Department of Biology, Dalhousie University, Halifax, Nova Scotia, CanadaB3H 4J11
Author for correspondence: Leo C. Vining. Tel: +1 902 494 2040. Fax: +1 902 494 3736. e-mail: leo.vining{at}dal.ca
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ABSTRACT |
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Keywords: phosphopantetheinyl transferase gene, jad genes, polyketide antibiotic, acyl carrier protein synthase
Abbreviations: ACP, acyl carrier protein; ACPS, holo-acyl carrier protein synthase; act, actinorhodin biosynthesis gene; ArCP, aryl carrier protein; CLF, chain-length factor; cml, chloramphenicol biosynthesis gene; fren, frenolicin biosynthesis gene; gra, granaticin biosynthesis gene; jad, jadomycin biosynthesis gene; KS, ketosynthase; NRPS, non-ribosomal peptide synthetase; PCP, peptidyl carrier protein; PKS, polyketide synthase; Ppan, phosphopantetheine; PPTase, phosphopantetheinyl transferase
The GenBank accession numbers for the jadM and jadNOX sequences reported in this paper are AF222693 and AY026363, respectively.
a Present address: Department of Medicine, McMaster University, Hamilton, Ontario, Canada.
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INTRODUCTION |
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The polyketide family of secondary metabolites shares a general biosynthetic mechanism with the fatty acids of primary metabolism. The carbon skeletons are formed by sequential condensation of activated low-molecular-mass acids, such as acetate, propionate and butyrate. The PKS condensation reaction is similar to that used by the fatty acid synthases (FAS) found in all organisms where fatty acids are required for the biosynthesis of lipids (Hopwood, 1997 ). Both PKS and FAS enzyme complexes require post-translational modification of their constituent acyl carrier proteins to become catalytically active. The inactive apo-proteins are converted to active holo-enzymes by esterifying a specific serine hydroxyl with the 4'-phosphopantetheine prosthetic group of coenzyme A (Lambalot et al., 1996
). Genes for the phosphopantetheinyl transferases of fatty acid synthase (ACPS) and enterobactin synthetase (EntD) in E. coli have been identified (Lambalot et al., 1996
), as has the gene for Sfp, responsible for activating surfactin synthetase in Bacillus subtilis (Quadri et al., 1998a
).
The jad cluster of genes for jadomycin production in S. venezuelae ISP5230 is made up of a core group, jadABCDE, encoding the PKS that assembles the parent polyketide (Han et al., 1994 ) and auxiliary genes encoding supplementary enzymes with tailoring (Yang et al., 1996
) or regulatory functions (Yang et al., 1995
). Among the tailoring group are jadE, encoding an oxidoreductase that reduces the C-10 ketone of the incipient angucycline ring system in jadomycins (Kulowski et al., 1999
), and jadF, which encodes another oxidoreductase that opens the angucycline B ring to facilitate insertion of isoleucine. Less clearly in the tailoring group is jadJ, which encodes an enzyme complex for carboxylating acetyl-CoA to malonyl-CoA and might be expected to serve a general metabolic purpose. However, the role of the gene in S. venezuelae appears to be linked to extension of the polyketide chain used for jadomycin B biosynthesis (Han et al., 2000
). Here, we report evidence that another gene, jadM, located unambiguously in the jad cluster, encodes a phosphopantetheinyl transferase that is functionally associated with biosynthesis of the polyketide-derived antibiotic.
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METHODS |
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DNA manipulation and transformation.
Plasmid DNA was isolated from E. coli by the alkaline lysis method (Sambrook et al., 1989 ). Bacteriophage
DNA was purified essentially as reported by Yamamoto et al. (1970)
. Genomic DNA was isolated as described by Hopwood et al. (1985)
. T4 DNA ligase and restriction enzymes were used as recommended by the suppliers. Competent cells of E. coli strains were prepared and transformed by the procedures of Sambrook et al. (1989)
. For conjugal transfer of plasmids from E. coli to streptomycetes, the protocol of Mazodier et al. (1989)
was followed.
Cloning and sequencing of jadM.
A 0·55 kb XhoI/SacI fragment of jadM was obtained from a segment of S. venezuelae ISP5230 genomic DNA cloned in Lambda LH7 (Han et al., 1994 ) and subcloned in pBluescript II SK(+) as pJV104 (McVey, 1998
). The plasmid insert, labelled with 32P, was used to probe a genomic library prepared in Lambda GEM-11 from a partial Sau3AI digest of S. venezuelae ISP5230 (Facey, 1994
) plated on LB agar in three 9 cm diameter Petri dishes, and incubated overnight to give approximately 106 plaques. Hybridization (Sambrook et al., 1989
) yielded eight labelled clones (Lambda LW1LW8), from which DNA was extracted, digested with XhoI and probed by Southern hybridization with the 0·55 kb XhoI/SacI fragment from the plasmid pJV104. A 7·0 kb labelled fragment was detected in DNA from six of the eight clones. This fragment from LW3 was inserted in the XhoI site of pBluescript II SK(+) to obtain the plasmid pJV401. From pJV401, a 3·3 kb XhoI/KpnI fragment containing jadM was subcloned in pBluescript II SK(+) to give pJV402. Nested overlapping deletions were introduced into the pJV402 insert with an ExoIII/S1 deletion kit (MBI Fermentas) and the cloned DNA was sequenced (GenBank accession numbers AF222693 and AY026363) by the dideoxynuceotide chain-termination method (Sanger et al., 1977
).
Sequence analysis and disruption.
The sequenced pJV402 insert was examined with ORF Finder (NCBI) and FramePlot 2.3 (Ishikawa & Hotta, 1999 ) to detect ORFs. BLASTX was used with individual ORF sequences to query GenBank nucleic acid and protein databases. Sequences were aligned and their relatedness was assessed with CLUSTAL W (Thompson et al., 1994
). The preferred target for insertional inactivation was an NruI site 85 bp downstream of the jadM start codon. However, this site was only 210 bp from the XhoI end of the cloned fragment, so to increase opportunities for double crossovers when the plasmid was introduced into S. venezuelae, the 3·4 kb XhoI/KpnI chromosomal sequence cloned in pJV402 was extended at the XhoI end by ligation to a 1·3 kb NruI(EcoRV)/XhoI fragment of pJV405 that contained S. venezuelae DNA adjoining jadM. The pJV405 insert had initially been subcloned in pBluescript II SK(+) as a 4·0 kb SacI fragment from Lambda LH7. One of the products (pJV404A) was digested with NruI and XhoI, then subcloned as a 1·3 kb fragment between the EcoRV and XhoI sites of pBluescript II SK(+) to give pJV405. Digestion of pJV405 with XhoI/KpnI and ligation with the 3·4 kb XhoI/KpnI fragment from pJV402 gave pJV406 (Fig. 1a
). Digestion of pJV406 with NruI, dephosphorylation with calf intestinal alkaline phosphatase and ligation with the 1·6 kb EcoRV cassette containing apr (retrieved from pJV225) yielded constructs with apr in alternative transcriptional orientations (opposite to jadM in pJV407A; similar in pJV407B). Linearizing pJV407A/B with EcoRI/EcoRV, blunting the 5·6 kb linear products with S1 nuclease and ligation to the promiscuous vector pJV326 linearized with BamHI and blunted with S1 nuclease, gave pJV408A/B (with the apr fragment in alternative orientations). The constructs were transferred conjugally from E. coli to S. venezuelae ISP5230 (Fig. 1b
).
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Jadomycin production and analysis.
Cultures of S. venezuelae ISP5230 were grown from a vegetative inoculum obtained by incubating a spore suspension (20 µl) in 25 ml MYM medium (Stuttard, 1982 ) in a 125 ml Erlenmeyer flask on a rotary shaker for 24 h at 30 °C. Portions (1 ml) of this culture were used to inoculate 25 ml Gal2I medium in 125 ml Erlenmeyer flasks. The Gal2I cultures, after incubation as above for 6 h, were supplemented with 0·75 ml of absolute ethanol and incubation was resumed for 48 h. Filtered broths were extracted with ethyl acetate and assayed by HPLC using Beckman System Gold equipment and software. Culture extracts (20 µl) in methanol were injected on a C18 reverse-phase silica column (50x4·6 mm) and eluted at a flow rate of 1 ml min-1 with a linear gradient from 100% solvent A (acetonitrile/water, 1:1) to 25, 50, 100 and 0% solvent B (100% acetonitrile) programmed to change after 3, 6·5, 7·5 and 10 min, respectively. Both solvents contained 0·1% (v/v) trifluoroacetic acid. Jadomycins in the eluate were monitored at 313 nm; jadomycin B had a retention time of 7·5 min.
Northern hybridization.
RNA was isolated from Gal2I cultures of S. venezuelae ISP5230 with the modified Kirby mix, followed by phenol/chloroform extraction and DNase I treatment (Kieser et al., 2000 ). It was fractionated and adsorbed on nylon membranes (GeneScreen & GeneScreen Plus, NEN Life Science) by the method of Sambrook et al. (1989)
. Northern hybridization followed NENs protocol for their products.
Bioassay of chloramphenicol.
The method was modified from that of Doull et al. (1986) . Spores of an S. venezuelae strain spread evenly on MYM agar in a 9 cm Petri plate were incubated at 30 °C for 4872 h. Plugs were removed aseptically with a cork borer, placed equidistantly on MYM agar in 9 cm Petri plates and incubated at 30 °C for 1214 h. They were then overlaid (2·5 ml per plate) with soft GNY agar (Malik & Vining, 1970
) seeded with 1·0% (v/v) of a Micrococcus luteus culture grown overnight in GNY liquid medium. The overlaid plates were incubated overnight and the plugs were examined for zones of inhibition.
Expression of jadM in E. coli.
A 1·0 kb XhoI/PvuII fragment containing jadM was retrieved from pJV406 (see Fig. 1a), blunt-ended with S1 nuclease and ligated (T4 DNA ligase) to pET-21(+), also linearized with BamHI and blunt-ended with S1 nuclease. Transforming E. coli BL21(DE3) with the ligation mixture yielded a colony from which pJV409 was isolated. Cultures of the transformant and E. coli BL21(DE3) harbouring pET-21(+) were grown to late-exponential phase in LB medium; the T7 polymerase promoter in pET-21(+) was induced with isopropylthio ß-galactopyranoside (1 mM final concentration) and cells harvested by centrifugation were resuspended in gel loading buffer (62·5 mM Tris/HCl, pH 6·8; 2%, w/v, SDS; 5%, v/v, 2-mercaptoethanol; 10%, v/v, glycerol; 0·025% bromphenol blue), lysed by heating at 100 °C for 35 min and analysed by SDS-PAGE (Laemmli, 1970
). Total proteins electrophoresed on a 4% (w/v) polyacrylamide stacking gel and a 15% (w/v) separating gel were visualized by staining the gel with 0·1% Coomassie brilliant blue R-250.
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RESULTS |
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Function of jadM
Database searches with BLASTX showed that the deduced amino acid sequence of jadM is 36% similar and 24% identical to the 237 amino acid sequence of HetI in Anabaena PCC7120. In this cyanobacterium, hetI, hetM and hetN have been implicated in the production of an unidentified secondary metabolite regulating heterocyst spacing (Black & Wolk, 1994 ). Sequence analysis indicates that HetN is an NAD(P)H-dependent enzyme similar to oxidoreductases associated with polyketide and fatty acid biosynthesis, and in addition suggests that HetM contains an ACP domain. Similarities between HetI and members (Spf, Gsp and Ent) of the phosphopantetheinyl transferase superfamily (Lambalot et al., 1996
; Walsh et al., 1997
; Silakowski et al., 1999
) led Lambalot et al. (1996)
to propose that HetI is a HetM-specific PPTase involved in synthesis of the secondary metabolic product influencing heterocyst formation. In addition to resembling the HetI sequence in Anabaena, the deduced amino acid sequence of jadM resembles the sequence of MtaA in Stigmatella aurantiaca (29% similar and 22% identical amino acids). This protein is a PPTase activating biosynthesis of the electron-transport inhibitor myxothiazol, probably by post-translational modification of MtaB, MtaG and the unique combination of PKSs and nonribosomal peptide synthetases (NRPSs) forming the biosynthetic machinery for making myxothiazol (Silakowski et al., 1999
). MtaA may also be responsible for transferring Ppan to proteins involved in the biosynthesis of a variety of secondary metabolites in S. aurantiaca.
Comparison of JadM with the phosphopantetheinyl transferase superfamily
Phosphopantetheinyl transferase activity was first detected in EntD from E. coli and Spf from B. subtilis. Acyl carrier protein synthetase (ACPS), which catalyses conversion of the inactive apo-form of a fatty acid synthase complex to the functional form, was the first PPTase for which the gene was cloned and characterized. Several genes involved in peptide secretion (entD in E. coli, sfp in B. subtilis and gsp in Bacillus brevis) have since been identified, and their products appear to represent a new class of proteins (Borchert et al., 1994 ). Through refinement of sequence alignments that indicated 1222% similarity with the ACPS peptide sequence, a PPTase superfamily that included the Sfp/Gsp/EntD group was identified (Lambalot et al., 1996
; Gehring et al., 1997
). Gsp is present in a locus required for gramicidin biosynthesis; EntD and Sfp were originally reported to activate enterobactin and surfactin synthetase, respectively, and Sfp was recently reported to modify the apo-form of heterologous recombinant proteins, including the PCP domain of Saccharomyces cerevisiae Lys2 (involved in lysine biosynthesis; Ehmann et al., 1999
) and the E. coli ACP domain (Quadri et al., 1998a
; Gokhale et al., 1999
). Lambalot et al. (1996)
identified two consensus motifs shared by PPTase family members, and further study implicated the conserved residues in enzymic reactions transferring the phosphopantetheinyl moiety of coenzyme A to the hydroxyl of conserved serines in the ACP domain of PKS and the PCP domain of NRPS (Reuter et al., 1999
).
To determine whether JadM contained the PPTase consensus, CLUSTAL W was used to align the sequence with members of the PPTase superfamily (Fig. 2). Highly conserved amino acids in the superfamily were present in JadM, and also in some residues considered important in Sfp-type PPTases (Reuter et al., 1999
). The crystal structure of Sfp enzymes indicates that the active site accommodates a magnesium ion that complexes with the pyrophosphate group in coenzyme A, the side chains of three acidic amino acids and one water molecule (Reuter et al., 1999
). These are highly conserved regions that may interact with PCP substrates. In Sfp the Mg2+-liganding residues Asp107 and Glu151 are highly conserved in every enzyme listed in Fig. 2
, including JadM. The
-phosphate of coenzyme A binds to Lys155 and His90. In JadM and all PPTases investigated, except 131454, Lys150 and His90 were highly conserved, while position 105 was invariably glycine for spatial reasons. The sequence motif Gly74-Lys75-Pro76 involved in binding the adenine base of coenzymeA was preserved in JadM, but with the Lys75 replaced by arginine. This is an acceptable substitution because Lys75 forms only hydrogen bonds and not a salt bridge with the main-chain carbonyls of Ile104, Lys155 and Gln156 (Reuter et al., 1999
). A CLUSTAL W alignment of the deduced amino acid sequence of jadM with the sequences of PPTases from other organisms supported assignment of the gene to the PPTase superfamily. The phylogenetic tree in Fig. 3
indicates that JadM, HetI and MtaA have a common ancestor, and that these three genes are more closely related to one another than to other members of the family. Comparisons of the deduced amino acid sequence of jadM with database sequences are consistent with such a conclusion.
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Disruption of jadM
To avoid restriction in S. venezuelae ISP5230, plasmid constructs pJV408A/B were passaged through the DNA methylation-deficient E. coli strain ET12567(pUZ8002) before being transferred from E. coli to S. venezuelae by interspecies conjugation (Mazodier et al., 1989 ). pUZ8002 is a large (about 70 kb) plasmid carrying the tra genes that facilitate conjugational transfer of a plasmid containing the oriT sequence. Single colonies of VS1075 and VS1076 transconjugants resistant to apramycin but sensitive to thiostrepton were selected. When genomic DNA was extracted from S. venezuelae ISP5230, VS1075 and VS1076, digested with EcoRV/BamHI, and probed by Southern hybridization with a 32P-labelled 3·2 kb BamHI/EcoRV fragment of pJV406 containing jadM, the S. venezuelae ISP5230 digest gave a single strong signal at 3·2 kb, whereas VS1075 and VS1076 digests gave a comparable signal at 4·8 kb (Fig. 4
). Use of the 1·6 kb apr cassette as a probe gave a single strong hybridization signal at 4·8 kb from the disrupted strains VS1075 and VS1076, as expected for double-crossover mutants. Cultures of VS1075 and VS1076 grew normally on minimal agar, but produced only 25% of the wild-type jadomycin B titre when grown in Gal2I liquid medium optimized for jadomycin B production and supplemented with ethanol. The low titre in both strains indicated that insertion of the apr cassette interfered with jadM expression, irrespective of apr orientation. A polar effect of the insertion cannot be excluded, but the AmR gene is accompanied by its promoter (Kaster et al., 1983
), and the apr cassette is not known to include a terminator that would interrupt transcription in both mutants. To determine if jadM was essential for chloramphenicol biosynthesis, the disrupted and wild-type strains were bioassayed after growth on MYM agar under conditions suitable for chloramphenicol production (Doull et al., 1986
). No decrease in the inhibition zone size was detected in strains VS1075 and VS1076, indicating that the jadM-disrupted mutants retained their ability to produce chloramphenicol.
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DISCUSSION |
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For the biosynthesis of type-II polyketide antibiotics in streptomycetes, each PKS complex must have a dedicated holo-ACP, which in turn requires a specific holo-ACP synthase. The latter enzymes function as integral components of the antibiotic biosynthesis pathway and differ from those that participate in fatty acid biosynthesis (Hopwood & Sherman, 1990 ; Hutchinson, 1995
). However, both fren and gra apo-ACPs could be phosphopantetheinylated in vitro by purified E. coli ACPS. When combined with ACP-deficient act ketosynthase and chain-length factor isolated from Streptomyces coelicolor A3(2), the holo-ACPs formed in vitro were fully functional in polyketide synthesis (Carreras et al., 1997
). Moreover, co-expression of actinorhodin and griseusin ACPs with ACPS in E. coli gave high titres of active holo-ACPS (Cox et al., 1997
), and E. coli ACPS efficiently modified post-translationally the apo-ACPs involved in biosynthesis of granaticin, frenolicin, oxytetracycline and tetracenomycin (Gehring et al., 1996
). These results imply that E. coli ACPS has broad substrate specificity. However, it will not recognize the apo-forms of several PCP and ArCP domains, including the apo-PCP domain of E. coli EntF and the apo-ArCP domain of E. coli EntB (Lambalot et al., 1996
; Gehring et al., 1996
; Quadri et al., 1998a
). Our evidence that an S. venezuelae ISP5230 mutant disrupted in jadM is unaffected in the production of chloramphenicol, an antibiotic now known to be biosynthesized via a non-ribosomal peptide synthetase (J. Y. He, N. Magarvey, M. Piraee, K. A. Aidoo & L. C. Vining, unpublished results) indicates that JadM is not required for this process, and could mean that it is not recognized by the PCP domain in the NRPS. There may be a separate PPTase in the cml cluster catalysing the conversion of apo-PCP to its holo-form. Since the jadM-disrupted mutants VS1075 and VS1076 grew normally on minimal agar, jadM is also not essential for fatty acid biosynthesis, and thus appears to be a jadomycin B pathway-specific PPTase.
Although this report is the first to describe the cloning and characterization from a streptomycete of a gene encoding a PPTase, more than 20 examples of PPTases, including ACPS, EntD and O195 of E. coli, Sfp of B. subtilis and Gsp of B. brevis, have been added to the group on the basis of sequence similarity since the superfamily was first recognized (Lambalot et al., 1996 ; Walsh et al., 1997
). However, only a few have been extensively characterized. Among these are PPT1 from the type-II fatty acid synthase of Brevibacterium ammoniagenes (Stuible et al., 1997
), PPT2, which activates mitochondrial ACP in Saccharomyces cerevisiae (Stuible et al., 1998
), Lys5 from the lysine biosynthesis system in S. cerevisiae (Ehmann et al., 1999
), MtaA from the polyketide synthase/polypeptide synthetase complex synthesizing mycothiazole in Stigmatella aurantiaca (Gaitatzis et al., 2001
) and PptT, which is required in the assembly of mycobactin, the peptide-polyketide siderophore of Mycobacterium tuberculosis (Gehring et al., 1997
; Quadri et al., 1998b
). The crystal structure of Sfp has indicated regions likely to be involved in interactions with the substrate of this PCP (Reuter et al., 1999
), and since most of these regions are highly conserved among PPTase family members, directed mutations may provide insights into substrate recognition and specificity by PCP, ACP and NRPS enzyme complexes. Already the crystallographic investigations have allowed a catalytic mechanism to be proposed (Parris et al., 2000
).
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Received 28 November 2000;
revised 9 February 2001;
accepted 21 February 2001.