Role of staphylococcal phage and SaPI integrase in intra- and interspecies SaPI transfer

SaPIbov2 is a member of the SaPI family of staphylococcal pathogenicity islands and is very closely related to SaPIbov1. Typically, certain temperate phages can induce excision and replication of one or more of these islands and can package them into special small phage-like particles commensurate with their genome sizes (referred to as the excision-replication-packaging [ERP] cycle). We have studied the phage-SaPI interaction in some depth using SaPIbov2, with special reference to the role of its integrase. We demonstrate here that SaPIbov2 can be induced to replicate by different staphylococcal phages. After replication, SaPIbov2 is efficiently encapsidated and transferred to recipient organisms, including different non-Staphylococcus aureus staphylococci, where it integrates at a SaPI-specific attachment site, att(C), by means of a self-coded integrase (Int). Phages that cannot induce the SaPIbov2 ERP cycle can transfer the island by recA-dependent classical generalized transduction and can also transfer it by a novel mechanism that requires the expression of SaPIbov2 int in the recipient but not in the donor. It is suggested that this mechanism involves the encapsidation of standard transducing fragments containing the intact island followed by int-mediated excision, circularization, and integration in the recipient.     

Specificity of staphylococcal phage and SaPI DNA packaging as revealed by integrase and terminase mutations

SaPI1 and SaPIbov1 are chromosomal pathogenicity islands in Staphylococcus aureus that carry tst and other superantigen genes. They are induced to excise and replicate by certain phages, are efficiently encapsidated in SaPI-specific small particles composed of phage virion proteins and are transferred at very high frequencies. In this study, we have analysed three SaPI genes that are important for the phage–SaPI interaction, int (integrase) terS (phage terminase small subunit homologue) and pif (phage interference function). SaPI1 int is required for SaPI excision, replication and packaging in a donor strain, and is required for integration in a recipient. A SaPI1 int mutant, following phage induction, produces small SaPI-specific capsids which are filled with partial phage genomes. SaPIbov1 DNA is efficiently packaged into full-sized phage heads as well as into SaPI-specific small ones, whereas SaPI1 DNA is found almost exclusively in the small capsids. TerS, however, determines DNA packaging specificity but not the choice of large versus small capsids. This choice is influenced by SaPIbov1 gene 12, which prevents phage DNA packaging into small capsids, and which is also primarily responsible for interference by SaPIbov1 with phage reproduction.     

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).     

Temperature-sensitive mutations affecting the replication of F-prime factors in Escherichia coli K 12

Summary More temperature-sensitive mutants affecting the replication of the F-gal⁺ episome of Escherichia coli K12 have been isolated. Eight of the mutations were located on F itself and three were located on the chromosome.The temperature sensitive F-gal⁺'s have been integrated into the chromosome to produce Hfr strains. These Hfr strains have transfer origins similar to Hfr Cavalli, and all show aberrant excision and transfer of elongated segments of the chromosome including the integrated F-gal to generate long merodiploids.The chromosomal mutations that govern the replication of F have been termed seg (for segregation). Wild-type F-gal⁺ can be integrated into seg cells at 42° C to give Hfrs, in a process analogous to integrative suppression in the formation of Hfrs from cells carrying mutations that are temperature-sensitive for chromosomal DNA replication (dnaA). A curious feature of an Hfr derived from a seg strain is that it also shows F-genote enlargement as well as normal transfer of chromosomal genetic marker. Preliminary transductional mapping data show that the mutation seg-2 is linked to the threonine locus (minute 0).     

Conditions and mutations affecting the maintenance and replication of plasmid DNA in Escherichia coli 15

Summary An Escherichia coli 15 strain has been constructed which contains, in addition to the plasmids inherent to E. coli 15 (P 1-like DNA and minicircular DNA), the colicinogenic factor E₁ (Col E₁). Whereas the P 1-like DNA of E. coli 15 is unaffected by the uptake of the colicin plasmid, the number of copies of minicircular DNA of E. coli 15 decreases and an equivalent amount of Col E₁ DNA becomes established in the cell. The ratio between these two small plasmids is dependent on the growth temperature. The mode of replication of minicircular DNA and Col E₁ DNA is very similar, but is different in various respects from that of the P 1-like plasmid: 1. Both small plasmids continue to replicate in the presence of chloramphenicol, whereas the replication of P 1-like DNA stops like the chromosomal DNA. 2. Rifampicin inhibits the synthesis of both small plasmids rather rapidly. The replication of P 1-like DNA continues during the remaining replication cycle of the chromosome in the presence of rifampicin. 3. The replication of Col E₁ DNA and of the minicircular DNA still proceeds at elevated temperatures (45–50°C), whereas little or no incorporation of ³H-thymidine into P 1-like DNA is observed at these temperatures. 4. Mutants have been obtained, which show altered properties in the maintenance and replication of the plasmids without being affected in the replication of the chromosomal DNA. In all these mutants the replication and (or) maintenance of the minicircular DNA of E. coli 15 and Col E₁ DNA is affected in the same way, but not that of the P 1-like plasmid.     

DNA replication in the autonomous parvoviruses

Both ends of the linear single-stranded parvoviral DNA genome contain short palindromic sequences which form duplex hairpins containingcis-acting information required for replication and encapsidation. DNA synthesis is primed directly by the 3′ end, and genomes are replicated through multimeric duplex intermediates by unidirectional, leading-strand synthesis. Unit-length genomes are excised from these concatemers, and their telomeres replicated, by the viral NS1 protein, which introduces a single-strand nick into specific origin sequences, becoming covalently attached to the 5′ end at the nick and providing a 3′ hydroxyl which primes synthesis of a new copy of the telomere. Progeny DNA synthesis requires ongoing replication and is dependent upon packaging.     

Infectivity of phage P2 DNA in presence of helper phage

Summary Phenol extracted deoxyribonucleic acid of temperate bacteriophage P2 infects E. coli strains C and K 12 with about equal efficiency. Infection occurs only if the bacteria exposed to P2 DNA are simultaneously infected with a related helper phage. Deoxyribonuclease completely destroys the infectivity of the DNA extract. The kinetics of the development of competence and the dependence of the number of infectious units on the multiplicity of infection of helper phage are compared with those of the DNA system. The molecular weight of P2 DNA was determined by sedimentation in a sucrose density gradient to be 2.20±0.2x10⁷.     

Cloning of DNA segments of phage 2C, which allows autonomous plasmid replication in Bacillus subtilis

The chromosome of Bacillus subtilis phage 2C is a 100-MDa double-stranded DNA molecule, containing hydroxymethyluracil in place of thymine and carrying redundant ends each encompassing 10% of the genome. 2C DNA was cleaved with EcoRI and HindIII, and cloned in the shuttle plasmids pSC 540 and pCP 115, both containing segments originating from B. subtilis and Escherichia coli plasmids. These chimaerical plasmids, carrying the chloramphenicol resistance gene, were unable to replicate in B. subtilis; this ability was restored, however, after the insertion of viral DNA segments. Physical maps of the recombinant plasmids were made; a large deletion of the E. coli-derived segment of pSC 540 was observed (which paralleled a loss of replication in this host), whereas addition of 2C DNA segments in pCP 115 was not accompanied by deletion (replication in E. coli was conserved in this case). Cloned viral segments mapped mostly, but not exclusively, within the redundant ends of 2C DNA. It is suggested that the thirteen recombinant clones carried the replication origin region of phage 2C DNA, and that these sequences originated within or close to the redundant extremities of the viral chromosome.     

Frameshift suppressor mutations affecting the major glycine transfer RNAs of Saccharomyces cerevisiae

Mutations have been identified in Saccharomyces cerevisiae glycine tRNA genes that result in suppression of +1 frameshift mutations in glycine codons. Wild-type and suppressor alleles of genes encoding the two major glycine tRNAs, tRNA(GCC) and tRNA(UCC), were examined in this study. The genes were identified by genetic complementation and by hybridization to a yeast genomic library using purified tRNA probes. tRNA(UCC) is encoded by three genes, whereas approximately 15 genes encode tRNA(GCC). The frameshift suppressor genes suf1+, suf4+ and suf6+ were shown to encode the wild-type tRNA(UCC) tRNA. The suf1+ and suf4+ genes were identical in DNA sequence, whereas the suf6+ gene, whose DNA sequence was not determined, was shown by a hybridization experiment to encode tRNA(UCC). The ultraviolet light-induced SU F1-1 and spontaneous SU F4-1 suppressor mutations were each shown to differ from wild-type at two positions in the anticodon, including a +1 base-pair insertion and a base-pair substitution. These changes resulted in a CCCC four-base anticodon rather than the CCU three-base anticodon found in wild-type. The RNA sequence of tRNA(UCC) was shown to contain a modified uridine in the wobble position. Mutant tRNA(CCCC) isolated from a SU F1-1 strain lacked this modification. Three unlinked genes that encode wild-type tRNA(GCC), suf20+, trn2, and suf17+, were identical in DNA sequence to the previously described suf16+ frameshift suppressor gene. Spontaneous suppressor mutations at the SU F20 and SU F17 loci were analyzed. The SU F20-2 suppressor allele contained a CCCC anticodon. This allele was derived in two serial selections through two independent mutational events, a +1 base insertion and a base substitution in the anticodon. Presumably, the original suppressor allele, SU F20-1, contained the single base insertion. The SU F17-1 suppressor allele also contained a CCCC anticodon resulting from two mutations, a +1 insertion and a base substitution. However, this allele contained an additional base substitution at position 33 adjacent to the 5' side of the four-base anticodon. The possible origin and significance of multiple mutations leading to frameshift suppression is discussed.     

Conjugative transfer and autonomous replication of a promiscuous IncQ plasmid in the cyanobacterium Synechocystis PCC 6803

Summary The promiscuous IncQ plasmid pKT210 (Cm^r, Sm^r) is efficiently transferred by transpecific conjugation from Escherichia coli to the facultatively heterotrophic cyanobacterium Synechocystis PCC6803 when mobilized by a helper plasmid coding for IncP transfer functions. The IncQ plasmid is stably maintained in the cyanobacterium as an autonomously replicating multicopy plasmid with no detectable structural alterations and can be recovered by transformation back to E. coli when using a mcrA mcrB host. Thus, the replicative host-range of IncQ plasmids extends beyond purple bacteria to the distinct procaryotic taxon of cyanobacteria, allowing the use of these small plasmids as convenient cloning vectors in Synechocystis PCC6803 and presumably also in cyanobacteria that are not amenable to genetic transformation. In contrast, an IncQ plasmid bearing the TRP1 gene of Saccharomyces cerevisiae failed to replicate when transferred to that yeast by transformation.     

Stimulation of adenovirus replication in simian cells in the absence of a helper virus by pretreatment of the cells with iododeoxyuridine.

Pretreatment of African green monkey kidney cells with 50 mu g of 5'-iododeoxyruidine (IUdR) per ml can modify their susceptibility to the replication of human adenovirus type 7 in the absence of simian virus 40 (SV40) although this enhancement of adenovirus replication is not as efficient as that of the helper SV40 virus. Since the number of infectious centers remains unchanged after IUdR pretreatment whereas the burst size of virus from each infected cell increases, the IUdR appears to allow each infected cell to produce more virus. Cell DNA synthesis appears to be stimulated in IUdR pretreated cells infected with adenovirus 7, but the host cell DNA synthesized is small enough to remain in the Hirt supernatant fluid. The modification of susceptibility to adenovirus replication and the changed pattern of cell DNA synthesis is stable for at least two additional cell passages of the pretreated cells.     

Genetic mapping of chromosomal mutations affecting the replication of the F-factor of Escherichia coli

Summary A genetic study has been performed on a set of mutations which prevent the replication of F-factors in Escherichia coli K-12. The gene affected is designated seg and has been located by transduction in the serB-thr segment of the chromosome. The seg gene does not appear to be related to the dnaC locus.     

An improved helper phage system for efficient isolation of specific antibody molecules in phage display

Phage display technology has been applied in many fields of biological and medical sciences to study molecular interactions and especially in the generation of monoclonal antibodies of human origin. However, extremely low display level of antibody molecules on the surface of phage is an intrinsic problem of a phagemid-based display system resulting in low success rate of isolating specific binding molecules. We show here that display of single-chain antibody fragment (scFv) generated with pIGT3 phagemid can be increased dramatically by using a genetically modified Ex-phage. Ex-phage has a mutant pIII gene that produces a functional wild-type pIII in suppressing Escherichia coli strains but does not make any pIII in non-suppressing E.coli strains. Packaging phagemids encoding antibody-pIII fusion in F⁺ non-suppressing E.coli strains with Ex-phage enhanced the display level of antibody fragments on the surfaces of recombinant phage particles resulting in an increase of antigen-binding reactivity >100-fold compared to packaging with M13KO7 helper phage. Thus, the Ex-phage and pIGT3 phagemid vector provides a system for the efficient enrichment of specific binding antibodies from a phage display library and, thereby, increases the chance of obtaining more diverse antibodies specific for target antigens.     

Eclipse-synchrony relationship in Escherichia coli strains with mutations affecting sequestration, initiation of replication and superhelicity of the bacterial chromosome

Initiation of replication from oriC on the Escherichia coli chromosomes occurs once and only once per generation at the same cell mass per origin. During rapid growth there are overlapping replication cycles, and initiation occurs synchronously at two or more copies of oriC. Since the bacterial growth can vary over a wide range (from three divisions per hour to 2.5 hours or more per division) the frequency of initiation should change in coordination with bacterial growth. Prevention of reinitiation from a newly replicated origin by temporary sequestration of the hemi-methylated GATC-sites in the origin region provides the molecular/genetic basis for the maintenance of the eclipse period between two successive rounds of replication. Sequestration is also believed to be responsible for initiation synchrony, since inactivation of either the seqA or the dam gene abolishes synchrony while drastically reducing the eclipse. In this work, we attempted to examine the functional relationship(s) between the eclipse period and the synchrony of initiation in E.coli strains by direct measurements of these parameters by density-shift centrifugation and flow-cytometric analyses, respectively. The eclipse period, measured as a fraction of DNA-duplication times, varied continuously from 0.6 for the wild-type E.coli K12 to 0.1 for strains with mutations in seqA, dam, dnaA, topA and gyr genes (all of which have been shown to cause asynchrony) and their various combinations. The asynchrony index, a quantitative indicator for the loss of synchrony of initiation, changed from low (synchronous) to high (asynchronous) values in a step-function-like relationship with the eclipse. An eclipse period of approximately 0.5 generation time appeared to be the critical value for the switch from synchronous to asynchronous initiation.