Requirements for the formation of plasmid-transducing particles of Bacillus subtilis bacteriophage SPP1.

We had previously proposed that the production of concatemeric plasmid DNA in plasmid-transducing SPP1 particles is a consequence of phage-directed rolling-circle-type replication of plasmid DNA. The production of such DNA was greatly enhanced when DNA/DNA homology was provided between phage and plasmid DNAs (facilitation of transduction). Here we present evidence that synthesis of concatemeric plasmid DNA can proceed after phage infection under conditions non-permissive for plasmid replication. We also propose that the naturally occurring homology between plasmid and phage is sufficient to account for the frequency of transduction observed in the absence of facilitating homology. Homology of greater than 47 bp gives the maximal facilitation of plasmid transduction. Recombination is not an essential part in the synthesis of concatemeric plasmid DNA.     

Recombination-dependent concatemeric plasmid replication.

The replication of covalently closed circular supercoiled (form I) DNA in prokaryotes is generally controlled at the initiation level by a rate-limiting effector. Once initiated, replication proceeds via one of two possible modes (theta or sigma replication) which do not rely on functions involved in DNA repair and general recombination. Recently, a novel plasmid replication mode, leading to the accumulation of linear multigenome-length plasmid concatemers in both gram-positive and gram-negative bacteria, has been described. Unlike form I DNA replication, an intermediate recombination step is most probably involved in the initiation of concatemeric plasmid DNA replication. On the basis of structural and functional studies, we infer that recombination-dependent plasmid replication shares important features with phage late replication modes and, in several aspects, parallels the synthesis of plasmid concatemers in phage-infected cells. The characterization of the concatemeric plasmid replication mode has allowed new insights into the mechanisms of DNA replication and recombination in prokaryotes.     

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.     

Recombination-dependent replication of plasmids during bacteriophage T4 infection

The replication of plasmids containing fragments of the T4 genome, but no phage replication origins, was analyzed as a possible model for phage secondary (recombination-dependent) replication initiation. The replication of such plasmids after T4 infection was reduced or eliminated by mutations in several phage genes (uvsY, uvsX, 46, 59, 39, and 52) that have previously been shown to be involved in secondary initiation. A series of plasmids that collectively contain about 60 kilobase pairs of the T4 genome were tested for replication after T4 infection. With the exception of those known to contain tertiary origins, every plasmid replicated in a uvsY-dependent fashion. Thus, there is no apparent requirement for an extensive nucleotide sequence in the uvsY-dependent plasmid replication. However, homology with the phage genome is required since the plasmid vector alone did not replicate after phage infection. The products of plasmid replication included long concatemeric molecules with as many as 35 tandem copies of plasmid sequence. The production of concatemers indicates that plasmid replication is an active process and not simply the result of passive replication after the integration of plasmids into the phage genome. We conclude that plasmids with homology to the T4 genome utilize the secondary initiation mechanism of the phage. This simple model system should be useful in elucidating the molecular mechanism of recombination-dependent DNA synthesis in phage T4.     

Replication and packaging of choleraphage phi 149 DNA.

The intercellular replication of the circularly permuted DNA of choleraphage phi 149 involves a concatemeric DNA structure with a size equivalent to six genome lengths. The synthesis of both monomeric and concatemeric DNAs during replication of phi 149 occurred in the cytoplasm. The concatemers served as the substrate for the synthesis of mature phage DNA, which was eventually packaged by a headful mechanism starting from a unique pac site in the concatemeric DNA. Packaging of DNA into phage heads involved binding of concatemeric DNA to the cell membrane. A scheme involving sequential packaging of five headfuls proceeding in the counterclockwise direction from the pac site is proposed. After infection under high-phosphate conditions, the concatemeric DNA intermediates were not formed, although synthesis of monomeric molecules was unaffected.     

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.     

Phage P22 helps phage MB78 to overcome blockage of DNA maturation in rifampicin-resistant host

Bacteriophage MB78, a virulent phage ofSalmonella typhimurium cannot grow in rifampicin-resistant mutant (rif-39) of the host having altered RNA polymerase. The temperate phage P22 which cannot multiply in presence of the virulent phage MB78 can, however, help MB78 to overcome replication inhibition in rif-39. The processing of concatemeric phage DNA to monomer is blocked in this nonpermissive host. Superinfection with P22 induces synthesis of at least five P22 specific polypeptides which help phage MB78 in the processing of the concatemeric DNA and maturation of phage particles.     

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.     

Influence of the growth rate on the macromolecular composition of Acinetobacter calcoaceticus in carbon-limited chemostat culture

Abstract Infectious phage particles can be formed in vitro when extracts of T1-infected cells are incubated with T1 DNA. The DNA packaging system is based on mixtures of complementing extracts from Escherichia coli sup⁰ cells infected with the amber mutants am 4 (gene 16) or am 10 (gene 13). Gene 16 mutants are defective in the formation of DNA-filled heads but make proheads; gene 13 mutants are defective in prohead formation. Three forms of DNA have been packaged: (1) endogenous concatemeric DNA present in mixtures of am 4 and am 10 mutant extracts; (2) concatemeric DNA; (3) virion DNA both when supplied exogenously to mixtures of am 4 · am 20 and am 10 · am 20 double mutant extracts ( am 20 inhibits T1 DNA synthesis). The reaction requires added ATP, Mg²⁺ and spermidine for optimum efficiency and produces about 1.5 × 10³ pfu/ μ g and about 1 × 10⁴ pfu/ μ g for exogenous concatemeric and virion DNA, respectively.     

Integration of phage Mu into the RP4::D3112A-plasmid in Escherichia coli cells

Hybrid plasmids obtained as a result of Mu phage insertions into the RP4::D3112 plasmid in Escherichia coli cells were studied. Stable maintenance of RP4::D3112 plasmid in E. coli cells was provided by using the D3112 phage genome with a point polar mutation in the A gene which prevented early genes' expression. The presence of D3112A- in the RP4 plasmid has been shown to have no effect on efficiency of phage Mu transposition into this plasmid. Moreover, RP4 and D3112 genomes were equivalent targets for Mu integration. The integration of transposable phage into genome of nonrelated phage can be used as one of the approaches to construct recombinant phage genomes in vivo in the absence of DNA homology.     

Replication of a plasmid containing two origins of bacteriophage f1

A chimeric plasmid was constructed that contains a tandem duplication of the bacteriophage f1 origin of DNA replication. This plasmid replicates stably in the absence of phage. When cells carrying this plasmid are infected with f1, two new plasmid-derived DNA species are generated: a smaller, chimeric plasmid containing only one f1 origin of replication, and a miniphage, the genome of which consists of the f1 fragment that was located between the two f1 origins of the original plasmid. These data support the hypothesis (Horiuchi, 1980) that the nucleotide sequence recognized for initiation of plus strand synthesis in f1 DNA replication is also the signal for its termination.     

Transfer of chimeric plasmids among Salmonella typhimurium strains by P22 transduction

Salmonella typhimurium bacteriophage P22 transduced plasmids having P22 sequences inserted in the vector pBR322 with high frequency. Analysis of the structure of the transducing particle DNA and the transduced plasmids indicates that this plasmid transduction involves two homologous recombination events. In the donor cell, a single recombination between the phage and the homologous sequences on the plasmid inserted the plasmid into the phage chromosome, which was then packaged by headfuls into P22 particles. The transducing particle DNA contained duplications of the region of homology flanking the integrated plasmid vector sequences and lacked some phage genes. When these defective phage genomes containing the inserted plasmid infected a recipient cell, recombination between the duplicated regions regenerated the plasmid. A useful consequence of this sequence of events was that genetic markers in the region of homology were readily transferred from phage to plasmid. Plasmid transduction required homology between the phage and the plasmid, but did not depend on the presence of any specific P22 sequence in the plasmid. When the infecting P22 carried a DNA sequence homologous to the ampicillin resistance region of pBR322, the vector plasmid having no P22 insert could be transduced. P22-mediated transduction is a useful way to transfer chimeric plasmids, since most S. typhimurium strains are poorly transformed by plasmid DNA.     

Involvement of the bacterial groM gene product in bacteriophage T7 reproduction. I. Arrest at the level of DNA packaging

The multiplication of bacteriophage T7 is blocked in Escherichia coli M. The genetic determinant of this ability (groM) to inhibit T7 growth was transferred to an E. coli K-12 recipient by means of conjugation. We determined at which precise step T7 maturation is blocked. Phage-directed protein and DNA synthesis as well as degradation of host DNA were not qualitatively affected. Instead of infective phages, only preheads were produced. These, however, were maturable in vitro. The newly synthesized phage DNA accumulated in a concatemeric form and matured from its tetrameric or longer forms (very fast sedimenting DNA) only into its dimeric form (fast-sedimenting DNA) or longer forms. The following step, i.e., the maturation of the dimeric to unit-length DNA, was not observed. Since the concatemeric form of T7 DNA accumulated in spite of the presence of maturable preheads, it is likely that the maturation process was blocked at the level of DNA packaging. As intermediates in the packaging process, we found some prehead-DNA complexes. We interpreted these as true assembly intermediates (or breakdown products thereof), since the attached DNA was still in its concatemeric form. This shows that the very first DNA packaging step, the binding of the progeny DNA to the preheads, was obviously not blocked. Rather, a later step, such as the filling of the preheads with T7 DNA or the stabilization of completely packaged particles (i.e., the final cutting of the concatemers into unit-size length), was inhibited.     

Gene X of bacteriophage f1 is required for phage DNA synthesis. Mutagenesis of in-frame overlapping genes

The gene II protein of bacteriophage f1 is a site-specific endonuclease required for initiation of phage viral strand DNA synthesis. Within gene II is another gene, X, encoding a protein of unknown function identical to the C-terminal 27% of the gene II protein, and separately translated from codon 300 (AUG) of gene II. By oligonucleotide mutagenesis, we constructed phage mutants in which this codon has been changed to UAG (amber) or UUG (leucine), and propagated them on cells carrying a cloned copy of gene X on a plasmid. The amber mutant makes no gene X protein, and cannot grow in the absence of the complementing plasmid; the leucine-inserting mutant can make gene X protein, and grows normally without the plasmid. Without gene X protein, phage DNA synthesis (particularly viral strand synthesis) is impaired. We discuss this finding in the context of other known in-frame overlapping genes (particularly genes A and A* of phage phi X174), many of which are also involved in the specific initiation of DNA synthesis, and suggest applications for the mutagenic strategy we employed.     

Generation of linear multigenome-length plasmid molecules in Bacillus subtilis.

Linear multigenome-length double and single stranded plasmid DNA was identified in a Bacillus subtilis ATP-dependent DNAase mutant strain (addA5) bearing plasmids pC194 or pBD95ts. Plasmid pBC30, a seg mutant of pC194, as well as some pUB110 derivatives with rearrangements external to the minimal replicon, produce high amounts of such a concatemeric DNA, even in Rec+ cells. The synthesis of this type of plasmid DNA occurs in the absence of an active plasmid-encoded Rep protein and is markedly affected in polA5 and recE4 genetic backgrounds. To account for these observations, we propose that the AddAB complex serves to prevent a sigma-type replication of plasmid DNA.     

Recombination between bacteriophage T4 and plasmid pBR322 molecules containing cloned T4 DNA

Reciprocal recombination between T4 DNA cloned in plasmid pBR322 and homologous sequences in bacteriophage T4 genomes leads to integration of complete plasmid molecules into phage genomes. Indirect evidence of this integration comes from two kinds of experiments. Packaging of pBR322 DNA into mature phage particles can be detected by a DNA--DNA hybridization assay only when a T4 restriction fragment is cloned in the plasmid. The density of the pBR322 DNA synthesized after phage infection is also consistent with integration of plasmid vector DNA into vegetative phage genomes. Direct evidence of plasmid integration into phage genomes in the region of DNA homology comes from genetic and biochemical analysis of cytosine-containing DNA isolated from mature phage particles. Agarose gel electrophoresis of restriction endonuclease-digested DNA, followed by Southern blot analysis with nick-translated probes, shows that entire plasmid molecules become integrated into phage genomes in the region of T4 DNA homology. In addition, this analysis shows that genomes containing multiple copies of complete plasmid molecules are also formed. Among phage particles containing at least one integrated copy, the average number of integrated plasmid molecules is almost ten. A cloning experiment done with restricted DNA confirms these conclusions and illustrates a method for walking along the T4 genome.     

Involvement of a Replicative DNA Helicase of Bacteriophage T4 in DNA Recombination

Bacteriophage T4 gene 41 encodes a replicative DNA helicase that is a subunit of the primosome which is essential for lagging-strand DNA synthesis. A mutation, rrh, was generated and selected in the helicase gene on the basis of limited DNA replication that ceases early. The survival of ultraviolet-irradiated phage and the frequency of genetic recombination are reduced by rrh. In addition, rrh diminishes the production of concatemeric DNA. These results strongly suggest that the gene 41 replicative helicase participates in DNA recombination.     

Regions of DNA sequence homology between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens

Summary Despite the fact that pTiC58 and pTiB6S3 functionally, have been shown to date to have only tumorigenicity and phage AP1 exclusion in common, many restriction fragments of the plasmids contain DNA sequences common to both. The bulk of this homologous DNA is concentrated in a few restriction endonuclease fragments and the remainder is organized in short discontinuous regions spread over many fragments. In pTiB6S3 the bulk of the homology is distributed throughout a 29x10⁶ dalton segment comprising 8 Sma I fragments. This region includes those sequences which are transferred to and transcribed in tumorigenic plant cells induced by B6-806 or closely related strains. The pattern of homology within this portion of the plasmid shows a region of low sequence homology (Sma I Fragment 3 b) apparently corresponding to the gene or genes coding for octopine synthesis in the plant tumor cells, surrounded by regions of high sequence homology. The extent of inter-plasmid homology then decreases with increasing distance from fragment 3b. The remainder of the homology is distributed throughout a segment of maximum size 21.5x10⁶ daltons comprising two Sma I fragments and cannot yet be definitely linked with any specific plasmid function.