Glycosylation of a Streptomyces coelicolor A3(2) cell envelope protein is required for infection by bacteriophage phi C31

Mutants of Streptomyces coelicolor A3(2) J1929 (Delta pglY) were isolated that were resistant to the Streptomyces temperate phage phi C31. These strains could be transfected with phi C31 DNA, but could not act as infective centres after exposure to phage. Thus, it was concluded that infection was blocked at the adsorption/DNA injection step. The mutants fell into three classes. Class I mutants were complemented by a gene, SCE87.05, isolated from the cosmid library of S. coelicolor A3(2). The product of SCE87.05 had good overall homology to a Mycobacterium tuberculosis hypothetical protein and regions with similarity to dolichol phosphate-D-mannose:protein O-D-mannosyltransferases. Concanavalin A (ConA) inhibited phi C31 infection of S. coelicolor J1929, and this could be partially reversed by the addition of the sugar, alpha-D-methyl-pyranoside. Moreover, glycosylated proteins from J1929, but not from the class I mutant DT1017, were detected using ConA as a probe in Western blots. Class I and II mutants were sensitive to phi C31hc, a previously isolated phage exhibiting an extended host range phenotype, conferred by h. A phage with the same phenotype, phi DT4002, was isolated independently, and a missense mutation was found in a putative tail gene. It is proposed that the phi C31 receptor is a cell wall glycoprotein, and that the phi C31h mutation compensates for the lack of glycosylation of the receptor.     

Cloning of whiG, a gene critical for sporulation of Streptomyces coelicolor A3(2).

In whiG mutants of Streptomyces coelicolor A3(2), aerial hyphae do not show any sign of sporulation. A library of S. coelicolor DNA was prepared in a phi C31 temperate phage vector (KC516), and one recombinant phage (KC750) that could restore the wild-type phenotype to a collection of whiG mutants when integrated into their genomes was found. Subcloning experiments with low- and high-copy-number Streptomyces plasmid vectors allowed partial localization of whiG in the cloned DNA and revealed that hypersporulation was associated with the presence of extra copies of whiG.     

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.     

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.     

Genetic characteristics and genome structure of Streptomyces coelicolor actinophages]

Actinophage phi C31 of Streptomyces coelicolor A3 (2) and two novel temperate actinophages phi C43 and phi C62 isolated from strains of blue actinomycetes group are homoimmune, serologically and functionally related. DNA molecules of phages phi C31, phi C43 and phi C62 have cohesive ends; sizes of DNAs of these phages and some mutants have been determined. The extent of homology between the DNAs of three phages is 93-96% as shown by heteroduplex analysis. The regions of non-homology are of a deletion-insertion type and of approximately 1500 base pairs in the length. Location of deletions in DNAs of mutant phages phi C31 vd and phi C31 c5 has been shown. Structural modifications in phage dnas have been found only to occur in the right part of molecules. Heteroduplex maps have been constructed for all phages studied.     

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.     

A triggered-suicide system designed as a defense against bacteriophages.

A novel bacteriophage protection system for Lactococcus lactis based on a genetic trap, in which a strictly phage-inducible promoter isolated from the lytic phage phi31 is used to activate a bacterial suicide system after infection, was developed. The lethal gene of the suicide system consists of the three-gene restriction cassette LlaIR+, which is lethal across a wide range of gram-positive bacteria. The phage-inducible trigger promoter (phi31P) and the LlaIR+ restriction cassette were cloned in Escherichia coli on a high-copy-number replicon to generate pTRK414H. Restriction activity was not apparent in E. coli or L. lactis prior to phage infection. In phage challenges of L. lactis(pTRK414H) with phi31, the efficiency of plaquing was lowered to 10(-4) and accompanied by a fourfold reduction in burst size. Center-of-infection assays revealed that only 15% of infected cells released progeny phage. In addition to phage phi31, the phi31P/LlaIR+ suicide cassette also inhibited four phi31-derived recombinant phages at levels at least 10-fold greater than that of phi31. The phi31P/LlaIR+-based suicide system is a genetically engineered form of abortive infection that traps and eliminates phages potentially evolving in fermentation environments by destroying the phage genome and killing the propagation host. This type of phage-triggered suicide system could be designed for any bacterium-phage combination, given a universal lethal gene and an inducible promoter which is triggered by the infecting bacteriophage.     

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.     

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 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.     

Phi SC623, a temperate actinophage of Streptomyces coelicolor Müller, and its relatives phi SC347 and phi SC681

Three species-specific, temperate actinophages of Streptomyces coelicolor Müller, phi SC623, phi SC347 and phi SC681, were compared with respect to host range, virion structure, antiserum cross-inactivation, DNA-restriction pattern, DNA hybridization, and DNA base composition. The restriction map of phi SC623 (57 kb) was established with eight restriction enzymes; the homologies of this phage with phi SC347 and phi SC681 suggested that it might be a hybrid phage composed of approximately equal parts homologous to one of the other two phages. No homology was detected between phi SC623 and R4, a temperate, wide-host-range phage which can also lysogenize S. coelicolor Müller.     

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.