Phospholipid metabolism in Pseudomonas BAL-31 infected with lipid-containing bacteriophage PM2.

Infection of Pseudomonas BAL-31 with the lipid-containing bacteriophage PM2 resulted in no detectable change in the rate of phosphatidylglycerol (PG) or phosphatidylethanolamine (PE) biosynthesis. An increase in the PG content of infected cultures was not seen until the cultures began to lyse, and this increase was in fact only a relative increase resulting from the extensive turnover of PE at the onset of culture lysis. Turnover studies revealed that the glycerol, phosphorus fatty acid, and ethanolamine moieties of PE turned over simultaneously at the time of lysis, and therefore made it unlikely that there was a PE to PG conversion during the latent period of the phage. The lipid found in the bacteriophage did not reflect a preferential selection for lipid synthesized before or after infection, but in fact reflected the composition of the host membrane at the time the phage were assembled. The use of a modified medium that allowed the cultivation of Pseudomonas BAL-31 as a prototroph and resulted in reliable lysis times of infected cultures led us to the conclusion that PM2 infection effects little change in host phospholipid metabolism, and that there is sufficient PG in the host cytoplasmic membrane to account for a full burst of phage. As a result of the reliable lysis times that we have achieved, we concluded that certain metabolic events, i.e., PE turnover, are lytic phenomena and must not be confused with events relevant to the biosynthesis and maturation of the phage.     

Infection of spheroplasts of Pseudomonas with DNA of bacteriophage PM2

Summary Spheroplasts of Pseudomonas BAL-31/PM2, obtained by treatment of the bacteria with lysozyme, can be infected with purified DNA from bacteriophage PM2. After 4 h of incubation the yield of progeny phage reaches a value of 10⁷-6×10⁷ plaque forming units/g PM2 DNA. The yield increases linearly with the concentration of DNA over at least 3 orders of magnitude.The biological activity of double-stranded circular PM2 DNA containing one or more single-strand breaks per molecule (component II), does not differ significantly from that of intact PM2 DNA (component I). Single-stranded PM2 DNA obtained by denaturation of component II, and the irreversible alkali-denatured form of component I are also infective.     

Calcium requirement for assembly of the lipid-containing bacteriophage PM2

The bacteriophage PM2 requires extracellular Ca²⁺ at concentrations greater than 3 · 10⁻⁴ M for the production of viable virus, whereas the host cell Pseudomonas BAL-31 grows normally in medium containing 3 · 10⁻⁵ M Ca²⁺ (low calcium). Virus attachment occurs normally in low calcium, the infected cultures partially lyse, but no infectious virus particles are released. Sucrose gradient analysis shows that lysates made in low calcium contain no PM2-like particles. The addition of calcium very late in the infectious cycle completely restores virus production to cultures infected in low calcium, whereas removal of calcium after infection prevents virus production. Our experiments indicate that Ca²⁺ is essential for some process late in the lytic cycle, such as the final assembly of stable, infectious PM2 particles.     

Effect of lipid alkyl chain perturbations on the assembly of bacteriophage PM2.

The lipid-containing bacteriophage PM2 can produce infectious virus in cultures infected at temperatures up to 31.5 degrees C, but not at 34 degrees C. Its host, Pseudomonas BAL-31, grows at 34 degrees C and cultures infected at that temperature undergo lysis. Sucrose-gradient analysis shows that 34 degrees C lysates contain no PM2-like particles. Temperature-shift experiments establish that the thermally sensitive process is late in infection when virus assembly is taking place. Adamantanone, a small hydrophobic molecule that perturbs membrane hydrocarbon zones, prevents the production of infective virus. Concentrations which prevent virus production have no effect on host-cell growth or stability of mature virions. Adamantanone exerts its effects late in the infectious cycle, and lysates amde in its presence contain no PM2-like particles. These experiments, carried out at 25 degrees C, indicate that adamantanone prevents the assembly of stable PM2 virus. Spin-label studies suggest that the lipid alkyl chains of the host-cell membrane are in an "ordered" state at temperatures below about 33 degrees C and undergo a transition to a "disordered" state above that temperature. Furthermore, the addition of adamantanone perturbs the hydrocarbon zones, producing a greater degree of disorder even below 25 degrees C. Our findings suggest that the cell membrane can function and grow with the lipid alkyl chains in either the "ordered" or "disordered" state, but that the "ordered" state must be maintanined for PM2 assembly to occur.     

Cation Fluxes and Permeability Changes Accompanying Bacteriophage Infection of Escherichia coli

Infection of Escherichia coli by bacteriophage T2 was accompanied by a rapid but transient increase in the rate of loss of small molecules from the bacterial cells. This transient leakage was studied with radioactive labels such as (42)K and (28)Mg. Bacteriophage-induced leakage was dependent on the ratio of phage to bacteria: the higher the multiplicity of infection, the greater the leakage. No leakage occurred at 4 C [when adsorption proceeds but injection of phage deoxyribonucleic acid (DNA) is blocked]. Leakage was caused by heavily irradiated phage as well as by normal phage; therefore, the intracellular functioning of the bacteriophage DNA was not required. This conclusion was supported by experiments which showed phage-induced leakage in the presence of chloramphenicol or sodium cyanide. Leakage could be prevented by infecting the bacteria with phage in the presence of high magnesium concentrations. Phage-induced leakage was terminated by a "sealing" reaction, after which potassium turnover by infected and uninfected cells was very similar. The sealing reaction occurred even in the presence of chloramphenicol, suggesting that the sealing is controlled by bacterial and not bacteriophage genes. We were not able to detect any effect of normal bacteriophage infection on the influx (active transport) of potassium and magnesium into the cells.     

Effect of lipid alkyl chain perturbations on the assembly of bacteriophage PM2

The lipid-containing bacteriophage PM2 can produce infectious virus in cultures infected at temperatures up to 31.5 °C, but not at 34 °C. Its host, Pseudomonas BAL-31, grows at 34 °C and cultures infected at that temperature undergo lysis. Sucrose-gradient analysis shows that 34 °C lysates contain no PM2-like particles. Temperature-shift experiments establish that the thermally sensitive process is late in infection when virus assembly is taking place.Adamantanone, a small hydrophobic molecule that perturbs membrane hydrocarbon zones prevents the production of infective virus. Concentrations which prevent virus production have no effect on host-cell growth or stability of mature virions. Adamantanone exerts its effects late in the infectious cycle, and lysates made in its presence contain no PM2-like particles. These experiments, carried out at 25 °C, indicate that adamantanone prevents the assembly of stable PM2 virus.Spin-label studies suggest that the lipid alkyl chains of the host-cell membrane are in an “ordered” state at temperatures below about 33 °C and undergo a transition to a “disordered” state above that temperature. Furthermore, the addition of adamantanone perturbs the hydrocarbon zones, producing a greater degree of disorder even below 25 °C. Our findings suggest that the cell membrane can function and grow with the lipid alkyl chains in either the “ordered” or “disordered” state, but that the “ordered” state must be maintained for PM2 assembly to occur.     

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.     

Isolation and characterization of a temperate bacteriophage from the ruminal anaerobe Selenomonas ruminantium

A temperate bacteriophage was obtained from an isolate of the ruminal anaerobe Selenomonas ruminantium. Clear plaques that became turbid on further incubation occurred on a lawn of host bacteria. Cells picked from a turbid plaque produced healthy liquid cultures, but these often lysed on storage. Mid-log-phase liquid cultures incubated with the bacteriophage lysed and released infectious particles with a titer of up to 3 X 10(7) PFU/ml. A laboratory strain of S. ruminantium, HD-4, was also sensitive to this bacteriophage, which had an icosohedral head (diameter, 50 nm) and a flexible tail (length, 140 nm). The bacteriophage contained 30 kilobases of linear, double-stranded DNA, and a detailed restriction map was constructed. The lysogenic nature of infection was demonstrated by hybridization of bacteriophage DNA to specific restriction fragments of infected host genomic DNA and by identification of a bacteriophage genomic domain which may participate in integration of the bacteriophage DNA. Infection of S. ruminantium in vitro was demonstrated by two different methods of cell transformation with purified bacteriophage DNA.     

Isolation and characterization of a temperate bacteriophage from the ruminal anaerobe Selenomonas ruminantium.

A temperate bacteriophage was obtained from an isolate of the ruminal anaerobe Selenomonas ruminantium. Clear plaques that became turbid on further incubation occurred on a lawn of host bacteria. Cells picked from a turbid plaque produced healthy liquid cultures, but these often lysed on storage. Mid-log-phase liquid cultures incubated with the bacteriophage lysed and released infectious particles with a titer of up to 3 X 10(7) PFU/ml. A laboratory strain of S. ruminantium, HD-4, was also sensitive to this bacteriophage, which had an icosohedral head (diameter, 50 nm) and a flexible tail (length, 140 nm). The bacteriophage contained 30 kilobases of linear, double-stranded DNA, and a detailed restriction map was constructed. The lysogenic nature of infection was demonstrated by hybridization of bacteriophage DNA to specific restriction fragments of infected host genomic DNA and by identification of a bacteriophage genomic domain which may participate in integration of the bacteriophage DNA. Infection of S. ruminantium in vitro was demonstrated by two different methods of cell transformation with purified bacteriophage DNA.     

Lysogenization of Escherichia coli him+, himA, and himD hosts by bacteriophage Mu.

The possible outcomes of infection of Escherichia coli by bacteriophage Mu include lytic growth, lysogen formation, nonlysogenic surviving cells, and perhaps simple killing of the host. The influence of various parameters, including host himA and himD mutations, on lysogeny and cell survival is described. Mu does not grow lytically in or kill him bacteria but can lysogenize such hosts. Mu c+ lysogenizes about 8% of him+ bacteria infected at low multiplicity at 37 degrees C. The frequency of lysogens per infected him+ cell diminishes with increasing multiplicity of infection or with increasing temperature over the range from 30 to 42 degrees C. In him bacteria, the Mu lysogenization frequency increases from about 7% at low multiplicity of infection to approach a maximum where most but not all cells are lysogens at high multiplicity of infection. Lysogenization of him hosts by an assay phage marked with antibiotic resistance is enhanced by infection with unmarked auxiliary phage. This helping effect is possible for at least 1 h, suggesting that Mu infection results in formation of a stable intermediate. Mu immunity is not required for lysogenization of him hosts. We argue that in him bacteria, all Mu genomes which integrate into the host chromosome form lysogens.     

Effect of lysogeny on transfection and transfection enhancement in Bacillus subtilis.

Strains of Bacillus subtilis 168 lysogenic for bacteriophage phi105 transfer with deoxyribonucleic acid (DNA) isolated from bacteriophage SPO2 at a higher efficiency than non-lysogenic strains. This enhancement of transfection was not the result of recombination between bacteriophages SPO2 and phi105. Superinfection marker rescue increased transfection with DNA from bacteriophage phi105 occurred simultaneously with the addition of the transfecting DNA. Again, this enhancement of transfection was not the result of recombination but rather a protection of the transfecting DNA by the superinfecting bacteriophage. The ability of the superinfecting bacteriophage to protect the transfecting DNA from inactivation was maximal when the bacteria were just becoming competent. Bacteriophage phi1 cannot replicate after the transfection of competent bacteria lacking a functional DNA replication system, whereas bacteriophage phi1 was able to replicate after infection of competent bacteria grown under comparable conditions. These observations support the hypothesis that GAPase and an inducible repair system play an important role in the development of competence.     

fii, a bacterial locus required for filamentous phage infection and its relation to colicin-tolerant tolA and tolB.

We describe mutations in a new bacterial locus, designated fii, which do not allow the filamentous bacteriophage f1 to infect bacteria harboring the F plasmid. Mutations at this locus do not affect the ability of F plasmid-containing bacteria to undergo conjugation or be infected by the F plasmid-specific RNA phage f2. The filamentous phage can still adsorb to the F sex pilus, but the DNA is unable to enter the bacteria. All fii mutants become tolerant to colicins E1, E2, and E3. Strains with amber mutations in fii also are unable to plaque P1, even though they can be infected with this phage. Mutations in fii also prevent infection of bacteria harboring the N plasmid by the filamentous bacteriophage IKe. The fii locus maps adjacent to tolA, mutants of which demonstrate tolerance to high levels of the E and K colicins. The three genes tolA, tolB, and fii are shown to reside on a 4.3-kilobase fragment of the Escherichia coli chromosome. Each gene has been cloned into a chimeric plasmid and shown to complement, in trans, mutations at the corresponding chromosomal locus. Studies in maxicells show that the product of fii appears to be a 24-kilodalton protein which copurifies with the cell envelope. The product of tolA has been identified tentatively as a 51-kilodalton protein. Data from cloning, Tn5 mutagenesis, and P1 transduction studies are consistent with the gene order sucA-fii-tolA-tolB-aroG near 17 min on the E. coli map.     

Replication of bacteriophage f1: A complex containing gene II protein in gene V mutant-infected bacteria

A dense complex has been isolated from bacteria infected with gene V amber mutant f 1 bacteriophage. The major protein in this complex is the f 1 bacteriophage-specific gene II protein. Other proteins in the complex include the f 1 bacteriophage coat protein and proteins which migrate on sodium dodecyl sulfate/polyacrylamide gel electrophoresis with the f1 bacteriophage-specific gene III, gene IV and X protein. A protein of approximately 20,000 M_r is also present in the complex. Examination of bacteria infected with gene V mutant f1 bacteriophage revealed the complex as a densely staining amorphous body which appears to be associated with the cytoplasmic membrane. Bacteria infected with f1 bacteriophage that contain amber mutations in genes other than gene V do not contain this complex.     

A novel lysis system in PM2, a lipid-containing marine double-stranded DNA bacteriophage

In this study we investigated the lysis system of the lipid-containing double-stranded DNA bacteriophage PM2 infecting Gram-negative marine Pseudoalteromonas species. We analysed wt and lysis-deficient phage-induced changes in the host physiology and ascribed functions to two PM2 gene products (gp) involved in lysis. We show that bacteriophage PM2 uses a novel system to disrupt the infected cell. The novelty is based on the following findings: (i) gp k is needed for the permeabilization of the cytoplasmic membrane and appears to play the role of a typical holin. However, its unique primary structure [53 aa, 1 transmembrane domain (TMD)] places it into a new class of holins. (ii) We have proposed that, unlike other bacteriophages studied, PM2 relies on lytic factors of the cellular origin for digestion of the peptidoglycan. (iii) gp l (51 aa, no TMDs) is needed for disruption of the outer membrane, which is highly rigidified by the divalent cations abundant in the marine environment. The gp l has no precedent in other phage lytic systems studied so far. However, the presence of open reading frame l-like genes in genomes of other bacterial viruses suggests that the same system might be used by other phages and is not unique to PM2.     

工业微生物的噬菌体感染与防治策略

以细菌为基础的生物技术在蓬勃发展的同时也不断受到噬菌体感染的威胁,噬菌体感染已成为微生物发酵过程中的一个顽疾,其实质是噬菌体与细菌之间复杂的共进化关系。在漫长的进化过程中,噬菌体已经形成了多种针对细菌抗性系统的逃逸机制。合理的工厂设计、菌株的轮换策略和传统的基因工程方法能在一定程度上降低噬菌体感染的风险,但仍然无法避免。基于CRISPR-Cas系统的防治策略仅需噬菌体的序列信息就可以理性设计噬菌体抗性菌株,且可以通过叠加效应不断增强菌种抗性,从而避免噬菌体的逃逸;群体感应信号分子则可以从整体水平上调节细菌的噬菌体抗性。这些新发现为噬菌体感染问题的解决带了新的希望,而噬菌体基因组编辑技术和合成生物学的快速发展则将进一步加深人们对噬菌体感染防治领域的认识。     

Rapid and sensitive detection method of a bacterium by using a GFP reporter phage

A rapid, sensitive, and convenient method for detecting a specific bacterium was developed by using a GFP phage. Here we describe a model system that utilizes the temperate Escherichia coli-restricted bacteriophage lambda, which was genetically modified to express a reporter gene for GFP to identify the colon bacillus E. coli in the specimen. E. coli infected with GFP phage was detected by GFP fluorescence after 4-6 hr of incubation. The results show that a few bacteria in a specimen can be detected under fluorescence microscopy equipped with a sensitive cooled CCD camera. When E. coli and Mycobacterium smegmatis were mixed in a solution containing GFP phage, only E. coli was infected, indicating the specificity of this method. The method has the following advantages: 1) Bacteria from biological samples need not be purified unless they contain fluorescent impurities; 2) The infection of GFP phage to bacteria is specific; 3) The fluorescence of GFP within infected bacteria enables highly sensitive detection; 4) Exogenous substrates and cofactors are not required for fluorescence. Therefore this method is suitable for any phage-bacterium system when bacteria-specific phages are available.     

Use of Miracil D to Suppress Bacterial Ribonucleic Acid and Protein Synthesis During Bacteriophage MS2 Infection

Under certain culture conditions, Miracil (35 mug/ml) halts the growth of uninfected Escherichia coli. Cellular ribonucleic acid (RNA) synthesis is almost completely suppressed, whereas deoxyribonucleic acid and protein synthesis are inhibited to a lesser extent. When the drug is added to host bacteria prior to infection with bacteriophage MS2, the phage adsorb to the cells, but penetration of the viral RNA is inhibited. Penetration may be achieved without further viral development by infection in the presence of chloramphenicol. If the bacteria are infected with MS2 in the presence of chloramphenicol, subsequently washed to remove the chloramphenicol, and then treated with Miracil at any time between 0 and 20 min postinfection, a second viral function is inhibited and the yield of progeny phage is reduced. Addition of the drug after 20 min postinfection does not inhibit the infection process. When Miracil is present from early times in infection, only a limited synthesis of both double- and single-stranded virus-specific RNA is observed. The viral RNA species thus produced do not appear to differ from those made in the absence of the drug. A comparison of the activities of the viral RNA synthetase produced during the course of infection in the presence and in the absence of Miracil suggests that a possible cause of the inhibition is the synthesis of an unstable enzyme in the presence of the drug.     

Comparison of the frequency of imi-r resistant Pseudomonas aeruginosa mutants generated by infection of the bacteria with various bacteriophage-transposons]

We compared the frequencies of imipenem-resistant (imi-r) mutants of a Pseudomonas aeruginosa laboratory strain PAO1 infected by the phages-transposons (PT) specific for this pseudomonad species. The frequency of imi-r mutants among the lysogenic bacteria that appeared after infection reflects the frequency of integrative (conserved) PT transposition into ompD2 gene responsible for synthesis of porin, the protein required for the passage of antibiotic through the cell membrane. After infection by either PT the proportion of imi-r mutants among the lysogenic bacteria was higher than that of spontaneous mutants. The imi-r mutants induced by PT infection form colonies that differ in morphology when grown on different media. The frequencies of imi-r mutants induced by all PT are similar, except for HW12, PM57, and PM62 assigned to a species of the group B3. The phages of this species induce imi-r mutants at a high frequency. Variations in frequencies and colony morphology of imi-r mutants are discussed.     

Interaction of Yersinia pestis bacteria with bacteriophage mu

Yersinia pestis cells are shown to be sensitive to bacteriophage Mu cts62 infection. Lysis of bacteria has been shown to be more efficient on solid nutrient medium than in LB broth. 10(-5) pfu per ml is the maximal concentration of bacteriophage particles yielded from the broth cultures of bacteria. Moi 0.1 has been used to obtain such yields of bacteriophage. Lysogenization of Yersinia pestis cells has not been achieved when the standard methods of bacteriophage infection have been used. It was accomplished by the conjugal transfer of plasmid RP4::MU cts62 to Yersinia pestis from Escherichia coli. The deficiency of Yersinia pestis in producing bacteriophage Mu cts62 mature particles during the lytic cycle of bacteriophage is discussed.