preservation of shape in bacterial rods under various conditions of growth and the mechanism of bacterial killing by mecillinam.     

Early Initiation of Deoxyribonucleic Acid Replication and Shortening of Generation Time Associated with Inhibition of Lateral Wall Formation by Mecillinam

The effects of mecillinam on the growth of rods of the pH-conditional morphology mutant MirM7 was studied. It has been found that mecillinam causes, coincident with transition to coccal shape, a balanced rise in the rate of viable count increase and the rate of macromolecular synthesis which lasts either until the cells enter a stationary growth phase or indefinitely, in the case of continuously diluted cultures. When the antibiotic is removed from cells which have already become coccoid, cells continue to grow at a faster rate until they resume the rod shape. No change in the per-cell rate of protein synthesis has been seen in untreated or mecillinam-treated cells before or after the change in growth rate. Studies with synchronously growing cells have shown that the antibiotic causes a shortening in the I period (initiation of deoxyribonucleic acid replication). Evaluation of the residual divisions in nalidixic acid-treated, exponential-phase cells has shown that mecillinam also shortens the D period (cell division). It is proposed that, in strain MirM7, inhibition of lateral wall elongation by the antibiotic allows the initiation of a new septum, though inhibition is still in progress. The initiation of a new septum is, in turn, responsible for both the early inibition of deoxyribonucleic acid replication and accelerated division. In the parental strain, MirA12, as well as in other sensitive gram-negative rods which divide, become cocci, and stop dividing after addition of the antibiotic, inhibition of lateral wall formation activates a feedback mechanism which prevents insertion of new septa (Satta et al., J. Bacteriol. 142:43-51, 1980). Consequently, no early initiation of deoxyribonucleic acid replication is observed, and the last division allowed by the antibiotic occurs in due time. This negative control is missing in MirM7.     

Chronic HIV-1 infection frequently fails to protect against superinfection

Reports of HIV-1 superinfection (re-infection) have demonstrated that the immune response generated against one strain of HIV-1 does not always protect against other strains. However, studies to determine the incidence of HIV-1 superinfection have yielded conflicting results. Furthermore, few studies have attempted to identify superinfection cases occurring more than a year after initial infection, a time when HIV-1-specific immune responses would be most likely to have developed. We screened a cohort of high-risk Kenyan women for HIV-1 superinfection by comparing partial gag and envelope sequences over a 5-y period beginning at primary infection. Among 36 individuals, we detected seven cases of superinfection, including cases in which both viruses belonged to the same HIV-1 subtype, subtype A. In five of these cases, the superinfecting strain was detected in only one of the two genome regions examined, suggesting that recombination frequently occurs following HIV-1 superinfection. In addition, we found that superinfection occurred throughout the course of the first infection: during acute infection in two cases, between 1-2 y after infection in three cases, and as late as 5 y after infection in two cases. Our results indicate that superinfection commonly occurs after the immune response against the initial infection has had time to develop and mature. Implications from HIV-1 superinfection cases, in which natural re-exposure leads to re-infection, will need to be considered in developing strategies for eliciting protective immunity to HIV-1.     

Cluster K mycobacteriophages: insights into the evolutionary origins of mycobacteriophage TM4

Five newly isolated mycobacteriophages--Angelica, CrimD, Adephagia, Anaya, and Pixie--have similar genomic architectures to mycobacteriophage TM4, a previously characterized phage that is widely used in mycobacterial genetics. The nucleotide sequence similarities warrant grouping these into Cluster K, with subdivision into three subclusters: K1, K2, and K3. Although the overall genome architectures of these phages are similar, TM4 appears to have lost at least two segments of its genome, a central region containing the integration apparatus, and a segment at the right end. This suggests that TM4 is a recent derivative of a temperate parent, resolving a long-standing conundrum about its biology, in that it was reportedly recovered from a lysogenic strain of Mycobacterium avium, but it is not capable of forming lysogens in any mycobacterial host. Like TM4, all of the Cluster K phages infect both fast- and slow-growing mycobacteria, and all of them--with the exception of TM4--form stable lysogens in both Mycobacterium smegmatis and Mycobacterium tuberculosis; immunity assays show that all five of these phages share the same immune specificity. TM4 infects these lysogens suggesting that it was either derived from a heteroimmune temperate parent or that it has acquired a virulent phenotype. We have also characterized a widely-used conditionally replicating derivative of TM4 and identified mutations conferring the temperature-sensitive phenotype. All of the Cluster K phages contain a series of well conserved 13 bp repeats associated with the translation initiation sites of a subset of the genes; approximately one half of these contain an additional sequence feature composed of imperfectly conserved 17 bp inverted repeats separated by a variable spacer. The K1 phages integrate into the host tmRNA and the Cluster K phages represent potential new tools for the genetics of M. tuberculosis and related species.     

Phenotypic transformation including host-range transition through superinfection of T-even phages

Mosaic genome design, considered evidence of horizontal gene transfer, is prominent in T-even phage tail fiber genes involved in host recognition. The possibility of direct gene transfer was assessed through superinfection with two virulent phages T2 and PP01, which caused host recognition shift. Two recombinant phages designated as TPr03 and TPr04 were isolated. PCR-restriction fragment length polymorphism analysis and sequence analysis suggested that 18% of the TPr03 and 38% of the TPr04 genome derived from PP01. Both isolates showed host ranges identical to PP01. The results suggested the possibility of generating various recombinant phages by intentional dual infections and of the occasional occurrence in nature of generation of phage showing new characteristics through superinfection, followed by the genomic recombination.     

Characterization of temperate bacteriophages of Leuconostoc oenos and evidence for two prophage attachment sites in the genome of starter strain PSU-1

The close relatedness between 17 Leuconostoc oenos bacteriophages, induced with mitomycin C from strains isolated in different geographic regions, was inferred from their morphology, DNA homology and protein composition. The genome of all the phages had cohesive end termini and ranged in size from 36.4 to 40.9 kb. According to the restriction patterns obtained by digestion with five enzymes, the phages were divided in six groups. Lysogenization of a spontaneous phage-cured derivative of Leuc. oenos strain PSU-1 was achieved with 16 phages and the analysis of the lysogens showed that the phage DNA integrates in the host chromosome in one or two sites. The att B loci were located on the macrorestriction Asc I and Not I fragments of the recipient strain. A survey of Leuc. oenos strains with a phage DNA probe confirmed the lysogenic nature of several, but not all of the original phage hosts. These results are discussed in the light of evidence for the instability of some lysogenic PSU-1 derivatives.     

Transducing phages of Pseudomonas aeruginosa related to phage F116L

New phages K104 and B26, which are relative to F116L by a number of biological characters, appeared to show general transducing activity. Phage K104 transduces all tested markers with higher frequency than the phage B26. Linkage of the bacterial markers pair ilv202--met28 durspectively. When recipient bacteria lysogenic for phages K104 and B26 are used, frequencies of transduction by phage F116L are decreased. In the presence of F116L prophage the frequency of transduction by phage B26 is 10-fold increased. Phages B26 and F116L do not grow on bacteria lysogenic for these phages. Phage F116L does not grow on the lawn of bacteria, lysogenic for phage K104, while phage B26 grows on the same lawn with the efficiency of plating about 10(-2).     

In-depth analysis of a heterosexually acquired human immunodeficiency virus type 1 superinfection: evolution, temporal fluctuation, and intercompartment dynamics from the seronegative window period through 30 months postinfection

Human immunodeficiency virus type 1 (HIV-1) superinfection refers to the acquisition of another strain by an already infected individual. Here we report a comprehensive genetic analysis of an HIV-1 superinfection acquired heterosexually. The infected individual was in a high-risk cohort in Tanzania, was exposed to multiple subtypes, and was systematically evaluated every 3 months with a fluorescent multi-region genotyping assay. The subject was identified in the window period and was first infected with a complex ACD recombinant strain, became superinfected 6 to 9 months later with an AC recombinant, and was monitored for >2.5 years. The plasma viral load exceeded 400,000 copies/ml during the first 9 months of infection but resolved to the set point of 67,000 copies/ml by 3 months after superinfection; the CD4 cell count was 377 cells/mul at 30 months. Viral diversity was evaluated with techniques designed to fully sample the quasi-species, permitting direct observation of the evolution, temporal fluctuation, and intercompartment dynamics of the initial and superinfecting strains and recombinants derived from them. Within 3 months of superinfection, seven different molecular forms were detected in gag and six were detected in env. The proportions of forms fluctuated widely over time in plasma and peripheral blood mononuclear cells, illustrating how challenging the detection of dually infected individuals can be. Strain-specific nested PCR confirmed that the superinfecting strain was not present until the 9 month follow-up. This study further defines the parameters and dynamics of superinfection and will foster appropriate studies and approaches to gain a more complete understanding of risk factors for superinfection and its impact on clinical progression, epidemiology, and vaccine design.     

Role of reticuloendotheliosis virus envelope glycoprotein in superinfection interference.

Cells expressing specific proviruses are resistant to superinfection by viruses of the same subgroup. To investigate the role of the reticuloendotheliosis virus (REV) envelope glycoprotein (env-gp) in the establishment of resistance to superinfection, we constructed plasmids that express either the wild-type env-gp or an env-gp derivative that lacks part of the transmembrane (TM) protein. After transfection, transient expression of the wild-type env gene resulted in syncytium formation in a mammalian cell line permissive for virus replication, whereas synthesis of the TM-defective env-gp did not result in syncytium formation. Several stable cell lines expressing either the normal or TM-defective env-gp were isolated. Expression of the normal env-gp in the absence of expression of other viral genes induced resistance to infection by REV. Immunofluorescence analysis of cells expressing the TM-defective env derivative and an examination of the glycosylation pattern of this peptide indicated that it is not translocated to the cell surface but resides primarily in the rough endoplasmic reticulum. However, these cells were also resistant to REV infection. Thus, interaction between the env derivative and the cellular component that functions as a receptor for the virus can occur in the endoplasmic reticulum and renders the cell immune to superinfection.     

Effect of ribonucleic acid phage superinfection on lysis-inhibited Escherichia coli

Groman, Neal B. (University of Washington, Seattle), and Grace Suzuki. Effect of ribonucleic acid phage superinfection on lysis-inhibited Escherichia coli. J. Bacteriol. 90:1007-1012. 1965.-Induced culturesof Escherichia coli K-12(lambda112)F(+) were superinfected with ribonucleic acid phage f2 at various times to test for the specificity of lysis inhibition and the concurrent inhibition of growth. When f2 superinfection occurred within 90 min after induction, lysis was observed in normally lysis-inhibited cultures. Later superinfections produced very little lysis. Following early superinfection, both lambda112 and f2 phages were produced in induced cells. When superinfection occurred during the period in which growth was inhibited, f2 production was totally inhibited. The inhibition of f2 was not due to its inability to adsorb, nor was it due to damage inflicted on cells by ultraviolet irradiation or to exhaustion of the medium. The data suggest that inhibition of lysis of induced K-12(lambda112)F(+) is phage-specific, whereas the accompanying inhibition of growth is nonspecific.     

苏云金芽孢杆菌两株溶原性噬菌体的生物学特性

研究苏云金芽孢杆菌(Bacillus thuringiensis)的溶原性及其噬菌体的生物学特性,从生产菌株MZ1中分离了两株溶原性噬菌体。MZ1经诱导后产生直径约为3mm和1mm的噬斑,分离获得属长尾噬菌体科的噬菌体MZTP01和MZTP02两株;分别对6株和7株不同亚种的Bt菌株具有侵染力;免疫血清与相应噬菌体的中和反应K值分别为45和326,且两者无相关抗原性。MZTP01抵抗酸、碱、紫外线和热的能力比MZTP02强,但抵抗有机溶剂的程度比MZTP02弱。MZTP01的潜伏期为80min,裂解量为55;MZTP02的潜伏期为40min,裂解量为175。核酸结构分析均表明为线性dsDNA分子。两基因组DNA的凝胶电泳表明分子量均在9.4~23kb之间,并被HindⅢ酶切分别产生8条和9条清晰条带。该菌株被证明为二元溶原菌,可能是造成生产损失的主要原因;为防治溶原性噬菌体提供了生物学信息。     

Lysogeny in Leuconostoc oenos.

Thirty strains of Leuconostoc oenos were exposed to mitomycin C to induce lysogenic bacteriophages. Lysis curves typical for lysogenic strains were obtained with 19 strains. Indicator strans were found for 17 of these phages. Five were characterized by electron microscopy, lytic spectrum, molecular masses of the proteins, sequencing of five N-terminal amino acids of the two major proteins and DNA analysis (restriction patterns, cross hybridization). The results revealed a very close relationship between the phages. Hybridization experiments between the DNAs of the temperate phages and the appropriate lysogenic strains revealed phage-related sequences in the DNA of the lysogenic strain.     

Mycobacteriophage Fruitloop gp52 inactivates Wag31 (DivIVA) to prevent heterotypic superinfection

Bacteriophages engage in complex dynamic interactions with their bacterial hosts and with each other. Bacteria have numerous mechanisms to resist phage infection, and phages must co‐evolve by overcoming bacterial resistance or by choosing an alternative host. Phages also compete with each other, both during lysogeny by prophage‐mediated defense against viral attack and by superinfection exclusion during lytic replication. Phages are enormously diverse genetically and are replete with small genes of unknown function, many of which are not required for lytic growth, but which may modulate these bacteria–phage and phage–phage dynamics. Using cellular toxicity of phage gene overexpression as an assay, we identified the 93‐residue protein gp52 encoded by Cluster F mycobacteriophage Fruitloop. The toxicity of Fruitloop gp52 overexpression results from interaction with and inactivation of Wag31 (DivIVA), an essential Mycobacterium smegmatis protein organizing cell wall biosynthesis at the growing cellular poles. Fruitloop gene 52 is expressed early in lytic growth and is not required for normal Fruitloop lytic replication but interferes with Subcluster B2 phages such as Hedgerow and Rosebush. We conclude that Hedgerow and Rosebush are Wag31‐dependent phages and that Fruitloop gp52 confers heterotypic superinfection exclusion by inactivating Wag31.     

Protection against retroviral diseases after vaccination is conferred by interference to superinfection with attenuated murine leukemia viruses.

Cell cultures expressing a retroviral envelope are relatively resistant to superinfection by retroviruses which bear envelopes using the same receptor. We tested whether this phenomenon, known as interference to superinfection, might confer protection against retroviral diseases. Newborn mice first inoculated with the attenuated strain B3 of Friend murine leukemia virus (F-MuLV) were protected against severe early hemolytic anemia and nonacute anemiant erythroleukemia induced by the virulent strain 57 of F-MuLV. Vaccinated animals were also protected as adults against acute polycythemic erythroleukemia induced upon inoculation with the viral complex containing the defective spleen focus-forming virus and F-MuLV 57 as helper virus. Animals were inoculated as newborns, which is known to induce immune tolerance in mice, and the rapid kinetics of protection, incompatible with the delay necessary for the immune response to develop, indicated that protection was not due to an immune mechanism but rather was due to the rapid and long-lasting phenomenon of interference. This result was confirmed by combining parental and envelope chimeric MuLV from different interference groups as vaccinal and challenge viruses. Although efficient protection could be provided by vaccination by interference, we observed that attenuated replication-competent retroviruses from heterologous interference groups might exert deleterious synergistic effects.     

New waterborne bacteriophages active on Yersinia enterocolitica.

Seven bacteriophages active on Yersinia enterocolitica (YE) were isolated from surface water samples collected in Granada, Spain. A comparison of the respective host ranges of these new phages and of reference phages used for YE phage typing showed that YE strains belonging to various phage types, grown at either 37 or 25 degrees C, expressed susceptibility to reference sewage water phages whereas susceptibility to new waterborne phages, as well as to reference phages from lysogenic YE, was only demonstrated in YE strains grown at 25 degrees C. A YE strain isolated by stool culture from a pig was lysogenic for a bacteriophage which behaved like waterborne phages and reference phages from lysogenic YE strains. The possibility that the isolation of waterborne bacteriophages might, in certain circumstances, reflect the presence of lysogenic YE was raised.     

Intersubtype human immunodeficiency virus type 1 superinfection following seroconversion to primary infection in two injection drug users

In this study, we describe two cases of human immunodeficiency virus type 1 (HIV-1) intersubtype superinfection with CRF01_AE and subtype B strains, which occurred in two injection drug users participating in a prospective cohort study in Bangkok, Thailand. In both cases, the superinfecting strain was detected by molecular and serologic analyses several weeks after complete seroconversion to the primary infection with a strain belonging to a different subtype. Superinfection occurred despite specific T-cell and humoral antibody responses to the primary virus. In both cases, cross-subtype immune responses were limited or absent prior to the second infection. These data show that, in some individuals, the quality and quantity of the immune response elicited by primary HIV-1 infection may not protect against superinfection. This finding has important implications for vaccine design. HIV-1 vaccines, at a minimum, will need to include potent, broadly protective, conserved immunogens derived from several group M subtypes.     

First complete-genome documentation of HIV-1 intersubtype superinfection with transmissions of diverse recombinants over time to five recipients

Human immunodeficiency virus type 1 (HIV-1) recombinants in the world are believed to be generated through recombination between distinct HIV-1 strains among coinfection or superinfection cases. However, direct evidence to support transmission of HIV-1 recombinants from a coinfected/superinfected donor to putative recipient is lacking. Here, we report on the origin and evolutionary relationship between a set of recombinants from a CRF01_AE/CRF07_BC superinfected putative donor and diverse CRF01_AE/CRF07_BC recombinants from five putative recipients. Interviews on sociodemographic characteristics and sexual behaviors for these six HIV-1-infected men who have sex with men showed that they had similar ways of partner seeking: online dating sites and social circles. Phylogenetic and recombination analyses demonstrated that the near-full-length genome sequences from six patients formed a monophyletic cluster different from known HIV-1 genotypes in maximum likelihood phylogenetic trees, were all composed of CRF01_AE and CRF07_BC fragments with two common breakpoints on env, and shared 4–7 breakpoints with each other. Moreover, 3’ half-genomes of recombinant strains from five recipients had identical/similar recombinant structures with strains at longitudinal samples from the superinfected donor. Recombinants from the donor were paraphyletic, whereas five recipients were monophyletic or polyphyletic in the maximum clade credibility tree. Bayesian analyses confirmed that the estimated time to the most recent common ancestor (tMRCA) of CRF01_AE and CRF07_BC strains of the donor was 2009.2 and 2010.7, respectively, and all were earlier than the emergence of recombinants from five recipients. Our results demonstrated that the closely related unique recombinant forms of HIV-1 might be the descendent of a series of recombinants generated gradually in a superinfected patient. This finding highlights the importance of early initiation of antiretroviral therapy as well as tracing and testing of partners in patients with multiple HIV-1 infection.     

Genome Sequence and Characteristics of Lrm1, a Prophage from Industrial Lactobacillus rhamnosus Strain M1

Prophage Lrm1 was induced with mitomycin C from an industrial Lactobacillus rhamnosus starter culture, M1. Electron microscopy of the lysate revealed relatively few intact bacteriophage particles among empty heads and disassociated tails. The defective Siphoviridae phage had an isometric head of approximately 55 nm and noncontractile tail of about 275 nm with a small baseplate. In repeated attempts, the prophage could not be cured from L. rhamnosus M1, nor could a sensitive host be identified. Sequencing of the phage Lrm1 DNA revealed a genome of 39,989 bp and a G+C content of 45.5%. A similar genomic organization and mosaic pattern of identities align Lrm1 among the closely related Lactobacillus casei temperate phages A2, ΦAT3, and LcaI and with L. rhamnosus virulent phage Lu-Nu. Of the 54 open reading frames (ORFs) identified, all but 8 shared homology with other phages of this group. Five unknown ORFs were identified that had no homologies in the databases nor predicted functions. Notably, Lrm1 encodes a putative endonuclease and a putative DNA methylase with homology to a methylase in Lactococcus lactis phage Tuc2009. Possibly, the DNA methylase, endonuclease, or other Lrm1 genes provide a function crucial to L. rhamnosus M1 survival, resulting in the stability of the defective prophage in its lysogenic state. The presence of a defective prophage in an industrial strain could provide superinfection immunity to the host but could also contribute DNA in recombination events to produce new phages potentially infective for the host strain in a large-scale fermentation environment.