Structural analysis of the actinophage phi C31 attachment site.

The lysogenisation of actinophage phi C31 in S. coelicolor J 1501 occurs by site-specific recombination. The DNA segments containing the attachment sites on the host chromosome, the phage genome and the two junctions created by the insertion of the prophage were cloned and the nucleotide sequences determined. The attachment sites (att) share an extremely short common sequence of three base pairs. Adjacent to the core sequences some direct- and inverted repeats were found as potential binding sites for proteins involved in site-specific recombination.     

Temperature-sensitive mutants of actinophage phi C31 of Streptomyces coelicolor A3(2)]

Temperature-sensitive mutants of phiC31 actinophage were isolated by the treatment with nitrosoquanidine and UV light. 20 cistron which control various stages of intracellular phage growth are identified on the basis of modified complementation tests. The sequence of ts markers and c-region is established and the relative distance between them is estimated according to the results of three factor crosses.     

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.     

Characterization of a late promoter from the Streptomyces temperate phage phi C31.

An operon expressed late in the lytic cycle of the Streptomyces temperate phage phi C31 was shown to be transcribed from an inducible promoter, phi lp (phage late promoter), which resembled the previously reported early promoters. mRNAs initiated at phi lp were processed at the 3' end (and possibly also the 5' end) of a tRNA(Thr)-like sequence, resulting in leaderless polycistronic mRNAs.     

Characterization of bacteriophage phi C69 of Saccharopolyspora erythraea and demonstration of heterologous actinophage propagation by transfection of Streptomyces and Saccharopolyspora

A bacteriophage, designated phi C69, isolated from a culture of Saccharopolyspora erythraea was characterized. The phage propagates on Sac. erythraea NRRL 2338 but does not infect 10 Streptomyces or 3 Micromonospora species tested. It infects Sac. erythraea NRRL 2359 but does not produce infectious phage particles in this host. phi C69 is approximately 40 kb in length and contains cohesive ends. A cos fragment containing ligated phage DNA ends was cloned in Escherichia coli. Restriction maps of the phage DNA and the cos fragment for several enzymes are shown. Transfection of both Sac. erythraea and Streptomyces lividans with phi C69 resulted in approximately equal titres of infectious phage particles produced from approximately the same number of regenerating cells. Transfection of Sac. erythraea with DNA from Streptomyces phages SH10 and KC404 also resulted in the production of infectious phage particles. The basis for differences among hosts in susceptibility to infection by various actinophages is discussed.     

Restriction mapping of the DNA of the Streptomyces temperate phage phi C31 and its derivatives

DNA of phi C31 propagated on Streptomyces lividans 66 contained no sites for the restriction enzymes BamHI, SalPI (=PstI) and XhoI; one for XbaI; three for HpaI; five for ClaI and KpnI; six for EcoRI; about 13 for HindIII; about 14 for BclI; and more than 15 for FspAI, HgiAI, SacI, SalGI and SmaI. A complete map of 20 sites (XbaI, HapI, ClaI, KpnI and EcoRI) was obtained using partial digestion and double digestion of DNA of the wild-type and deletion and insertion mutants. The total molecular size was estimated to be 41.2 kb.     

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 expression of Streptomyces and Escherichia coli drug-resistance determinants cloned into the Streptomyces phage phi C31

Lysogens obtained by infecting Streptomyces albus G with a phi C31-pBR322 chimaeric prophage or its delta W12 deletion derivative had increased tetracycline resistance. The ability of the delta W12 derivative to transduce tetracycline resistance was inactivated by inserting a viomycin resistance determinant (vph) into the BamHI site of the pBR322 tet gene, and restored by excising the vph gene. Another deletion mutant (delta W17) of the chimaera, carrying an intact tet gene, was normally unable to transduce tetracycline resistance. This inability was correlated with the finding, by Southern hybridisation analysis, that the att site required for insertion of phi C31 prophage into the host chromosome was located within the delta W17 deletion. Use of phi C31 lysogenic recipient permitted the integration of the att-deleted phage, presumably by homologous recombination, giving tetracycline-resistant double lysogens. This technique was extended to S. coelicolor A3(2) in the detection of derivatives of the att-deleted phage into which a thiostrepton-resistance determinant (tsr) had been inserted in vitro. Phage released from double lysogens were mainly recombinants. One such recombinant is a PstI vector for DNA cloning, able to accommodate up to 6 kb of introduced DNA.     

Survival of the temperate actinophage φC31 and Streptomyces lividans in soil and the effects of competition and selection on lysogens

Abstract Lysogenic infections were demonstrated in microcosms of sterile soil inoculated with Streptomyces lividans and the φC31 derivative, KC301, in free state or via lysogenized hosts. Intermittent soil mixing caused liberation of KC301 due to lysis of germinating lysogenized and uninfected spores. The presence of lysogenized host ensured that KC301 was maintained at a constant density. The lysogen S. lividans TK24 (KC301) achieved a population density lower than that of its non-lysogenized counterpart. Thiostrepton in the soil did not select for the thiostrepton resistance gene KC301. The long-term survival in soil of a temperate actinophage was demonstrated.     

The streptomyces genome contains multiple pseudo-attB sites for the (phi)C31-encoded site-specific recombination system

The integrase from the Streptomyces phage (phi)C31 is a member of the serine recombinase family of site-specific recombinases and is fundamentally different from that of lambda or its relatives. Moreover, (phi)C31 int/attP is used widely as an essential component of integration vectors (such as pSET152) employed in the genetic analysis of Streptomyces species. phiC31 or integrating plasmids containing int/attP have been shown previously to integrate at a locus, attB, in the chromosome. The DNA sequences of the attB sites of various Streptomyces species revealed nonconserved positions. In particular, the crossover site was narrowed to the sequence 5'TT present in both attP and attB. Strains of Streptomyces coelicolor and S. lividans were constructed with a deletion of the attB site ((Delta)attB), and pSET152 was introduced into these strains by conjugation. Thus, secondary or pseudo-attB sites were identified by Southern blotting and after rescue of plasmids containing DNA flanking the insertion sites from the chromosome. The sequences of the integration sites had similarity to those of attB. Analysis of the insertions of pSET152 into both attB(+) and (Delta)attB strains indicated that this plasmid can integrate at several loci via independent recombination events within a transconjugant.     

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.     

Two genes involved in the phase-variable phi C31 resistance mechanism of Streptomyces coelicolor A3(2).

The phage growth limitation (Pgl) system of Streptomyces coelicolor confers resistance to phi C31 and its homoimmune phages. The positions of the pgl genes within a 16-kb clone of S. coelicolor DNA were defined by subcloning, insertional inactivation, and deletion mapping. Nucleotide sequencing and functional analysis identified two genes, pglY and pglZ, required for the Pgl+ (phage-resistant) phenotype. pglY and pglZ, which may be translationally coupled, are predicted to encode proteins with M(r)S of 141,000 and 104,000, respectively. Neither protein shows significant similarity to other known proteins, but PglY has a putative ATP/GTP binding motif. The pglY and pglZ genes are cotranscribed from a single promoter which appears to be constitutive and is not induced by phage infection.