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.     

Escherichia coli detection by GFP-labeled lysozyme-inactivated T4 bacteriophage

Escherichia coli has been used as an indicator of the fecal contamination of water and food, identifying potential health hazards. In this study, an E. coli-specific bacteriophage, T4, was used to detect E. coli bacteria. The T4 phage small outer capsid (SOC) protein was used to present green fluorescent protein (GFP), an easily detectable marker protein, on the phage capsid. To inactivate phage lytic activity, we used the T4e(-) phage, which does not produce the lysozyme responsible for host cell lysis. Infection of E. coli K12 cells with the GFP-labeled T4e(-) phage (T4e(-)/GFP) enabled the visualization and distinction of E. coli K12 cells from T4 phage-insensitive cells, Pseudomonas aeruginosa. Prolonged incubation of E. coli K12 cells with the T4e(-)/GFP phage did not lead to cell lysis. Propagation of T4e(-)/GFP in host cells increased the intensity of green fluorescence, making the distinction of E. coli cells from other cells simple and effective. This method enables the rapid, conclusive quantitation of E. coli cells within an hour.     

Infectivity of different forms of lambda bacteriophage DNA in transfection of calcinated Escherichia coli]

Infectivity of linear lambdaDNA molecules is proved to be about a hundred times higher in calcinated E. coli K12 (lambai434) than in E. coli K12(lambda-): the levels of transfection were 1-3-10(7) and 1-2-10(5) infective centers per 1 mug DNA, respectively. In E. coli JC 5743 rec B21 defective for exonucleases I and V the level of transfection was 1-3-10(6). High infectivity of linear lambdaDNA in lysogenic cells cannot be explained by a helping effect of phage particles spontaneously liberated by these cells. It can be caused by recombinations of inserted lambdaDNA molecules with prophage or by the low activity of some nucleases in the lysogenic cells. Covalently closed and "Hershey" ring forms of lambdaDNA penetrate the calcinated cells as readily as linear molecules do but the infectivity of the former ones is proved to be very low.     

Repression of lambda-Associated Enzyme Synthesis After lambda(vir) Superinfection of Lysogenic Hosts.

Lisio, Arnold L. (National Institutes of Health, Bethesda, Md.), and Arthur Weissbach. Repression of lambda-associated enzyme synthesis after lambda(vir) superinfection of lysogenic hosts. J. Bacteriol. 90:661-666. 1965.-Phage lambda(vir) is a multiple mutant of lambda which is capable of overcoming the immunity of a host lysogenic for lambda, and initiating normal vegetative replication of the superinfecting phage genome. Superinfection of Escherichia coli K-112 (lambda(22)) with lambda(vir) results in a normal phage yield, lysis time, and H(3)-thymine incorporation compared with infection of the sensitive host, K-112 (S). However, the production of the lambda phage-specific early protein, lambda-exonuclease, after superinfection of E. coli K-112 (lambda(22)) with lambda(vir) is only 25 to 50% of that obtained from corresponding infection of a nonlysogenic host, E. coli K-112 (S). This repression of lambda-exonuclease synthesis is dependent on the C(1) cistron of the prophage and is overcome if the lysogenic host cells are induced prior to superinfection. The data are interpreted as evidence for partial repression of lambda(vir) by the host immunity.     

Expression of the genome of Mu-like phage D3112 specific for Pseudomonas aeruginosa in Escherichia coli and Pseudomonas putida cells

The behavior of Escherichia coli cells carrying RP4 plasmid which contains the genome of a Mu-like D3112 phage specific for Pseudomonas aeruginosa was studied. Two different types of D3112 genome expression were revealed in E. coli. The first is BP4-dependent expression. In this case, expression of certain D3112 genes designated as "kil" only takes place when RP4 is present. As a result, cell division stops at 30 degrees C and cells form filaments. Cell division is not blocked at 42 degrees C. The second type of D3112 genome expression is RP4-independent. A small number of phage is produced independently of RP4 plasmid but this does not take place at 42 degrees C. No detectable quantity of the functionally active repressor of the phage was determined in E. coli (D3112). It is possible that the only cause for cell stability of E. coli (D3112) or E. coli (RP4::D3112) at 42 degrees C in the absence of the repressor is the fact of an extremely poor expression of D3112. In another heterologous system, P. putida both ways of phage development (lytic and lysogenic) are observed. This special state of D3112 genome in E. coli cells is proposed to be named "conditionally expressible prophage" or, in short, "conex-phage", to distinguish it from a classical lysogenic state when stability is determined by repressor activity. Specific blockade of cell division, due to D3112 expression, was also found in P. putida cells. It is evident that the kil function of D3112 is not specific to recognize the difference between division machinery of bacteria belonging to distinct species or genera. Protein synthesis is needed to stop cell division and during a short time period this process could be reversible. Isolation of E. coli (D3112) which lost RP4 plasmid may be regarded as an evidence for D3112 transposition in E. coli. Some possibilities for using the system to look for E. coli mutants with modified expression of foreign genes are considered.     

Evidence of septic system failure determined by a bacterial biochemical fingerprinting method

AIMS: To provide evidence of septic system failure by comparing two faecal indicator bacteria, enterococci and Escherichia coli, from defective septic tanks and adjacent creeks. METHODS AND RESULTS: A biochemical fingerprinting method was used to type and compare enterococci and E. coli strains from 39 septic tanks with creek water samples. Phenotypic diversity of enterococci (0.5 +/- 0.3) and E. coli (0.5 +/- 0.3) in septic tanks were significantly lower than those found in water samples (0.8 +/- 0.1, P < 0.0001 for enterococci and 0.9 +/- 0.1, P < 0.0001 for E. coli). Among 1072 enterococci isolates tested from septic tanks, 203 biochemical phenotypes (BPTs) were found of which 98 BPTs from 33 septic tanks were identical to several water samples. Similarly, among 621 E. coli isolates tested from septic tanks, 159 BPTs were found of which 53 BPTs from 26 septic tanks were also identical to water samples. The number of the latter bacteria was significantly (P = 0.01) higher in water samples collected from downstream compared with that of upstream in the study area. A high similarity between the populations of both indicator bacteria was also found between defective septic tanks and downstream water samples further indicating the contamination of both creeks by defective septic systems. CONCLUSIONS: Biochemical fingerprinting of faecal indicator bacteria is a useful and rapid method to provide direct evidence for septic system failure. Combination of both faecal indicator bacteria (enterococci and E. coli) provides a better judgement of the performance of a septic system. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to provide direct evidence of septic system failure by identifying the presence of specific bacterial types in septic tanks and surface waters. Based on our findings, we suggest that the performance evaluation of a septic system should be accompanied by direct analysis of faecal indicator bacteria.     

Isolation and characteristics of mutation pfm affecting the penetration of phage Mu into Escherichia coli K-12 cells

A mutant of Escherichia coli K-12 strain with the destroyed process of establishment of lysogenic state for phage Mu in the course of zygotic induction has been obtained. The mutation revealed, designated pfm (penetration factor for Mu), interferes with adsorption of phage Mu to the surface of E. coli K-12 cells. On the basis of data obtained, there is every reason to believe that the phage Mu DNA connection with the membrane components of the bacterial cell provides optimizing condition for the primary integrative transposition of phage Mu at the stage of Mu DNA introduction into the cell.     

Isolation and Characterization of Specialized φ80 Transducing Phages Carrying Regions of the Salmonella typhimurium trp Operon

We have isolated a series of nondefective phi80 specialized transducing phage which carry segments of the Salmonella typhimurium trp operon. These phage were obtained from a lysogenic derivative of a merozygote constructed by transferring an S. typhimurium trp episome into an Escherichia coli strain which lacks the normal phi80 attachment site. The deoxyribonucleic acid (DNA) from one such phage was purified and employed in DNA-ribonucleic acid (RNA) hybridization studies. The results obtained show that, under our hybridization conditions, heterologous hybridization is less efficient than homologous hybridization. It was also observed that not all S. typhimurium trp messenger RNA can readily anneal to E. coli trp operon DNA. Heterologous hybrids consisting of S. typhimurium trp messenger RNA and E. coli trp operon DNA were estimated to have a dissociation constant 10-fold larger than that of homologous hybrids.     

Analysis of Twin-Arginine Translocation Pathway Homologue in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

We investigated the relationship between expression of the O side chain of outer membrane lipopolysaccharide (LPS) and infection by a Shiga toxin 2 (Stx2)-converting phage in normal and benign strains of Escherichia coli. Of 19 wild-type E. coli strains isolated from the feces of healthy subjects, those with low-molecular-weight LPS showed markedly higher susceptibility to lytic and lysogenic infection by Stx2 phages than those with high-molecular-weight LPS. All lysogens produced infectious phage particles and Stx2. The Stx-negative E. coli O157:H7 strain ATCC43888 with an intact O side chain was found to be resistant to lysis by an Stx2 phage and lysogenic infection by a recombinant Stx2 phage, whereas a rfbE mutant deficient in the expression of the O side chain was readily infected by the phage and yielded stable lysogens. The evidence suggests that an O side chain deficiency leads to the creation of new pathotypes of Shiga toxin-producing E. coli (STEC) within the intestinal microflora.     

Deoxyribonucleic Acid Hybridization Analysis of the Defective Bacteriophage Carried by Strain 15 of Escherichia coli

Evidence from several laboratories indicates that strain 15 of Escherichia coli is lysogenic for a defective phage. When lysates from induced cultures were centrifuged in CsCl, three bands were obtained. In order of decreasing density, these bands contained tailless particles, complete phages, and a second band of complete phages, in a ratio of 65.7:28.6:5.7. Reassociation rate measurements were used to establish that the molecular weights of the deoxyribonucleic acid (DNA) species from the phages in the first two bands are similar. A smaller genome is postulated in the complete phages from the minor band. Hybridization experiments revealed extensive homology between the DNA species from all three phage bands, thus suggesting that the complete and tailless particles are not different at the genetic level. The DNA from each phage band was also shown to hybridize almost completely with DNA from either E. coli 15T(-) or a reportedly cured derivative of 15T(-). In contrast, only about 25% of each phage DNA was able to react with DNA from E. coli strains B and K-12 C-600.     

Para-aminobenzoic acid inhibits a set of SOS functions in Escherichia coli K12

PABA - Vitamin H1 of group B, has obtained increasing fundamental interest as a very potent natural antimutagen after a series of our publications since 1979. In the first set of our experiments, we studied PABA in the assays with the alkylating agent N-methyl-N-nitrosourea (MNU). Mutagenic efficiency of this agent was suppressed up to 10-fold when PABA was administered into Escherichia coli cells concurrently with the mutagen or prior to the mutagenic treatment. NMR spectrometric and UV-spectrophotometric measurements did not reveal an interaction between the direct acting MNU and PABA, typical for some N-nitroso compounds and phenolics. PABA suppressed the error-prone DNA repair pathway induced by UV-irradiation. PABA decreased MNU-induced phage lambda lysogenic induction more than two orders of magnitude. PABA inhibited the thermal shift up to 400-fold in phage lambda from the permissive to non-permissive temperature in E. coli mutant tif-1 and decreased about two-fold W-reactivation of UV-damaged phage lambda. Chloramphenicol treatment of the cells just after the mutagenic treatment prevented the occurrence of PABA specific activity. The results suggest that PABA affects the SOS DNA repair pathway and the mutagenic response of E. coli. PABA appears to be an effective bioantimutagen reducing mutagenesis by modulating the error-prone DNA repair (SOS) response.     

Conditionally replicating plasmid vectors that can integrate into the Klebsiella pneumoniae chromosome via bacteriophage P4 site-specific recombination.

P4 is a satellite phage of P2 and is dependent on phage P2 gene products for virion assembly and cell lysis. Previously, we showed that a virulent mutant of phage P4 (P4 vir1) could be used as a multicopy, autonomously replicating plasmid vector in Escherichia coli and Klebsiella pneumoniae in the absence of the P2 helper. In addition to establishing lysogeny as a self-replicating plasmid, it has been shown that P4 can also lysogenize E. coli via site-specific integration into the host chromosome. In this study, we show that P4 also integrates into the K. pneumoniae chromosome at a specific site. In contrast to that in E. coli, however, site-specific integration in K. pneumoniae does not require the int gene of P4. We utilized the alternative modes of P4 lysogenization (plasmid replication or integration) to construct cloning vectors derived from P4 vir1 that could exist in either lysogenic mode, depending on the host strain used. These vectors carry an amber mutation in the DNA primase gene alpha, which blocks DNA replication in an Su- host and allows the selection of lysogenic strains with integrated prophages. In contrast, these vectors can be propagated as plasmids in an Su+ host where replication is allowed. To demonstrate the utility of this type of vector, we show that certain nitrogen fixation (nif) genes of K. pneumoniae, which otherwise inhibit nif gene expression when present on multicopy plasmids, do not exhibit inhibitory effects when introduced as merodiploids via P4 site-specific integration.     

Lytic and lysogenic infection of diverse Escherichia coli and Shigella strains with a verocytotoxigenic bacteriophage

A verocytotoxigenic bacteriophage isolated from a strain of enterohemorrhagic Escherichia coli O157, into which a kanamycin resistance gene (aph3) had been inserted to inactivate the verocytotoxin gene (vt2), was used to infect Enterobacteriaceae strains. A number of Shigella and E. coli strains were susceptible to lysogenic infection, and a smooth E. coli isolate (O107) was also susceptible to lytic infection. The lysogenized strains included different smooth E. coli serotypes of both human and animal origin, indicating that this bacteriophage has a substantial capacity to disseminate verocytotoxin genes. A novel indirect plaque assay utilizing an E. coli recA441 mutant in which phage-infected cells can enter only the lytic cycle, enabling detection of all infective phage, was developed.     

Noise magnetic fields abolish the gap junction intercellular communication suppression induced by 50 hz magnetic fields

Previously, we have reported that exposure to 50 Hz coherent sinusoidal magnetic fields (MF) for 24 h inhibits gap junction intercellular communication (GJIC) in mammalian cells at an intensity of 0.4 mT and enhances the inhibition effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) at 0.2 mT. In the present study, we further explored the effects of incoherent noise MF on MF-induced GJIC inhibition. GJIC was determined by fluorescence recovery after photobleaching (FRAP) with a laser-scanning confocal microscope. The rate of fluorescence recovery (R) at 10 min after photobleaching was adopted as the functional index of GJIC. The R-value of NIH3T3 cells exposed to 50 Hz sinusoidal MF at 0.4 mT for 24 h was 30.85 +/- 14.70%, while the cells in sham exposure group had an R-value of 46.36 +/- 20.68%, demonstrating that the GJIC of NIH3T3 cells was significantly inhibited by MF exposure (P < .05). However, there were no significant differences in the R-values of the sham exposure, MF-plus-noise MF exposure (R: 49.58 +/- 19.38%), and noise MF exposure groups (R: 46.74 +/- 21.14%) (P > .05), indicating that the superposition of a noise MF alleviated the suppression of GJIC induced by the 50 Hz MF. In addition, although MF at an intensity of 0.2 mT synergistically enhanced TPA-induced GJIC inhibition (R: 24.90 +/- 13.50% vs. 35.82 +/- 17.18%, P < .05), further imposition of a noise MF abolished the synergistic effect of coherent MF (R: 32.51 +/- 18.37%). Overall, the present data clearly showed that although noise MF itself had no effect on GJIC of NIH3T3 cells, its superposition onto a coherent sinusoidal MF at the same intensity abolished MF-induced GJIC suppression. This is the first report showing that noise MF neutralizes 50 Hz MF-induced biological effect by using a signaling component as the test endpoint.     

Site-specific recombination between cloned attP and attB sites from the Haemophilus influenzae bacteriophage HP1 propagated in recombination-deficient Escherichia coli.

Plasmids were constructed which contain both attP and attB DNA segments derived from the insertion sites of the lysogenic bacteriophage HP1 and its host, Haemophilus influenzae. Similar plasmids containing the two junction segments (attL and attR regions) between the phage genome and the lysogenic host chromosome were also prepared. The formation of recombinant dimer plasmids was observed when attP-attB plasmids were propagated in Escherichia coli HB101 (recA), while plasmids containing the junction segments did not form recombinant dimers. Deletion of the phage DNA segment adjacent to the attP site from the attP-attB constructions eliminated detectable recombination, suggesting that this sequence contains the gene encoding the HP1 integrase. No plasmid recombination was observed in strains of E. coli defective in integration host factor. This suggests that integration host factor is important in the expression or activity of the system which produces the site-specific recombination of sequences derived from HP1 and H. influenzae. Further, it suggests that a protein functionally analogous to E. coli integration host factor may be present in H. influenzae.     

The Shiga-toxin VT2-encoding bacteriophage varphi297 integrates at a distinct position in the Escherichia coli genome

The plaque-forming VT2-encoding lambdoid bacteriophage varphi297 was isolated from a Belgian clinical Escherichia coli O157:H7 isolate. PCR walking, starting from the int gene of phage varphi297, demonstrated that the varphi297 prophage integrated in the yecE gene of a lysogenic E. coli K12 strain. This integration site, in E. coli K12 and in the original clinical O157:H7 isolate, was confirmed by PCR using primers flanking this site. The excisionase protein of phage varphi297 is identical to the excisionase of VT1-encoding phage VT1-Sakai, while the integrases, which are 82% identical, show significant sequence divergence in the central and C-terminal region. This can explain the different integration sites of both prophages. The activity of the integrase was proven by its ability to mediate the integration of a suicide plasmid, carrying the attachment site of varphi297, at the appropriate position in the E. coli chromosome.     

Evidence for a New Endonuclease Synthesized by λ Bacteriophage

Infection of nonlysogenic Escherichia coli CR34(S) (Thy(-)) with bacteriophage lambda C(I)857 resulted in the formation of twisted circular double-stranded phage deoxyribonucleic acid (DNA; species I). When such infected bacteria were incubated in the absence of thymine, there was a significant decrease in the amount of species I DNA after 60 min of incubation. A similar loss of species I lambda DNA during incubation in a thymine-deficient medium was also observed after infection of the endonuclease I-deficient strain, E. coli 1100(S) (Thy(-)). This destruction of twisted, circular lambda DNA in thymine-deprived cells did not occur in the presence of chloramphenicol nor in lysogenic E. coli CR34 carrying a noninducible lambda prophage. It is therefore concluded that the endonuclease which attacks this circular configuration of lambda DNA is newly synthesized after infection and is directed by the phage chromosome.     

Detection of Escherichia coli with fluorescent labeled phages that have a broad host range to E. coli in sewage water

Escherichia coli is used as an indicator microorganism in public health. The conventional way to detect E. coli requires several days to produce a result, because it requires incubation of cells. Therefore a rapid and sensitive detection method is needed. T4e-/GFP phage, characterized by suppression of lysozyme and fusion of GFP (green fluorescent protein) to its SOC (small outer capsid) protein, was constructed, and it was shown to be able to detect E. coli K12 sensitively within several hours. However, because the host range of T4 phage to E. coli present in sewage water and sea water is narrow, this phage cannot be used to detect E. coli in environmental water. Two phages named IP008 and IP052, which have a broad host range to E. coli present in sewage influent, were screened from sewage influent. Mixture of these two phages produced clear plaques on 50% of E. coli screened from sewage influent. To use these phages as a tool for detection of E. coli, gfp was inserted into gene e, which encodes a lytic enzyme, and thus lytic-activity-suppressed phages were constructed (IP008e-/GFP and IP052e-/GFP). However, the fluorescent intensity of E. coli cells infected with IP008e-/GFP and IP052e-/GFP was not enough for visualization of the cell. Therefore, in addition to the insertion of gfp into gene e, fusion of GFP to SOC of IP008e-/GFP and IP052e-/GFP was conducted to produce IP008e-/2xGFP and IP052e-/2xGFP. E. coli cells infected with IP008e-/2xGFP and IP052e-/2xGFP showed much stronger fluorescence intensity than E. coli cells infected by IP008e-/GFP and IP052e-/GFP. It is anticipated that, using these GFP-labeled phages, a broad range of E. coli present in sewage influent water can be detected rapidly.     

Transposition of the phage D3112 genome in Escherichia coli cells

It has been demonstrated that the genome of phage D3112 of Preudomonas aeruginosa can be transposed into Escherichia coli chromosome as a component of the hybrid plasmid RP4 TcrKms::D3112. Also, transposition of D3112 from E. coli (D3112) chromosome into RP4 plasmid occurs. The phage stimulates the chromosome mobilizing activity of RP4 plasmid, similar to other transposons. E. coli (RP4::D3112) cells were previously shown to form no colonies at 30 degrees C. Auxotrophic mutants and mutants incapable of utilizing different carbohydrates were found among E. coli clones survived after a long incubation at 30 degrees C (at frequencies approximately 10(-3) - 10(-4). These mutants inherited stably the capability to produce D3112 phage. E. coli auxotrophic mutants have arisen indeed as a consequence of phage integration into the E. coli chromosome, since prototrophic transductants derived from these mutants after their treatment with generalized transducing P1 phage have lost the ability to produce D3112 phage. Clones with mutations in Km or Tc genes of RP4 plasmid, occurring at high frequencies (about 3%) were found after introduction of RP4 into E. coli (D3112). These mutant RP4 plasmids carry insertions of D3112 genomes. Clones of E. coli which lost mutant plasmids still produce D3112 and retain their initial auxotrophic mutations.