The effect of the IncP-2-group plasmid on the growth of Pseudomonas aeruginosa bacteriophages

The influence of plasmids of the IncP-2 group on development of bacteriophages of Pseudomonas aeruginosa was studied. Six different types of phage growth inhibition conferred by natural plasmids of the IncP-2 group were found. All these plasmids were shown to have no effect on adsorption and injection of phage DNA into cells, only blocking intracellular phage development. The differences between phage inhibition mechanisms were shown by comparison of efficiency of colony formation by cells containing different plasmids, in the presence of different phages. The presence of the RpL11 plasmid reduces the frequency of lysogenization with G101 phage but not with B3 phage. The mutants of pMG53 plasmid having modified phage inhibition spectrum were obtained. It was inferred that inhibition of different phages is under control of different loci of this plasmid. The mutants of phage B3 overcoming inhibition by plasmids were obtained. It was supposed that the plasmids act at least at three different sites of the phage B3 genome.     

Influence of the Chitosan Oligomer on the Phage Particles and Reproduction of Phage 1-97A in the Culture of Bacillus thuringiensis

The causes of bacteriophage 1-97A inactivation by the chitosan oligomer with a polymerization degree of 15 and the influence of the oligomer on the phage reproduction in the culture of Bacillus thuringiensissubsp. galleriae, strain 1-97, were studied. The study of the inactivation kinetics showed that, in 1 h, virtually all chitosan was bound to the phage particles, causing, as evidenced by electron microscopy, DNA release from the phage head, destruction of the phage particles, and agglutination of the phage particles or of their tails in the region of the basal plate. High-polymeric chitosan caused more pronounced destruction of the phage particles than the oligomer. It was established that chitosan prevented the production of complete phage particles. One of the mechanisms of such an influence may be the production in the presence of chitosan of phage particles devoid of DNA.     

A second function of the S gene of bacteriophage lambda

Infection of Escherichia coli by bacteriophage lambda caused an immediate inhibition of uptake by members of all three classes of E. coli active transport systems and made the inner membrane permeable to sucrose and glycine; however, infection stimulated alpha-methyl glucoside uptake. Phage infection caused a dramatic drop in the ATP pool of the cell, but the membrane did not become permeable to nucleotides. Infection by only one phage per cell was sufficient to cause transport inhibition. However, adsorption of phage to the lambda receptor did not cause transport inhibition; DNA injection was required. The inhibition of transport caused by lambda phage infection was transient, and by 20 min after infection, transport had returned to its initial level. The recovery of transport activity appeared to require a lambda structural protein with a molecular weight of 5,500. This protein was present in wild-type phage and at a reduced level in S7 mutant phage but was missing in S2 and S4 mutant phage. Cells infected with S7 phage had a partial recovery of active transport, whereas cells infected with S2 or S4 phage did not recover active transport. Neither the inhibition of transport caused by phage infection nor its recovery were affected by the protein synthesis inhibitors chloramphenicol and rifampin.     

Isolation of the Bacteriophage Lambda Receptor from Escherichia coli

A factor which inactivates the phage lambda can be extracted from Escherichia coli. This factor is a protein and is located in the outer membrane of the bacterial envelope. It is found in extracts of strains which are sensitive to phage lambda, but not in extracts of strains specifically resistant to this phage. We conclude that this factor is the lambda receptor, responsible for the specific adsorption of the phage lambda to E. coli cells. A partial purification of the lambda receptor is described. Inactivation of the phage by purified receptor is shown to be accompanied by the release of deoxyribonucleic acid from the phage.     

Effect of ozone on bacteriophages in the phage--host-cell system

The antiviral action of ozone was studied using Escherichia coli K-12 AB1157 virulent phage T4 and Pseudomonas aeruginosa PAO1 temperate phage SM as models depending on the phage state during the action: a free phage, a phage in the presence of sensitive host cells, or a vegetative phage. Bacteriophages T4 and SM were found to be much more sensitive to ozone as compared to the bacterial strains AB1157 and PAO1. The latter protected phage particles against the activation by ozone at a concentration which effectively inactivated these phages in the absence of the bacteria. Ozone also exerted an inhibiting effect on vegetative phage SM, and the degree of inhibition decreased with the termination of intracellular growth stages.     

Factors Influencing the Adsorption of Bacteriophage 2 to Cells of Pseudomonas aeruginosa

Phage 2 adsorbed to Pseudomonas aeruginosa strain BI in 5 mM Tris buffer, providing that cations like Na(+), Mg(2+), or Ca(2+) were present. Adsorption was observed over a broad pH range, reaching a maximum level around pH 7.5, which coincided with the pH required for maximal activity of the phage 2-associated slime polysaccharide depolymerase. Mutants of strain BI and other strains of P. aeruginosa possessing slime layers that were devoid of phage 2 depolymerase substrate were incapable of adsorbing phage 2. On the other hand, those strains containing substrate for the phage 2 depolymerase in the slime layer were capable of adsorbing phage 2. The same relationship of phage depolymerase-substrate interaction to phage adsorption was observed with Pseudomonas phage 8, which possesses a depolymerase that differs in its specificity from the phage 2 depolymerase. The receptor-like activity of purified slime containing the specific substrate for the phage-associated depolymerase was demonstrable by its ability to inactivate phage. However, receptor-like activity or phage inactivation was not observed with those slimes that were devoid of the depolymerase substrate.     

Roles of Exopolysaccharides and Lipopolysaccharides in the Adsorption of the Siphovirus Phage NM8 to Rhizobium meliloti M11S Cells

The exopolysaccharides produced by Rhizobium meliloti M11S inhibited nonspecifically the adsorption of phage NM8 by coating the cells. But lipopolysaccharides (LPS) had a specific inhibitory effect. Only the polysaccharide moiety of LPS, composed of glucose, glucosamine, galactose, 3-deoxy-D-manno-octulosonic acid (KDO), and large amounts of sialic acid, inhibited phage adsorption; neither the lipid A moiety nor a cellular glucan was involved. Rhizobium strains lacking sialic acids did not bind phage NM8. Inhibition of phage binding by lectin specific for N-acetylneuraminic acid demonstrated that phage NM8 bound to sialic acids. Preincubation of the phage with monosaccharides showed that inactivation of phage was very stereospecific for N-acetylneuraminic acid. Phage adsorption was also strongly inhibited by N-acetylglucosamine, which is not present in the LPS. Therefore, the receptor for phage NM8 appears to be a saccharide site, probably involving the acetyl groups of sialic acids. Received: 8 March 1996 / Accepted: 29 June 1996     

Mapping of the c-region of phage phi80

A set of c-mutants of the phage phi80 is isolated. These mutants fit into three genes. According to plaque morphology and frequency of lysogenization of mutants, the genes were named cI, cII and cIII as it was previously done for phage lambda. Their order, determinated by mutant phage crosses, is cIII-sus326-cI-cII-sus250. Sus326 is a mutation in the gene 15, so it is probably an analogue of the N gene of the phage lambda. Thermoinducible mutants of the phage phi80 cts11 and cts12 correspond to the mutant types cItsB and cItsA of the phage lambda and they complement each other. Thus, it is supposed that phi80 phage repressor molecules consist of few protein subunits.     

Structural and energetic basis of infection by the filamentous bacteriophage IKe

Filamentous phage use the two N‐terminal domains of their gene‐3‐proteins to initiate infection of Escherichia coli. One domain interacts with a pilus, and then the other domain binds to TolA at the cell surface. In phage fd, these two domains are tightly associated with each other, which renders the phage robust but non‐infectious, because the TolA binding site is inaccessible. Activation for infection requires partial unfolding, domain disassembly and prolyl isomerization. Phage IKe infects E. coli less efficiently than phage fd. Unlike in phage fd, the pilus‐ and TolA‐binding domains of phage IKe are independent of each other in stability and folding. The site for TolA binding is thus always accessible, but the affinity is very low. The structures of the two domains, analysed by X‐ray crystallography and by NMR spectroscopy, revealed a unique fold for the N‐pilus‐binding domain and a conserved fold for the TolA‐binding domain. The absence of an activation mechanism as in phage fd and the low affinity for TolA probably explain the low infectivity of phage IKe. They also explain why, in a previous co‐evolution experiment with a mixture of phage fd and phage IKe, all hybrid phage adopted the superior infection mechanism of phage fd.     

A Survey of the bacteriophage WO in the endosymbiotic bacteria Wolbachia

Bacteriophages are common viruses infecting prokaryotes. In addition to their deadly effect, phages are also involved in several evolutionary processes of bacteria, such as coding functional proteins potentially beneficial to them, or favoring horizontal gene transfer through transduction. The particular lifestyle of obligatory intracellular bacteria usually protects them from phage infection. However, Wolbachia, an intracellular alpha-proteobacterium, infecting diverse arthropod and nematode species and best known for the reproductive alterations it induces, harbors a phage named WO, which has recently been proven to be lytic. Here, phage infection was checked in 31 Wolbachia strains, which induce 5 different effects in their hosts and infect 25 insect species and 3 nematodes. Only the Wolbachia infecting nematodes and Trichogramma were found devoid of phage infection. All the 25 detected phages were characterized by the DNA sequence of a minor capsid protein gene. Based on all data currently available, phylogenetic analyses show a lack of congruency between Wolbachia or insect and phage WO phylogenies, indicating numerous horizontal transfers of phage among the different Wolbachia strains. The absence of relation between phage phylogeny and the effects induced by Wolbachia suggests that WO is not directly involved in these effects. Implications on phage WO evolution are discussed.     

Dynamic relocalization of phage phi 29 DNA during replication and the role of the viral protein p16.7

We have examined the localization of DNA replication of the Bacillus subtilis phage phi 29 by immunofluorescence. To determine where phage replication was localized within infected cells, we examined the distribution of phage replication proteins and the sites of incorporation of nucleotide analogues into phage DNA. On initiation of replication, the phage DNA localized to a single focus within the cell, nearly always towards one end of the host cell nucleoid. At later stages of the infection cycle, phage replication was found to have redistributed to multiple sites around the periphery of the nucleoid, just under the cell membrane. Towards the end of the cycle, phage DNA was once again redistributed to become located within the bulk of the nucleoid. Efficient redistribution of replicating phage DNA from the initial replication site to various sites surrounding the nucleoid was found to be dependent on the phage protein p16.7.     

Orientation of the DNA in the filamentous bacteriophage f1

The filamentous bacteriophage f1 consists of a molecule of circular single-stranded DNA coated along its length by about 2700 molecules of the B protein. Five molecules of the A protein and five molecules of the D protein are located near or at one end of the virion, while ten molecules of the C protein are located near or at the opposite end. The two ends of the phage can be separated by reacting phage fragments, which have been generated by passage of intact phage through a French press, with antibody directed against the A protein (Grant et al., 1981a). By hybridizing the DNA isolated from either end of ³²P-labeled phage to specific restriction fragments of fl replicative form I DNA, we have determined that the single-stranded DNA of the filamentous bacteriophage f1 is oriented within the virion. For wild-type phage, the DNA that codes for the gene III protein is located at the A and D protein end and that which corresponds to the intergenic region is located close to the C protein end of the particle. The intergenic region codes for no protein but contains the origins for both viral and complementary strand DNA synthesis. Analysis of the DNA orientation in phage in which the plasmid pBR322 has been inserted into different positions within the intergenic region of fl shows that the C protein end of all sizes of filamentous phage particles appears to contain a common sequence of phage DNA. This sequence is located near the junction of gene IV and the intergenic region, and probably is important for normal packaging of phage DNA into infectious particles. There appears to be no specific requirement for the origins of viral and complementary strand DNA synthesis to be at the end of a phage particle.     

Localization and functional role of the pseudomonas bacteriophage 2 depolymerase.

The adsorption apparatus of phage 2 consits of a symmetrical base plate of snowflake appearance, composed of six droplike spikes 7.0 to 7.5 nm in length with a maximum diameter of 4.5 to 5.0 nm. The spikes are attached by their narrow ends to a central ring 7.0 to 7.5 nm in diameter. Phage 2 deopolymerase, a phage 2-induced hydrolytic enzyme, was found to be a structural protein of phage 2 or in close association with the base plate. Pdp1, a phage 2 mutant, possesses a polypeptide that is antigenically similar to the depolymerase, but devoid of hydrolytic activity. This polypeptide was found to be located in the region of the base plate of pdp1. Treatment of intact cells of strain BI with purified phage 2 depolymerase inhibited the adsorption of phage 2. When phage receptor-containing fractions of slime glycolipoprotein and lipopolysaccharide were hydrolyzed by the depolymerase, amino sugars were released, and the phage-inactivating activities of these fractions were lost. The depolymerase was also observed to induce the lysis of strain BI cells in hypotenic medium. The phage 2 depolymerase appears to play a role in adsorption and release of phage.     

The inhibitory effect of dithiothreitol on the assembly of the filamentous phage fd

Assembly of the filamentous phage fd is preceded by the formation of a complex between the viral single-stranded (ss) DNA and the virally coded gene 5 protein (gene 5 protein-ssDNA complex). The presence of 5 mM dithiothreitol in the growth medium prevents phage production; however, phage infection, phage DNA replication and phage genome expression are still observed. In contrast, the gene 5 protein-ssDNA complex is not formed in the presence of dithiothreitol in vivo, although the complex is not affected by the disulfide reducing agent in vitro. Furthermore, host lipid composition is altered by growth in the presence of dithiothreitol. The zwitterionic lipid, phosphatidylethanolamine, increases while the cationic phospholipid content, cardiolipin and phosphatidylglycerol, decreases. This suggests a role for lipids or membranous structures in the process of gene 5 protein-ssDNA complex formation.     

Interchangeability of the adsorption proteins of bacteriophages Ff and IKe.

The wild-type adsorption protein (g3p) of filamentous phage IKe cannot be exchanged with its analogous protein in the related Ff (M13, fd, and f1) phage particles. Deletion mutants of the protein, however, are assembled into Ff phage particles. These hybrid Ff phage particles bearing deleted IKe g3p attach to N pili, thus conserving the host attachment property of the protein but not its infection-initiating function. This means that the attachment specificity is determined by IKe g3p independently of other phage components in contact with it. Infection initiation function, the process in which phage DNA is released into the host, in contrast seems to require either more complex structural features of the protein (for example, a certain oligomeric structure) provided only in the original particle, or a concerted action of g3p with another particle component, not replaceable by its homologous counterpart in the related phage.     

Control of the Replication Complex of Bacteriophage P22

A replication complex for the vegetative synthesis of the deoxyribonucleic acid (DNA) of the temperate phage P22 previously has been described. This complex is an association of parental phage DNA, most of the newly synthesized phage DNA made during pulses with (3)H-thymidine, and other cell constituents, and has a sedimentation rate in neutral sucrose gradients of at least 1,000S. The complex is one of the intermediates, intermediate I, in the synthesis and maturation of phage P22 DNA after infection or induction. Evidence supporting the replicative nature of intermediate I is presented. Phage replication is repressed in lysogenic bacteria. On superinfection of P22 lysogens with nonvirulent phage, little association of the input phage DNA with a rapidly sedimenting fraction is demonstrable. However, after induction with ultraviolet light, the superinfecting parental phage DNA quickly acquires the rapid sedimentation rate characteristic of intermediate I; phage DNA synthesis follows; and progeny phages are produced. Infection with a virulent mutant of P22 produces progeny phages in lysogens. Its DNA associates with intermediate I. In mixed infection with the virulent phage, replication of nonvirulent phage P22 is still repressed, even though the virulent replicates normally. The nonvirulent input DNA does not associate with intermediate I. The repressor of the lysogenic cell prevents replication by interfering with the physical association of template material with intermediate I. A phage function is required for association of phage template with the replication machinery.     

Natural variability and diurnal fluctuations within the bacteriophage population of the rumen.

To investigate the impact of nutritional and environmental factors on bacteriophage activity in the rumen, it is first valuable to determine the extent of natural variations and fluctuations in phage populations from different animal species, and from animals located together and separately, and variation in animals over time. Differences in phage populations between sheep on different diets, between sheep and goats, and within the rumen over time were investigated by using pulsed-field gel electrophoresis and comparing total phage DNA in ruminal fluid. It was found that no two individuals had similar DNA banding patterns, even when similarly fed and penned together, indicating there is considerable individual diversity in phage populations between animals. Despite these individual differences, the quantities, but not the banding patterns, of phage DNA were similar for animals within groups but varied between groups, suggesting that nutritional factors may influence overall phage activity in the rumen. In sheep fed once daily, a distinct diurnal variation in the phage population was observed. Two hours postfeeding, total phage DNA dropped to its lowest level. The phage population then increased, reaching a maximal level 8 to 10 h postfeeding before declining over the next 4 h to reach a stable concentration for the rest of the cycle. The general trend in phage DNA concentration appeared similar to previously recorded diurnal fluctuations in ruminal bacterial populations in cattle fed once daily.     

Late gene function in bacteriophage T4 in the absence of phage DNA replication

Under certain conditions the late genes of coliphage T4 may function in the absence of phage DNA replication. Quasi-late gene function is the function of certain late genes in the absence of both phage DNA replication and the product of the maturation gene 55. It does not depend on how phage DNA synthesis is prevented. Replication-uncoupled late gene function is late gene function from unreplicated DNA in the absence of phage ligase, and is still under the control of gene 55. It is most efficient if phage DNA replication is prevented by a mutation in the phage gene (43) for DNA polymerase. Both quasi-late gene function and replication-uncoupled late gene function are enhanced by the presence of mutations controlling a phage exonuclease (gene 46 or 47).     

The mechanism of the resistance of methicillin-resistant Staphylococcus aureus to phages from the International Collection

The resistance of methicillin-resistant staphylococci to phage 85 is due to the presence of a certain system restriction modification in microbial cells. The loss of the capacity for restricting phage DNA by the cell as the consequence of the loss of the mec determinant is not accompanied by the loss of its capacity for modifying phage DNA.     

Inhibition of Host Deoxyribonucleic Acid Synthesis by T4 Bacteriophage in the Absence of Protein Synthesis

The requirement for phage protein synthesis for the inhibition of host deoxyribonucleic acid synthesis has been investigated by using a phage mutant unable to catalyze the production of any phage deoxyribonucleic acid. It has been concluded that the major pathway whereby phage inhibit host syntheses requires protein synthesis. The inhibition of host syntheses by phage ghosts is not affected by inhibitors of protein synthesis.