Generation of transducing particles in Staphylococcus aureus.

Transduction of plasmid pC194 and bacteriophage phi 11de varied inversely with the multiplicity of infection. As the multiplicity of infection decreased from 10(-1) to 10(-5) PFU/CFU, the transduction frequency of pC194 increased 10(4)-fold; the transduction frequency of phi 11de increased 300-fold with a 100-fold decrease in multiplicity of infection. Physical and genetic analysis of the transduced DNA showed that pC194 resided in the phage particle as a random, circularly permuted linear concatemer. In DNA prepared from phage that cotransduced pC194 and phi 11de, pC194 resided in the transducing phage primarily as a linear multimer of 15.8 kilobases, or about 5.4 pC194 monomers. The pC194 multimer was randomly inserted into the phi 11 genome.     

Cloning of restriction fragments of DNA from staphylococcal bacteriophage phi 11.

EcoRI fragments of Staphylococcus aureus bacteriophage phi 11 DNA were cloned in vector plasmid pSA2100 in S. aureus. The clones were analyzed in marker rescue experiments with suppressor- and temperature-sensitive mutants of phi 11 to correlate the genetic and physical map. Several mutants could be identified on the physical map, and a clone containing fragment EcoRI-B of phi 11 DNA expressed immunity to phage infection. In addition, it was found that recombinant plasmids containing phi 11 DNA sequences can be transferred by high-frequency transduction after phage phi 11 infection of host cells.     

Selective plasmid transduction in Bacillus pumilus.

The inducible temperate bacteriophage phi75 and a clear-plaque-forming variant, phi75C1, mediated transduction of a 4.4 X 10(6)-dalton multicopy Bacillus pumilus plasmid, pPL10, at frequencies of 10(-5) to 10(-6) transductants per plaque-forming unit. phi75- and phi75C1-mediated transduction of several chromosome markers tested did not occur at a detectable frequency. phi75-mediated plasmid transducing activity resides in particles that are similar to infectious particles in sedimentation velocity and buoyant density.     

Characterization of interspecific plasmid transfer mediated by Bacillus subtilis temperate bacteriophage SP02.

Plasmid pPL1010 is a 7.0-kilobase derivative of plasmid pUB110 that harbors the cohesive end site of the bacteriophage SP02 genome. Plasmid pPL1017 is a 6.8-kilobase derivative of plasmid pC194 that contains the immunity region of bacteriophage phi 105 and the cohesive end site of bacteriophage SP02. These plasmids are transducible by bacteriophage SP02 at a frequency of 10(-2) transductants per PFU among mutant derivatives of Bacillus subtilis 168 and have been transferred to other strains of B. subtilis and B. amyloliquefaciens by means of bacteriophage SP02-mediated transduction, with frequencies ranging from 10(-5) to 10(-7) transductants per PFU. The introduced plasmids were stably maintained in nearly all new hosts in the absence of selective pressure. An exception was found in B. subtilis DSM704, which also harbored three cryptic plasmids. Plasmids pPL1010 and pPL1017 were incompatible with a 7.9-kilobase replicon native to strain DSM704. Furthermore, plasmid pPL1017 was processed by strain DSM704 into a approximately 5.3-kilobase replicon that was compatible with the resident plasmid content of strain DSM704. The use of bacteriophage SP02-mediated plasmid transduction has allowed the identification of Bacillus strains that are susceptible to bacteriophage SP02-mediated genetic transfer but cannot support bacteriophage SP02 lytic infection.     

Mutations in Prophage φ11 That Impair the Transducibility of Their Staphylococcus aureus Lysogens for Methicillin Resistance

Methicillin resistance (mec) is not transduced into Staphylococcus aureus 8325-4, but is transduced into this host after it has been lysogenized with phage phi11 and has acquired the penicillinase plasmid pI524 by a separate transduction (Cohen and Sweeney, 1970, 1973). Strain 8325-4 is competent for transformation of typical plasmid or chromosomal markers and for mec only if it is lysogenic for phi11 or a related prophage (Sj?str?m et al., 1974, 1975). A mutant strain of phi11 that was temperature sensitive (Ts) for vegetative multiplication did not mediate competence for transformation of its 8325-4 lysogen if the lysogen had been grown at a nonpermissive temperature (Sj?str?m and Philipson, 1974). We isolated four Ts mutants of phi11 that did not mediate transducibility of their 8325-4(pI524) lysogens for mec after growth at nonpermissive temperatures (40 to 42 degrees C). Transduction of typical plasmid or chromosomal markers was not affected. These phi11-Ts mutants mediated normal competence of their lysogens for transformation of a tetracycline resistance plasmid. Similarly, phi11-Ts mutants that rendered their lysogens temperature sensitive for transformation did not depress the frequency of transduction of mec. These two types of phi11-Ts mutants fell into two different genetic complementation groups that differed in the physiology of deoxyribonucleic acid synthesis and in the time of expression of the mutations during a single-burst growth cycle at a nonpermissive temperature. A virulent mutant of phi11, which plaqued with 100% efficiency on 8325(phi11), also failed to condition strain 8325-4 for transducibility of mec but retained the ability to confer competence for transformation of a tetracycline resistance plasmid. Different genetic loci and physiological functions are involved in phi11 mutations that affect transducibility of mec and those that affect competence for transformation of markers generally in S. aureus 8325-4.     

Genetic map of the Staphylococcal bacteriophage phi11.

Ten sus mutants of the staphylococcal bacteriophage phi 11, each a representative from a different complementation group, have been used in three-factor cross experiments. The results of these crosses indicate a circular genetic map for phi 11. Functional studies of the mutants have been limited to electron microscopic examinations of lysates after prophage induction (or infection). One gene is an early gene, five genes are concerned with tail formation, and three are concerned with head formation. The tenth gene is possibly a head gene. The contribution by phi 11 to the genomic content of the plasmid-phage hybrid phi 11 de has been investigated. Phi 11 de contains most of the late genes and appears to be missing a continuous phi 11 segment that includes the early gene flanked by two late genes.     

Regions of incompatibility in single-stranded DNA bacteriophages phi X174 and G4.

The intracellular presence of a recombinant plasmid containing the intercistronic region between the genes H and A of bacteriophage phi X174 strongly inhibits the conversion of infecting single-stranded phi X DNA to parental replicative-form DNA. Also, transfection with single-stranded or double-stranded phi X174 DNA of spheroplasts from a strain containing such a "reduction" plasmid shows a strong decrease in phage yield. This phenomenon, the phi X reduction effect, was studied in more detail by using the phi X174 packaging system, by which plasmid DNA strands that contain the phi X(+) origin of replication were packaged as single-stranded DNA into phi X phage coats. These "plasmid particles" can transduce phi X-sensitive host cells to the antibiotic resistance coded for by the vector part of the plasmid. The phi X reduction sequence in the resident plasmid strongly affected the efficiency of the transduction process, but only when the transducing plasmid depended on primosome-mediated initiation of DNA synthesis for its conversion to double-stranded DNA. The combination of these results led to a model for the reduction effect in which the phi X reduction sequence interacted with an intracellular component that was present in limiting amounts and that specified the site at which phi X174 replicative-form DNA replication takes place. The phi X reduction sequence functioned as a viral incompatibility element in a way similar to the membrane attachment site model for plasmid incompatibility. In the DNA of bacteriophage G4, a sequence with a similar biological effect on infecting phages was identified. This reduction sequence not only inhibited phage G4 propagation, but also phi X174 infection.     

Bacteriophage SPO2-mediated plasmid transduction in transpositional mutagenesis within the genus Bacillus.

A single copy of the Streptococcus faecalis transposon Tn917, located in the Bacillus subtilis chromosome, was able to transpose onto the SPO2 cos plasmid pPL1017, which codes for chloramphenicol resistance and contains the bacteriophage phi 105 immunity region. Selection for pPL1017::Tn917 chimeras was performed by SPO2-mediated plasmid transduction of transposon-borne resistance to macrolide-lincosamide-streptogramin B antibiotics (MLSr). The transposition of Tn917 onto plasmid pPL1017 occurred with a frequency of 10(-5) and was dependent on the presence of a subinhibitory dose of erythromycin. Twelve chimeras were subjected to genetic and physical analyses. Two Cams transductants harbored plasmids whose chloramphenicol acetyltransferase genes had been insertionally inactivated by Tn917. Several transpositions in the vicinity of the phi 105 immunity region were detected. However, all of the 300 MLSr, Camr transductants screened were immune to phi 105 infectious activity. One pPL1017::Tn917 chimera, pLK200, was transferred by SPO2 plasmid transduction into the Bacillus amyloliquefaciens prototrophic strain DSM7. Plasmid pLK200 was effective in the mutagenesis of the DSM7 chromosome and yielded auxotrophs at a frequency of 0.5 to 5.3%. Generation of auxotrophs was also dependent on the presence of a subinhibitory dose of erythromycin. Forty-four auxotrophs representing at least nine amino acid requirements were recovered.     

Temperate Bacillus subtilis bacteriophage phi 3T: chromosomal attachment site and comparison with temperate bacteriophages phi 105 and SPO2.

The temperate Bacillus subtilis bacteriophage phi 3T contains within its genome a locus, designated thyP3, that encodes for a protein with thymidylate synthetase activity. Bacteriophage phi 3T is different from the two previously characterized temperate phages, phi 105 and SPO2, in: heteroimmunity, response to bacteriophage antisera, endonuclease digestion pattern, induction in the presence of 6-(p-hydroxyphenylazo)-uracil, and effect on the lytic cycle of bacteriophage phi 1. The mean burst size of phi 3T is 56. The dose response curve with bacteriophage phi 3T DNA is linear for transfection and transformation to the Thy+ phenotype. The inserted prophage has been mapped by PBS1 transduction; it is between chromosomal markers ilvA8 and gltA in the terminus of the chromosome. Thus thyP3 maps at a site separate from, but between, the bacterial markers thyA and thyB when thyP3 is in the prophage state.     

Role of lysogenizing phages in the spread of drug-resistance plasmids in a staphylococcal population

The frequency of the transduction of plasmids rms5, rms7, pT127, pC194, pS194 and pUB101 by phages belonging to serological group B (80, 52, 52A, 53, 85, phi 11, S2) in two systems was compared. In system 1 phages for transduction were obtained from plasmid-containing lysogenic donors in the process of induction with mitomycin C; in system 2 phages for transduction were obtained by their multiplication in plasmid-containing nonlysogenic donors. In system 1 the transduction of plasmids rms5, rms7, pT127, pS194 by phage 52A was found to occur with a greater (by 3-5 orders) frequency than in system 2 (the frequency of transduction was 10(-2) to 10(-4), and 10(-6) to 10(-8) respectively). A similar situation was observed with plasmids rms5 and rms7 and phage 52; plasmid pT127 and phage 53; but not observed with plasmids rms5 and rms7 and phages 80, phi 11 and S2; plasmids pC194 and pS194 and phage 53; plasmid pUB101 and phages 52A, 80 and phi 11; plasmids pC194, pS194 and pT127 and phage 85.     

Transduction of Pseudomonas aeruginosa with a mutant of bacteriophage E79.

A mutant of the virulent bacteriophage E79 was isolated which mediated generalized transduction in Pseudomonas aeruginosa. Variable recovery of transductants as a result of phage killing was avoided by the use of recipients carrying the IncP-2 plasmid R38, and transduction frequencies of 4 X 10(-6) to 1 X 10(-5) per plaque-forming unit were obtained. Linkage studies have indicated that the coinheritance frequencies are less than would be expected from the published molecular weight of E79 deoxyribonucleic acid (120 X 10(6). By using recipients carrying R38, low-frequency transduction by wild-type E79 and two other virulent phages, F8 and phi 16, was demonstrated.     

Potential for transduction of plasmids in a natural freshwater environment: effect of plasmid donor concentration and a natural microbial community on transduction in Pseudomonas aeruginosa

Transduction of Pseudomonas aeruginosa plasmid Rms149 by the generalized transducing bacteriophage phi DS1 was shown to occur during a 9-day incubation of environmental test chambers in a freshwater reservoir. Plasmid DNA was transferred from a nonlysogenic plasmid donor to a phi DS1 lysogen of P. aeruginosa that served both as the source of the transducing phage and as the recipient of the plasmid DNA. When the concentration of donors introduced into the chambers was varied while the recipient concentration in each chamber was at a level equivalent to natural concentrations of P. aeruginosa, the concentration of plasmid-containing donor cells introduced was shown to affect the frequency of transduction significantly. Transduction was observed both in the absence and in the presence of the natural microbial community. The presence of the natural community resulted in a rapid decrease in the numbers of the introduced donors and recipients and a decrease in the number of transductants recovered. These results demonstrate the potential for naturally occurring transduction in aquatic environments and indicate that donor load may be an important parameter in assessing this potential.     

Potential for transduction of plasmids in a natural freshwater environment: effect of plasmid donor concentration and a natural microbial community on transduction in Pseudomonas aeruginosa.

Transduction of Pseudomonas aeruginosa plasmid Rms149 by the generalized transducing bacteriophage phi DS1 was shown to occur during a 9-day incubation of environmental test chambers in a freshwater reservoir. Plasmid DNA was transferred from a nonlysogenic plasmid donor to a phi DS1 lysogen of P. aeruginosa that served both as the source of the transducing phage and as the recipient of the plasmid DNA. When the concentration of donors introduced into the chambers was varied while the recipient concentration in each chamber was at a level equivalent to natural concentrations of P. aeruginosa, the concentration of plasmid-containing donor cells introduced was shown to affect the frequency of transduction significantly. Transduction was observed both in the absence and in the presence of the natural microbial community. The presence of the natural community resulted in a rapid decrease in the numbers of the introduced donors and recipients and a decrease in the number of transductants recovered. These results demonstrate the potential for naturally occurring transduction in aquatic environments and indicate that donor load may be an important parameter in assessing this potential.     

Thymineless bacteriophage induction in Staphylococcus aureus. I. High-frequency transduction with lysates containing a bacteriophage related to bacteriophage phi 11.

A thymine-requiring mutant of Staphylococcus aureus, strain 8325 (PI258)thy, undergoes prophage induction and lysis after thymine starvation. Four different phages were isolated from the lysate in low titers, among which was a phage designated phi 14, which differs from phage phi 11 in its immunity locus. The thymineless induced lysates of strain 8325(PI258)thy transduce the penicillinase plasmid at high frequency (10(-1), whereas transduction of chromosomal markers is inefficient. A plasmic-cured derivative of strain 8325(PI258)thy is also lysed by thymine starvation and be used for high-frequency transduction of other plasmids. Reconstitution of a strain of S. aureus that responds to thymine starvation was only partially successful, but this system can effectively be used to transduce plasmids or plasmid derivatives.     

Genes and regulatory sequences of bacteriophage phi X174

Fragments of the DNA of bacteriophage phi X174 were inserted in the plasmids pACYC177 and pBR322, in order to test the in vivo effects of separate phage genes and regulatory sequences. The phi X174 inserts were identified by recombination and complementation with phage mutants, followed by restriction enzyme analysis. The genes B, C, F and G can be maintained stably in the cell even when there is efficient expression of these viral genes. Recombinant plasmids with the complete genes D and E can only be maintained when the expression of these genes is completely blocked. Expression of complete H and J genes could not yet be demonstrated. The intact gene A was apparently lethal for the host cell, as it was never found in the recombinants. The genes F and G are expressed, even when they are not preceded by one of the well characterized viral or plasmid promoter sequences. Screening of the nucleotide sequence of phi X174 gives two promoter-like sequences just in front of the two genes. Viral sequences with replication signals (the phi X174 (+) origin of replication, the initiation site for complementary strand synthesis and the incompatibility sequence) appeared to be functional also when inserted in recombinant plasmids. A plasmid with the phi X (+) origin can be forced to a rolling circle mode of replication. The A protein produced by infecting phages works in trans on the cloned viral origin. The (-) origin can function as initiation signal for complementary strand synthesis during transduction of single-stranded plasmid DNA. The intracellular presence of the incompatibility sequence on a plasmid prevents propagation of infecting phages.     

Transformation of Bacillus subtilis and Escherichia coli by a hybrid plasmid pCD1.

The gene thyP3 from Bacillus subtilis bacteriophage phi 3T was cloned in the plasmid pMB9. The resulting chimeric plasmid, pCD1, is effective in transforming both Escherichia coli and Bacillus subtilis to thymine prototrophy. The activity of the thyP3 gene product, thymidylate synthetase, was assayed and found to be 9 times greater in a transformed strain of Escherichia coli than in a phi 3T lysogen of Bacillus subtilis. The physical location of restriction sites has been determined for two related plasmids pCD1 and pCD2. Hybridization studies clearly indicate that the plasmid gene responsible for Thy+ transformation is the gene from the bacteriophage phi 3T. The lack of restriction in this transformation process is consistent with our previous studies using bacterial DNA in heterospecific exchanges indicating that the nucleotide sequence surrounding the gene is the dominant factor in determining interspecific transformation.     

High-Frequency Plasmid Transduction by Lactobacillus gasseri Bacteriophage φadh

The temperate bacteriophage adh mediates plasmid DNA transduction in Lactobacillus gasseri ADH at frequencies in the range of 10^-8 to 10^-10 transductants per PFU. BglII-generated DNA fragments from phage adh were cloned into the BclI site of the transducible plasmid vector pGK12 (4.4 kb). Phage adh lysates induced from Lactobacillus lysogens harboring pGK12 or the recombinant plasmids were used to transduce strain ADH to chloramphenicol resistance. The transduction frequencies of recombinant plasmids were 10²- to 10⁵-fold higher than that of native pGK12. The increase in frequency generally correlated with the extent of DNA-DNA homology between plasmid and phage DNAs. The highest transduction frequency was obtained with plasmid pTRK170 (6.6 kb), a pGK12 derivative containing the 1.4- and 0.8-kb BglII DNA fragments of adh. DNA hybridization analysis of pTRK170-transducing phage particles revealed that pTRK170 had integrated into the adh genome, suggesting that recombination between homologous sequences present in phage and plasmid DNAs was responsible for the formation of high-frequency transducing phage particles. Plasmid DNA analysis of 13 transductants containing pTRK170 showed that each had acquired intact plasmids, indicating that in the process of transduction a further recombination step was involved in the resolution of plasmid DNA monomers from the recombinant pTRK170::adh molecule. In addition to strain ADH, pTRK170 could be transduced via adh to eight different L. gasseri strains, including the neotype strain, F. Gasser 63 AM (ATCC 33323).     

Transductional selection of cloned bacteriophage phi 105 and SP02 deoxyribonucleic acids in Bacillus subtilis.

The Bacillus subtilis temperate bacteriophages phi 105 and SP02 are incapable of transduction of the small, multicopy drug resistance plasmids pUB110 and pCM194. Cloning endonuclease-generated fragments of phi 105 or SP02 DNA into each of the plasmids renders the chimeric derivatives susceptible to transduction specifically by the phage whose deoxyribonucleic acid is present in the chimera. The majority of phage deoxyribonucleic acid fragments identified that render plasmids transducible by phi 105 or SP02 appear to be internal fragments, not fragments containing the cohesive ends. However, the highest overall transduction frequency was observed in SP02-mediated transduction of a derivative of pUB110 containing a 1.6-megadalton EcoRI fragment that likely contains the SP02 cohesive ends (plasmid pPL1010). The transducing activity present in a phi 105 transducing lysate had a buoyant density slightly greater than infectious particles, whereas the majority of transducing particles in an SP02(pPL1010) transducing lysate had a buoyant density slightly less than infectious particles. Although no detectable change in plasmid structure resulted from transduction by phi 105 or SP02, deoxyribonucleic acid isolated from a purified SP02(pPL1010) transducing lysate contained no detectable monomeric pPL1010, but did contain a form of pPL1010 of higher molecular weight than the monomer.     

Plasmid involvement in the formation of a spontaneous bacteriophage insensitive mutant of Lactococcus lactis

Lactococcus lactis subsp. lactis biovar. diacetylactis DPC721 is a spontaneous bacteriophage insensitive mutant of strain DPC220, isolated after challenge with an industrial bacteriophage, phi D1. Plasmid analysis demonstrated that the bacteriophage insensitivity was associated with the absence of two native DPC220 plasmids (pAH82 and pAH33), and the presence of a novel plasmid (pAH90) in DPC721. The plasmids were transferred by conjugative mobilization to a plasmid free background where it was confirmed by restriction mapping that pAH90 is a co-integrate formed by the precise recombination of pAH82 and pAH33. The resistance phenotype encoded by pAH90 was also active against two bacteriophage homologous for the plasmid-free strain. Plasmid pAH90 was shown to encode at least two independent resistance mechanisms, including an adsorption-inhibition mechanism and a restriction and modification system. The adsorption-inhibition mechanism encoded by the co-integrate plasmid was specific for one of the phage used in this study.     

Small Staphylococcus aureus plasmids are transduced as linear multimers that are formed and resolved by replicative processes

The molecular processes involved in the transduction of small staphylococcal plasmids by a generalized transducing phage, phi 11, have been analysed. The plasmids are transduced in the form of linear concatemers containing only plasmid DNA; plasmid-initiated replication is required for their generation but additive interplasmid recombination is not. Concatemers are probably generated by the interaction of one or more phage functions with replicating plasmid DNA. Insertion of any restriction fragment of the phage into the plasmid causes an approximately 10(5)-fold increase in transduction frequency, regardless of the size or genetic content of the fragment. The resulting transducing particles (Hft particles) contain mostly pure linear concatemers composed of tandem repeats of the plasmid::phage chimera, and their production requires active plasmid-initiated replication. The high frequency of transduction is a consequence of homologous recombination between the linear chimeric and phage concatemers, which has the effect of introducing an efficient pac site into the former. Following introduction into lysogenic recipient bacteria, the transducing DNA is first converted to the supercoiled form, then processed to monomers by a mechanism that requires the active participation of the plasmid replication system.