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Journal of Bacteriology, November 2002, p . 6081-6083, Vol . 184, No . 21
A Gene Encoding a Homologue of Dolichol Phosphate-ß-D-Mannose Synthase Is Required for Infection of Streptomyces coelicolor A3(2) by Phage
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| ABSTRACT |
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We have shown previously that a gene encoding a homologue to the eukaryotic dolichol-phosphate-D-mannose, protein O-D-mannosyltransferase, was required for
C31 infection of Streptomyces coelicolor . Here we show that a gene encoding the homologue to dolichol-phosphate-mannose synthase is also essential for phage sensitivity . These data confirm the role of glycosylation in the phage receptor for
C31 in S . coelicolor .
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The temperate phage
C31 has been used to develop phage vectors for genetic manipulation of Streptomyces species, many of which are commercially useful producers of antibiotics and other bioactive secondary metabolites (4, 8) .
C31 has a moderately broad host range and is able to infect around a third of the Streptomyces species tested (9, 18) . The earliest event in phage infection is the recognition by phage tail proteins of some component on the host cell surface (7, 11, 15) . While this initial step is often reversible, at some point a commitment to infection occurs in which a series of molecular changes in the phage results in the injection of DNA into the cell . Studies of the molecules on the cell surface recognized by the
C31 coat proteins will lead to a better understanding of the Streptomyces cell wall and to further development of phage vectors .
We have indicated previously that glycosylation of a Streptomyces coelicolor cell envelope protein is required for infection of the cell by
C31 (5) . Mutants of S . coelicolor strain J1929 (
pglY and therefore sensitive to
C31 [1]) that are unable to support plaque formation by
C31c25, a clear-plaque mutant of
C31, were isolated . The S . coelicolor mutants were unable to form infective centers after contact with phage but could release phage particles after being transfected with phage DNA . These observations indicated that the mutants are blocked early in phage infection, probably at the stage of receptor binding . The S . coelicolor mutants fell into three classes, designated I, II, and III . A gene isolated from the S . coelicolor ordered cosmid library, SCE87.05, complemented the class I mutants but not those of class II or III . SCE87.05 has significant similarity to the eukaryotic dolichol-phosphate-D-mannose (Dol-P-Man), protein O-D-mannosyltransferases (protein mannosyltransferase), suggesting that the receptor for
C31 infection is a glycoprotein generated through an O glycosylation pathway . This suggestion was supported by observations that the plant lectin concanavalin A could inhibit phage infection and that glycoproteins could not be detected by concanavalin A in Western blots of proteins from a mutant defective in SCE87.05 . A mutant phage,
C31hc, could form plaques on class I and class II mutants of S . coelicolor but not on class III mutants . It was proposed that
C31 normally recognizes a cell wall glycoprotein but that
C31hc recognizes the unglycosylated protein . Thus, like the class I mutants, the class II mutants are predicted to be defective in the glycosylation pathway and the class III mutants are predicted to lack the protein target of glycosylation . Here we have tested part of this model with experiments to investigate the role of a gene, SC6D7.16, encoding a homologue of Dol-P-Man synthase, proposed to be required for the protein glycosylation pathway .
Dol-P-Man catalyzes the transfer of mannose from GDP-mannose to Dol-P in fungi and in mammals (10, 17) . Dol-P-Man is then the substrate for the transfer of the mannose to a serine or threonine by the protein glycosyltransferase in the O- glycosylation pathway . The protein sequences of the fungal and mammalian Dol-P-Man synthases were used to search the S . coelicolor genome sequence (http://www.sanger.ac.uk/Projects/S_coelicolor/) . The closest homologue was the predicted product of SC6D7.16 (Fig . 1) . The cosmid SC6D7 was therefore introduced by protoplast transformation into two of the class II phage-resistant mutants, DT1029 and DT2021, and into the S . coelicolor class III mutant, DT2017, isolated previously (5) . Seven individual transformants of each strain were tested for sensitivity to phage by using a plate assay . This was performed by preinoculating one-half of an R1M plate with
C31c25 and then streaking spores from the transformants across the plate from the phage-free half to the phage-containing half . SC6D7 conferred sensitivity to phage
C31c25 in approximately 50% of the transformants of DT1029 and DT2021 (the class II mutants) . This incomplete complementation appears to be typical of complementation by cosmids, and it is believed that the mutant and wild-type alleles undergo homogenotization (5, 13) . To confirm the complementation, we constructed a plasmid, pDT16, that carried only SC6D7.16 . PCR was used to amplify the open reading frame and flanking DNA from cosmid SC6D7, and the PCR product was inserted into the integrating vector pSET152 (3) . This plasmid was introduced by conjugation into a class I strain, DT1017; the class II strains DT1028, DT1029, DT1035, DT2008, and DT2021; and the class III mutant DT2017 . Seven individual transconjugants from each mating were tested for sensitivity to phage by using the plate assay . Phage sensitivity was restored to all the transconjugants tested from the class II mutants DT1029, DT1035, and DT2021, and this was confirmed in a plaque assay (Fig . 2) . The class II mutants DT1028 and DT2008 remained phage resistant (data not shown) . As expected, pDT16 did not restore phage sensitivity to
C31c25 in either the class I or class III mutant .
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To confirm the requirement for SC6D7.16 in phage infection, we constructed an S . coelicolor strain, J1929::pDT15, containing a targeted insertion in SC6D7.16 . A DNA fragment internal to SC6D7.16 was amplified by using primers DT23 and DT24, inserted into the suicide vector pSET151 (3), and introduced into the phage-sensitive strain J1929 (1) by conjugation . Integration of this construct by insert-directed homologous recombination into S . coelicolor J1929 should give rise to a disrupted version of SC6D7.16 . All seven transconjugants tested were resistant to
C31 by the plate assay . Spores were amplified and used for plaque assays with
C31c25 and were resistant to infection, although a few very cloudy plaques could be observed at high phage titers (Fig . 2) . As expected, introduction of pDT16 encoding the wild-type allele of SC6D7.16 into J1929::pDT15 resulted in phage sensitivity (data not shown) . In addition, J1929::pDT15 spores could support plaque formation by
C31hc (data not shown) . This phenotype is consistent with a class II phage resistance phenotype (5) .
These observations indicate that the protein product of SC6D7.16 is essential for
C31 infection of S . coelicolor . SC6D7.16 is a homologue of the eukaryotic Dol-P-Man synthases (Fig . 1) . In eukaryotes, Dol-P-Man provides the mannosyl residues in glycosylphosphatidylinositols and in N, O, and C glycosylation . A knockout of the single-copy gene DPM1, which codes for Dol-P-Man synthase, resulted in complete loss of protein mannosylation in Saccharomyces cerevisiae (12) . Prokaryotes do not have dolichol . They contain instead other polyprenols, in particular undecaprenol phosphate (14) . There is little information on the polyprenols in the Streptomyces cell envelope; however, the closely related bacteria Mycobacterium tuberculosis and Mycobacterium smegmatis contain a variety of polyprenol phosphates, which are covalently attached to mannose (Pol-P-Man) (6) . The polyprenol phosphates in M . smegmatis have been shown to be used for several biosynthetic pathways for cell wall components, including mycolic acids, arabinogalactan, arabinomannan, and lipoarabinomannan (2, 6) . Pol-P-Mans in mycobacteria are thought to be synthesized by Ppm1, a functional analogue to the eukaryotic Dol-P-Man synthase and the closest homologue to SC6D7.16 (Fig . 1) .
The data presented here, together with previous findings (5), indicate that SCE87.05 and SC6D7.16, which encode a putative glycosyltransferase and a putative Pol-P-Man synthase, respectively, are required for the synthesis of the
C31 receptor in S . coelicolor . Similar to the roles of equivalent proteins in eukaryotes, these enzymes probably catalyze two steps in a protein glycosylation pathway in S . coelicolor . The observation that not all of the class II mutants are complemented by SC6D7.16 suggests that DT2008 and DT1028 are defective in other genes, possibly those encoding other enzymes in the glycosylation pathway . It is clear from these studies that these proteins are not essential for the growth of S . coelicolor, implying that they are not exclusively required components of cell wall biosynthesis . The role of protein glycosylation is not known either in the mycobacteria or in Streptomyces, but it will hopefully become clearer once the targets for glycosylation have been characterized .
| ACKNOWLEDGMENTS |
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We are grateful to Helen Kieser for providing cosmid SC6D7 .
We thank the BBSRC for funding (project grant 42/P09260) .
| FOOTNOTES |
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* Corresponding author . Mailing address: Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, United Kingdom . Phone: 44 115 9709778 . Fax: 44 115 9709906 . E-mail: Maggie.smith{at}nottingham.ac.uk .
| REFERENCES |
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C31 resistance mechanism of Streptomyces coelicolor A3(2) . J . Bacteriol . 177:4681-4689.
C31 . Mol . Microbiol . 41:601-610.
C31 and determination of its cohesive ends . FEMS Microbiol . Lett . 62:347-353.
can functionally replace the receptor-recognizing part of a long tail fiber protein of the unrelated bacteriophage T4 . J . Bacteriol . 171:4378-4384.
C31: nucleotide sequence, analysis and functional cloning . Mol . Gen . Genet . 213:269-277.
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