MB78, a virulent bacteriophage of Salmonella typhimurium

The isolation and some properties of a virulent bacteriophage of Salmonella typhimurium, MB78, which is morphologically, serologically, and physiologically unrelated to P22, are reported. The phage has a noncontractile long tail with partite ends. It cannot multiply in minimal medium in the presence of citrate. MB78-infected cells are, however, killed in such medium. This phage cannot grow in rifampin-resistant mutants of the host. The latent period of growth of this phage is much shorter than that of P22. Both sieA and sieB genes of the resident P22 prophage are required to exclude the superinfecting MB78 phage, whereas all temperate phages related to P22 are excluded by either one or both of the genes individually. Restriction endonuclease cleavage patterns of P22 and MB78 are distinctly different. The absence of homology between the two phages P22 and MB78 suggests that MB78 is not related to phage P22.     

Replication,maturation and physical mapping of bacteriophage MB78 genome

Bacteriophage MB78 is a virulent phage ofSalmonella typhimurium. The viral DNA is 42 kb in size and seems to be circularly permuted. We show that viral DNA replication is through concatemeric DNA formation which is subsequently converted into full length DNA through headful packaging. A restriction map of MB78 DNA for six restriction endonucleases e.g.BgIII,PvuII, ECORI, ClaI, SalI and SmaI has been constructed. The yield of certain fragments in less than molar amount is explained in terms of permutation and the headful mechanism of packaging. The packaging site (pac site) has been suggested.     

Maturation of bacteriophage 9NA DNA is influenced by the fatty acid composition of the host cell membrane

The fatty acid composition of the membrane of the conditional auxotroph fabB2 can be altered by allowing the cells to grow at non-permissive temperature (37°C) in the presence of a cis-unsaturated fatty acid. The phage 9NA, a virulent phage ofSalmonella typhimurium, can not multiply in fabB2. Synthesis and maturation of the phage DNA are differentially affected by variation in the fatty acid composition of the cell membrane. The replicating DNA associates with the membrane complex, the site of DNA synthesis. The association is comparatively weak in oleic, claidic, palmitoleic, palmitelaidic and linolelaidic acid enriched cells. When the cells are grown in the presence of palmitoleic acid, a large pool of concatemeric phage DNA accumulates in the cytoplasm within 10 min of infection. The conversion of concatemeric DNA to monomeric one i.e., mature phage length DNA, is inhibited in such cells. The presence of concatemeric DNA can be visualized by electron microscope. Such a situation is not observed when the cells are grown in media supplemented with other types of unsaturated fatty acids. The mechanism by which the host cell membrane lipid controls phage development is yet to be worked out.     

Rescue of abortive T7 gene 2 mutant phage infection by rifampin.

Infection of Escherichia coli with T7 gene 2 mutant phage was abortive; concatemeric phage DNA was synthesized but was not packaged into the phage head, resulting in an accumulation of DNA species shorter in size than the phage genome, concomitant with an accumulation of phage head-related structures. Appearance of concatemeric T7 DNA in gene 2 mutant phage infection during onset of T7 DNA replication indicates that the product of gene 2 was required for proper processing or packaging of concatemer DNA rather than for the synthesis of T7 progeny DNA or concatemer formation. This abortive infection by gene 2 mutant phage could be rescued by rifampin. If rifampin was added at the onset of T7 DNA replication, concatemeric DNA molecules were properly packaged into phage heads, as evidenced by the production of infectious progeny phage. Since the gene 2 product acts as a specific inhibitor of E. coli RNA polymerase by preventing the enzyme from binding T7 DNA, uninhibited E. coli RNA polymerase in gene 2 mutant phage-infected cells interacts with concatemeric T7 DNA and perturbs proper DNA processing unless another inhibitor of the enzyme (rifampin) was added. Therefore, the involvement of gene 2 protein in T7 DNA processing may be due to its single function as the specific inhibitor of the host E. coli RNA polymerase.     

Restriction-modification system in bacteriophage MB78

Restriction-modification system is present in bacteria to protect the cells against phage infection. Interestingly, the bacteriophage MB78, a virulent phage of Salmonella typhimurium possesses restriction-modification system. Permissive host transformed with plasmid having the genomic fragment of MB78 carrying the putative restriction-modification genes severely restrict the growth of the phage 9NA. Growth of phage MB78 is also restricted to some extent. However, the temperate phage P22 is not restricted at all. Cloning of the the putative restriction-modification genes has been done in both orientations in different vectors. The clones carrying the genes in the same orientation as that of the lacZ in pUC19 are mostly unstable. However, those are stable when cloned in opposite orientation. Viability of the transformants is strain-, orientation-, and medium-dependent. The two genes have also been cloned individually/separately. Hosts carrying only the modification gene do not restrict growth of phages while the hosts carrying only the restriction gene do. The former produces stable transformants while the latter produces very unstable transformants which were viable only upto 36 h or so. The colonies carrying modification gene were normal looking while those carrying the restriction gene were tiny, flat, and looked distressed resembling very much the clones carrying bacterial restriction-modification system. Amplification of the genes and subsequent cloning in expression vector will be carried out for characterization of the enzymes.     

Molecular cloning, sequencing, and expression of two late proteins of bacteriophage MB78

Bacteriophage MB78, a virulent phage of Salmonella typhimurium, does not allow other phages, such as P22 and 9NA, to grow in its presence. A detailed physical map of this phage has been constructed in our laboratory. In an ongoing effort to understand the genetics of this interesting phage, various genes were characterized. Here, we report cloning, sequencing, and expression of two late proteins, coded in a SalI-HindIII fragment (SH9), by using the minicell expression system. Further, we performed a kinetic study of phage proteins by infection the host LT2 cells and compared the proteins produced, with proteins obtained by the minicell expression system. Both sets of proteins run exactly parallel and migrated as 14- and 15-kDa proteins on a polyacrylamide gel. The synthesis of these two proteins started 15 min after infection with MB78 and was prominent after 45 min. One of the proteins exhibited 57% homology to the structural protein of mycobacteriophage L5.     

Molecular genetic analysis of bacteriophage P22 gene 3 product, a protein involved in the initiation of headful DNA packaging.

Bacteriophage P22 DNA packaging events occur in processive series on concatemeric phage DNA molecules. At the point where such series initiate, the DNA is recognized at a site called pac, and most molecular left ends are generated within six short regions called end sites, which are present in a 120 base-pair region surrounding the pac site. The bacteriophage P22 genes 2 and 3 proteins are required for successful generation of these ends and DNA packaging during progeny virion assembly. Mutants lacking the 162-amino-acid gene 3 protein replicate DNA and assemble functional procapsids. In this report we describe the nucleotide changes and DNA packaging phenotypes of a number of missense mutations of gene 3, which give the phage a higher than normal frequency of generalized transduction. In cells infected by these mutants, more packaging events initiate on the host chromosome than in wild-type infections, so the mutations are thought to affect the specificity of packaging initiation. In addition to having this phenotype, these mutations affect the process of phage DNA packaging in detectable ways. They may: (1) alter the target site specificity for packaging; (2) make target site recognition more promiscuous; (3) affect end site utilization; (4) alter the pac site; and (5) cause apparent random DNA packaging series initiation on phage DNA.     

Physical mapping and characterization of bacteriophage 9NA genome

The DNA of bacteriophage 9NA, a virulent phage ofSalmonella typhimurium, is linear, double stranded, circularly permuted and is approximately 56 kilobase pairs long. The 9NA genome is partially methylated. A physical map of the DNA has been constructed using the restriction endonucleasesBamHI,BglII,SmaI andPvuII. The putative packaging end (‘pac’ end) and the direction of packaging of the concatemeric DNA has been postulated.     

Participation of the host protein(s) in the morphogenesis of bacteriophage P22

Summary Spontaneous mutants of S. typhimurium resistant to thiolutin are conditionally non-permissive for phage P22 development (Joshi and Chakravorty 1979). At 40° C non-infective phage particles are produced. Phage development in two nonpermissive hosts (18/MC4 and 153/MC4) has been studied in detail. The steps at which the phage morphogenesis is interfered with differ in the two mutants. The electron micrograph of the particles produced in the mutant 18/MC4 reveals the presence of normal-looking particles; these particles contain phage DNA, adsorb to the permissive host but fail to inject their DNA. The particles produced in the mutant 153/MC4 which fail to adsorb to the host are found to be tail fibre-less. These observations indicate the involvement of host protein(s) in phage P22 morphogenesis.     

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.     

Characterization of phage isolates from a phage-carrying culture of Leuconostoc oenos 58N

Summary During large-scale cultivation of Leuconostoc oenos strain 58N, growth inhibition was detected and attributed to the presence of the virulent phage P581. To determine if this phage originated from a temperate phage, L. oenos 58N was exposed to mitomycin C, and this treatment led indeed to release of phages (P58II). Further examination of the lytic potential of phages P581 and P58II revealed that these two phages were able to lyse the same strains of L. oenos with the exception of the original host strain, which was only sensitive to P581. Results of DNA/DNA hybridization experiments failed to show homology between the DNA of phage P58II and the chromosomal DNA of L. oenos 58N. A phage-free culture of L. oenos 58N could be obtained after repeated subculture. These results indicate that the original L. oenos 58N was in a special type of phage-carrier state. Phages P58I and P58II were compared on the basis of morphology, lytic spectra, restriction enzyme analysis, DNA homology, genome size and protein structure and proved to be identical. It is assumed that P58I arose from the phage-carrier culture of L. oenos 58N and became virulent by some mutational event.Offprint requests to: E. K. Arendt     

A 14-kilodalton inner membrane protein of Vibrio cholerae biotype e1 tor confers resistance to group IV choleraphage infection to classical vibrios.

Choleraphage phi 149 differentiates the two biotypes, classical and el tor, of Vibrio cholerae. This phage cannot replicate in V. cholerae biotype el tor cells because the concatemeric DNA intermediates produced are unstable and cannot be chased to mature phage DNA. A V. cholerae biotype el tor gene coding for a 14,000-Da inner membrane protein which destabilizes the concatemeric DNA intermediates by hindering their binding to the cell membrane has been identified. Presumably, a 22,000-Da V. cholerae biotype el tor protein might also have a role in conferring phage phi 149 resistance to cells belonging to the biotype el tor. A nucleotide sequence homologous to the 1.2-kb V. cholerae biotype el tor DNA coding for both the 14,000- and 22,000-Da proteins is present in all strains of classical vibrios but is not transcribed. The nucleotide sequence of the gene coding for the 14,000-Da protein has been determined.     

Genes affecting progression of bacteriophage P22 infection in Salmonella identified by transposon and single gene deletion screens

Bacteriophages rely on their hosts for replication, and many host genes critically determine either viral progeny production or host success via phage resistance. A random insertion transposon library of 240,000 mutants in Salmonella enterica serovar Typhimurium was used to monitor effects of individual bacterial gene disruptions on bacteriophage P22 lytic infection. These experiments revealed candidate host genes that alter the timing of phage P22 propagation. Using a False Discovery Rate of < 0.1, mutations in 235 host genes either blocked or delayed progression of P22 lytic infection, including many genes for which this role was previously unknown. Mutations in 77 genes reduced the survival time of host DNA after infection, including mutations in genes for enterobacterial common antigen (ECA) synthesis and osmoregulated periplasmic glucan (OPG). We also screened over 2000 Salmonella single gene deletion mutants to identify genes that impacted either plaque formation or culture growth rates. The gene encoding the periplasmic membrane protein YajC was newly found to be essential for P22 infection. Targeted mutagenesis of yajC shows that an essentially full‐length protein is required for function, and potassium efflux measurements demonstrated that YajC is critical for phage DNA ejection across the cytoplasmic membrane.     

The molecular structure of the transducing particles of Salmonella phage P22. II. Density gradient analysis of DNA

Summary CsCl density gradient analysis showed that the DNA of plaque forming particles ofSalmonella phageP22 is lighter than the host DNA. The DNA of transducing phages exhibits an intermediate density, but close to host DNA. BU labelling of DNA synthesized in the cells after phage infection resulted in a density increase of transducing DNA of about 0.004 gxcm^-3, whereas infectious DNA increased by about 0.045 gxcm^-3. Shearing of isolated DNA molecules from unlabelledP22 lysates demonstrated that transducing DNA consists of two pieces of DNA of different density: 90% stem from the bacterial host whereas 10% are phage DNA and therefore responsible for the BU lable in transducing phages.     

A Salmonella Phage-P22 Mutant Defective in Abortive Transduction

In the course of a lytic infection the Salmonella phage P22 occasionally encapsulates bacterial DNA instead of phage DNA. Thus, phage lysates include two classes of viral particles. Phage particles carrying bacterial DNA are referred to as transducing particles and deliver this DNA to a host as efficiently as particles carrying phage DNA. Once injected, the transduced DNA can either recombine with the recipient chromosome to form a ``complete'''' transductant, or it can establish itself as an expressible, nonreplicating genetic element and form an ``abortive'''' transductant. In this work, we describe a P22-phage mutant with reduced ability to form abortive transductants. The mutation responsible for this phenotype, called tdx-1, was found as one of two mutations contributing to the high-transducing phenotype of the P22-mutant HT12/4. In addition, the tdx-1 mutation is lethal when combined with an erf-am mutation. The tdx-1 mutation has been mapped to a region of the P22 genome that encodes several injected proteins and may involve more than one mutant locus. The phenotypes of the tdx-1 mutation suggest that the Tdx protein(s) normally assist in the circularization of the P22 genome and also contribute to the formation of DNA circles thought to be required for abortive transduction.     

Absence of a protein constituent and occurrence of an oversized DNA genome in a high density mutant particle of phage P22

Summary Mutants of P22 phage with abnormal density in CsCl solution (P22ndc phage) were analyzed in detail for this report. Two dimensional polyacrylamide gel electrophoresis revealed that wild-type P22ndc ⁺ phage virions contained a new protein (gpU) in addition to nine already identified proteins, while P22ndc lacked gpU. The molecular weight of gpU was essentially the same as that of gp5 (45 500), and one mature virion of phage P22ndc ¹ contained as many as 30–50 molecules of gpU. As P22ndc is a plaque-forming phage, gpU cannot be essential for the growth and assembly of P22 phage. Both genetical and biochemical analysis of the phage DNA in the virion revealed that P22ndc phage contained 2%–4% longer DNA than wild type P22ndc ⁺. A model is presented to account for the formation of P22ndc phage.     

Cloning and characterization of a gene encoding 22 kDa functional protein of bacteriophage MB78

Functional protein of MB78 bacteriophage having apparent molecular weight of 22 kDa is expressed from 1.7 kb HindIII G fragment. The nucleotide sequence of this fragment showed two open reading frames of 222 and 196 codons in tail-to-tail orientation separated by a 62-nucleotide intercistronic region. The ORF of 22 kDa protein is present in opposite orientation, i.e. in the complementary strand, preceded by a strong ribosomal binding site and a promoter sequence. Another ORF started from the beginning of the fragment whose promoter region and translational start site lies in the 0.45kb HincII U fragment which is located next to the HindIII G fragment, that has the sequence for DNA bending. 3'end of the fragment has high sequence homology to the EaA and EaI proteins of bacteriophage P22, a close relative of MB78 phage.