Dispensable sequences and packaging constraints of DNA from the Streptomyces temperate phage phi C31

Deletion mutants of the temperate Streptomyces phage phi C31 were selected by two methods: resistance to the chelating agent sodium pyrophosphate, and plating of a phi C31::pBR322 hybrid phage on Streptomyces albus G to obtain large plaque mutants. The deletions defined a 7.7 kilobase (kb) segment of the phi C31 genome which is inessential for plaque formation, in addition to a shorter segment including the repressor gene. Analysis of deletions and insertions suggested that the minimum size of the phi C31 genome allowing plaque formation is 37.5 kb (91% of the wild-type length of 41.2 kb), and the maximum is at least 42.4 kb (103%). These results indicate that it should be possible to introduce up to 10 kb of foreign DNA into a suitably developed phi C31 vector.     

The restriction mapping of c gene deletions in Streptomyces bacteriophage phi C31 and their use in cloning vector development

In addition to 20 previously mapped restriction sites in the DNA of phi C31, we have determined eight sites for SphI, four for EcoRV, and two for SstII; there are none for BglII or SstI. Nine sites were in a 12-kb segment of DNA containing no previously mapped sites. Deletions causing clear-plaque morphology were located in this part of the DNA, in a 3-kb interval between an EcoRV and an SphI site at the centre of the DNA molecule. One of the deletions (delta C3) was obtained in a previously described phi C31c+::vph (viomycin phosphotransferase) derivative containing two PstI sites separated by 3.9-kb of inessential DNA. After in vitro PstI treatment, plaque-forming phages lacking the 3.9-kb fragment were obtained from the c+ phage but not from its delta C3 derivative. Thus a 36.2-kb genome, but not one of 34.4 kb, was able to give infectious virions. PstI-generated DNA fragments of up to 8 kb can be inserted in vitro into the delta C3 derivative with retention of the vph selective marker. With the insertion of a 6.03-kb PstI fragment of plasmid SCP2, the latter phage became a potential vector (with loss of vph) for BamHI-generated DNA fragments of up to 9 kb. In the course of this work, several ClaI sites in phi C31::pBR322 bifunctional replicons were shown to be lost when the DNA was propagated in a dam+ Escherichia coli strain. This will allow the use of such replicons for the cloning of ClaI-generated DNA fragments of up to 6.7 kb.     

Genetic mapping and characteristics of the actinophage phi C31 deletion mutants of Streptomyces coelicolor A3(2) incapable of lysogenization

Actinophage phi C31 deletion c mutants with impaired ability to make repressor were genetically studied. Genetic crosses indicate that the c28 deletion mutant is situated with the c-region of the phi C31 genetic map. Based on the results of a qualitive test for recombination between several c mutants, a scheme of their order relative to deletion mutants was presented. The approximate distances between eight c mutants have been represented in units of the physical DNA map estimation. Genetic studies of actinophage lyg deletion mutants which cannot lysogenize sensitive cultures were carried out. Mutants failed to lysogenize upon mixed infection with lyg+ phages. The absence of the effect of lyg+ gene in trans suggests that lyg deletions cause a structural defect in an integration site of the phage. Preliminary data on alignment of lyg positions on physical and genetic maps of phi C31 phage have been obtained. According to evidence from genetic crosses, lyg mutation has been located in the right half of the phi C31 genome.     

New derivatives of the Streptomyces temperate phage phi C31 useful for the cloning and functional analysis of Streptomyces DNA

The thiostrepton resistance gene (tsr) of Streptomyces azureus, and a synthetic oligonucleotide adapter sequence, were introduced into the DNA of attP-site-deleted phage phi C31-based cloning vectors. The DNA of two of the new derivatives, KC515 and KC516, contains single sites for the enzymes BamHI, BglII, PstI, PvuII, SstI (two sites close together) and XhoI, available for the insertion of DNA of up to 4 kb. The two vectors also contain a cloned, promoterless viomycin phosphotransferase gene (vph) from Streptomyces vinaceus. When an internal segment of the Streptomyces coelicolor glycerol (gyl) operon was inserted at the appropriate position and in the correct orientation next to vph, it could bring about in vivo recombination leading to fusion of vph of the chromosomally located gyl operon, resulting in glycerol-regulated expression of viomycin resistance. Two other new phi C31 derivatives, KC505 and KC518, are PstI and BamHI replacement vectors, respectively, for 2-8-kb DNA fragments, and allow simple screening for the presence of inserted DNA.