![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Bio-X Life Science Research Center and School of Life Science & Biotechnology, Shanghai Jiaotong University, Shanghai 200030, People's Republic of China,1 College of Pharmacy, Oregon State University, Corvallis, Oregon 97331-3507,2 Department of Chemistry, University of Washington, Seattle, Washington 98195-17003
Received 3 November 2004/ Accepted 21 March 2005
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Streptomyces hygroscopicus subsp. jinggangensis 5008 (S. hygroscopicus 5008 or strain 5008 hereafter), isolated from the Jinggang Mountain area of China in 1974 (27), produces at least two antibiotics of agricultural importance. Jingangmycin, a weakly basic water-soluble aminocyclitol antibiotic, which was later proven to be identical to validamycin A (Fig. 1) produced by S. hygroscopicus var. limoneus IFO 12703 (13), has been widely used as a prime control reagent against sheath blight disease of rice plants and dumping-off of cucumber seedlings in China and many other eastern Asian countries. Upon treatment with validamycin, normal extension of the main hyphae is switched to an abnormal branching at the tips and further development of the growing fungi is severely repressed (23). Furthermore, voglibose, a valiolamine derivative produced from validamycin by bioconversion and chemical modifications (10), is widely used for the treatment of diabetes. The other antibiotic, jingsimycin, is an acidic polypeptide similar to saramycetin and has activity against various fungi.
|
-glucosidases. Based on feeding experiments with isotopically labeled precursors, 2-epi-5-epi-valiolone, 5-epi-valiolone, valienone, and validone were suggested to be the intermediates of the validamycin A biosynthetic pathway by Floss and coworkers (7) (Fig. 1). The incorporation patterns of various putative intermediates seemed to be different between the validamycin-producing S. hygroscopicus var. limoneus and the acarbose-producing Actinoplanes sp. strain SE50/110, but 2-epi-5-epi-valionone was found to be efficiently incorporated into both compounds, suggesting that both biosynthetic pathways share the same initial cyclization reaction catalyzed by common enzymes with similar activities. AcbC, an enzyme closely related to 3-dehydroquinate synthetases (AroB proteins), was proven to be involved in the cyclization of D-sedoheptulose 7-phosphate to 2-epi-5-epi-valionone in acarbose biosynthesis (25). The encoding gene (acbC) was thus used as a heterologous probe for the cloning of the validamycin biosynthetic genes from S. hygroscopicus 5008, which is reported in this paper. The direct involvement of valA, an acbC homologue, in validamycin biosynthesis was confirmed in vivo by gene inactivation as well as in vitro by biochemical characterization of the reaction catalyzed by the ValA protein heterologously overexpressed in E. coli.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Cloning techniques.
Plasmid and total DNA was isolated from Streptomyces strains according to Kieser et al. (15). Restriction enzymes, T4 DNA ligase, Taq polymerase, and alkaline phosphatase were purchased from various companies (New England Biolabs, Takara, and MBI Fermentas). The gel recovery kit (Shanghai Watson and QIAGEN) was used for DNA recovery from agarose gels. For the generation of cosmid libraries, total DNA samples were partially digested with MboI, dephosphorylated with calf intestinal alkaline phosphatase, and size-fractionated by sedimentation analysis using a sucrose gradient (9). DNA fragments between 30 and 40 kb were mixed in a 1:1 molar ratio with BamHI-digested cosmid vector pHZ1358 and ligated at ca. 200 µg ml–1 DNA. Packaging was done with 
packaging mixes prepared according to Sambrook et al. (22).
DNA probes, PCR primers, and Southern hybridization.
An NdeI-EcoRI fragment carrying the acbC gene from Actinoplanes sp. strain SE50/110 producing acarbose was excised from plasmid pAS8/7 (25) and used as a heterologous probe. The two oligonucleotide primers used for PCR amplification were ValA-F (5'-GGATCCACATATGACCATGACCAAG-3') and ValA-R (5'-GAATTCACACCCCCATGTCC-3'). For Southern hybridization experiments, S. hygroscopicus 5008 genomic DNA was cleaved with restriction enzymes, separated on 0.8% agarose gels, and transferred onto Hybond-N+ nylon membrane (Amersham-Pharmacia). -[32P]dCTP-labeled radioactive probes using a random priming kit (Roche) were used for both Southern blots and in situ colony hybridization.
Construction of pHZ2236 for targeted deletion of 30-kb region within the ca. 70-kb contig.
Complete digestion of cosmid 3G8 DNA by BamHI and religation resulted in the construction of pHZ2234, in which an internal ca. 30 kb (marked by a solid bar in Fig. 2) of 3G8 was found to be deleted and the two 2.1-kb flanking fragments were connected. Then a 1.4-kb BamHI fragment carrying aac(3)IV (apramycin resistance gene) was inserted between the two 2.1-kb fragments with the same transcriptional direction as valA, which generated pHZ2236 for subsequent conjugation.
|
Antibiotic assay.
Production of validamycin by S. hygroscopicus 5008 and its derivatives was detected using a bioassay and high performance liquid chromatography (HPLC). For the bioassay, 1 milliliter of fermentation supernatant extracted with chloroform was mixed with 14 ml of melted agar (8 g of agar, 1 liter of water). An agar plug with Pellicularia sasakii (Shanghai Jiaotong University Stock Collection, Shanghai Jiaotong University, Shanghai), which is sensitive to validamycin, was transferred to the center of the agar plate and after 24 h incubation at 30°C the diameter of the colony was measured, which is inversely related to the inhibitory potency. For HPLC analysis, the strains were cultured in 40 ml of FM-II fermentation medium in 250-ml baffled flasks at 37°C and 220 rpm for 6 days. The fermentation broth was centrifuged at 12,000 rpm for 5 min followed by chloroform extraction. The extracted supernatant was directly loaded onto a Nucleosil C18 column (250 mm by 4.6 mm, Sigma-Aldrich) for HPLC analysis (Waters 220). The mobile phase (0.005 M sodium phosphate buffer-acetone, 97:3) was applied with the flow rate of 1 ml min–1 at room temperature. The elute was monitored at 210 nm with a Waters 996 photodiode array detector and the data were analyzed with a Waters Millennium Chromatography Manager.
Sequence analysis.
DNA sequencing was done at Shanghai Sangon Ltd. using pUC18 as the vector. Sequencing reactions were carried out using the Amersham Thermosequenase sequencing kit containing fluorescent dye terminators and an Applied Biosystems model 377 automated DNA sequencer. Sequence analysis was performed with the Lasergene DNA analysis tools (DNASTAR) (Madison, MI). Open reading frames and ribosome binding sites were predicted with FramePlot (11). Nucleotide and amino acid sequence comparisons against public databases were done using the BLAST program (1).
Cloning and heterologous overexpression of recombinant His6-tagged ValA.
The valA gene was amplified by PCR with Platinum Pfx DNA polymerase (Invitrogen) using the cosmid clone 3G8 as the template and primers ValA-F3, 5'-GAAGATCTGCATATGACCAAGCAGAGTTCCTTATCC-3' (BglII and NdeI), and ValA-R2, 5'-GGAATTCTCACACCCCCATGTCCACGGCACCG-3' (EcoRI). PCR amplification was done in a Thermocycler (Eppendorf, Mastercycler gradient) under the following conditions: 33 cycles of 90 s at 95°C, 45 s at 60°C, and 45 s at 72°C. The PCR products were digested with BglII and EcoRI, and subsequently ligated into BamHI- and EcoRI-digested pRSET-B. The constructs were transformed into E. coli XL-1-Blue and plated on LB agar plates containing 100 µg ml–1 ampicillin.
The plasmid DNA was isolated and introduced by heat-pulse transformation into E. coli BL21Gold(DE3)/pLysS (Stratagene), which was then plated onto LB agar plates containing 100 µg ml–1 ampicillin and 25 µg ml–1 chloramphenicol. The transformants were grown in 20 ml LB medium containing ampicillin and chloramphenicol at 37°C to an optical density at 600 nm of 0.6. Isopropyl-ß-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM and the incubation was continued at 28°C for 24 h. The cells were harvested by centrifugation at 3,500 rpm for 15 min and stored frozen at –80°C until further use.
Preparation of cell extracts and purification of His6-tagged ValA.
Cells were thawed and resuspended in disruption buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). The suspension was sonicated three times for 25 s each and cell debris was removed by centrifugation at 10,000 rpm for 10 min. The protein solution was applied to an Ni-nitrilotriacetic acid spin column (QIAGEN) and centrifuged at 2,000 rpm for 2 min. The column was washed with washing buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0, and 50 mM NaH2PO4, 300 mM NaCl, 100 mM imidazole, pH 8.0). The His6-tagged protein was eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 500 mM imidazole, pH 8.0) and dialyzed for 24 h against 1 liter of dialysis buffer (20 mM potassium phosphate [pH 7.4], 0.05 mM CoCl2, 2 mM KF and 0.5 mM dithiothreitol). Protein concentration was measured by the Bradford protein microassay with bovine serum albumin as the standard.
Enzyme assay.
The enzyme assay was carried out at 30°C for 3 to 12 h in a 100-µl volume of 20 mM potassium phosphate (pH 7.4), 0.05 mM CoCl2, 2 mM KF, 1 mM NAD+, 5 mM sedoheptulose 7-phosphate, and 50 µl of protein solution (2.4 mg/ml protein). The reaction progress was monitored by thin-layer chromatography analysis. The reaction mixture was lyophilized and the reaction products were extracted with methanol. This extract was then dried and a few drops of SIGMA-SIL-A (SIGMA) were added. The solvent was removed in a flow of argon gas and the products were reextracted with n-hexane and injected for gas chromatography-mass spectroscopy (GC-MS) (Hewlett Packard 5890 series II gas chromatograph).
Nucleotide sequence accession number.
The DNA and deduced protein sequences reported in this paper have been deposited in GenBank under accession number AY753181.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The BamHI digestion patterns of the six cosmids resulted in a contig spanning ca. 70 kb, without apparent genomic rearrangement. The different hybridization signal originating from BamHI-digested 15H4 was found to have resulted from the regeneration of a BamHI (designated B' in Fig. 2) site between the cloning site (BamHI) of the vector and an accidental MboI site of the insert during library construction, as this fragment was found to lie at the very end of the cloned fragment in cosmid 15H4.
Cosmid 3G8 contains genes involved in validamycin biosynthesis.
One of the six pHZ1358-derived (26) bifunctional cosmids, 3G8 (Fig. 2), carrying a tsr gene suitable for selection in Streptomyces and a 34.2-kb insert under the control of pIJ101 origin of replication (6), was proven to be extremely unstable (ca. 98% loss after one round of nonselective growth on SFM medium) in strain 5008. For targeted gene replacement, ca. 30 kb of the strain 5008 DNA insert containing the acbC homologue in cosmid 3G8 was replaced by a 1.4-kb apramycin resistance (Aprr) determinant, aac(3)IV, which resulted in pHZ2236 for subsequent conjugation.
pHZ2236 was transferred by conjugation from E. coli ET12567(pUZ8002) into strain 5008. About 10–8 exconjugants per donor were obtained which were initially selected to be Aprr. Surprisingly, all four randomly selected exconjugants were found to be sensitive to thiostrepton. A Southern transfer of total DNA (Fig. 3B) from the four Aprr Thios exconjugants (YU-1-1 to YU-1-4) together with wild-type 5008 was probed with the labeled 5.6-kb insert from pHZ2236 (Fig. 3A). As expected, the ca. 30-kb fragment in the 5008 chromosome was found to be replaced by a 1.4-kb fragment in the YU-1 mutants, resulting in a fusion of the 10-kb leftward BamHI fragment with the BglII end of the 1.4-kb aac(3)IV to form a new 11.4-kb BamHI fragment (Fig. 3A), which is distinguishable from the 10-kb BamHI fragment of the wild-type 5008 in Fig. 3B.
|
Sequencing analysis of a DNA fragment including the acbC homologue.
The common 6-kb BamHI fragment shared by the five cosmids (4G8, 3G8, 17F2, 20E1, and 13G5, Fig. 2) including the acbC homologue was cloned into pBluescript II SK(+) and sequenced. The overall G+C content of the sequenced region was 67.9%, lower than that of S. coelicolor A3(2), which showed an overall G+C content of 72.1%. FramePlot (11) analysis revealed three open reading frames (valA, valB, and valC) transcribed in the same direction, with valA separated from valB by 5 bp, while valB and valC overlap by 3 bp, suggesting that they are transcribed into one polycistronic mRNA (Fig. 2).
The valA gene seems to encode a polypeptide of 412 amino acids in length, with a putative ribosome binding site (GTGA) 10 bp preceding the putative translational start codon (ATG). The nucleotide sequence of valA has 59.5% identity with the Actinoplanes acbC gene, while the deduced ValA protein has 48% identity with the AcbC protein (25) (Fig. 4). The ValA protein also showed significant similarity (37% identity) to the AroB protein from Emericella nidulans (Fig. 4), which is known to be responsible for the cyclization of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) to dehydroquinate (4).
|
There is an incomplete open reading frame (tentatively designated ORF1) at the 5' end of the fragment, which is transcribed in the opposite direction and shows 31% identity to the N-terminal amino acids of dTDP-4-dehydrorhamnose reductase, RmlD, whose known function is to reduce dTDP-6-deoxy-L-xylo-4-hexulose to dTDP- L-rhamnose in Geobacillus stearothermophilus (19).
DNA replacement in valA is abolished validamycin biosynthesis.
Direct evidence for the involvement of valA in the biosynthesis of validamycin came from the replacement of a 563-bp DNA fragment internal to valA with aac(3)IV (Fig. 5A). This was performed by using a pHZ1358-derived plasmid (pJTU519, detailed in Materials and Methods), in which aac(3)IV (apramycin resistance gene) was sandwiched between sequences of 1.33 kb flanking to the left, and 1.5 kb flanking to the right of the 563-bp DNA to be deleted (Fig. 5A). JXH-1, a 5008 derivative, was obtained after introduction of pJTU519 into wild-type strain 5008 by conjugation from E. coli ET12567(pUZ8002), initial selection by thiostrepton, and further screening for the Thios Aprr phenotype. Total DNA from the two independent Thios Aprr exconjugants (JXH-1-1 and JXH-1-2) and from the wild-type strain 5008 was used as template for PCR amplification using two oligonucleotide primers (ValA-F and ValA-R) (Fig. 5B).
|
Characterization of ValA activity using heterologously overexpressed protein.
In order to confirm the function of its gene product as a 2-epi-5-epi-valiolone synthase, valA was heterologously expressed in E. coli. A 1.24-kb DNA fragment containing the valA gene was amplified from cosmid 3G8 by PCR, introducing BglII and NdeI restriction sites with the forward primer and an EcoRI site with the reverse primer. The PCR product was subcloned into the expression vector pRSET-B as a BglII/EcoRI fragment, transformed into E. coli XL-1-Blue. The correct plasmids were subsequently transformed into E. coli BL21Gold(DE3)pLysS. Expression of valA under the control of the T7 promoter was induced by isopropyl-ß-D-thiogalactopyranoside (IPTG), which gave rise to a 48-kDa soluble polyhistidine-tagged protein. Affinity purification on a Ni-nitrilotriacetic acid spin column (QIAGEN) or a BD TALON column gave a protein that was >80% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 6A).
|
The product was extracted from the lyophilized reaction mixture by methanol, converted to its trimethylsilylated derivative, and analyzed by GC-MS (Fig. 7). An isotopically labeled authentic sample, 2-epi-5-epi-[6-2H2]valiolone, chemically synthesized from D-mannose (25) was used for comparison (Fig. 7A). The enzyme product was detected as a tetratrimethylsilyl derivative of 2-epi-5-epi-valiolone [m/z 480 (M+)] with major fragment ions at m/z 335, 276, 217, 147, and 73 (Fig. 7D). This fragmentation pattern is consistent with that of the authentic sample, which showed m/z 482 (M+), 335, 278, 217, 147, and 73 (Fig. 7C).
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A first effort by heterologous expression in Streptomyces lividans 66, based on the proposed biosynthetic pathway (7) that implied that the validamycin gene cluster may possess a limited number of genes, failed in an initial trial. A second attempt using radioactively labeled strD encoding dTDP-glucose synthase of the streptomycin biosynthetic gene cluster of Streptomyces griseus as a probe also yielded no signal, which suggests that the activation of glucose in validamycin biosynthesis is catalyzed by an enzyme different from StrD, which seems to be specific for 6-deoxyhexose pathways.
The success of using the acbC gene from the acarbose cluster of Actinoplanes to probe for the validamycin biosynthetic gene cluster was not surprising, as both compounds contain the same valienamine moiety in their structures, whose initial biosynthetic step involves cyclization of D-sedoheptulose 7-phosphate to form 2-epi-5-epi-valionone. The significant sequence homologies between ValA and ValC of the validamycin pathway and AcbC and AcbM (40) of the acarbose pathway strongly support the idea that they have similar functions. This suggests that 2-epi-5-epi-valiolone in validamycin biosynthesis has the same fate of being phosphorylated (by ValC) as was demonstrated in acarbose biosynthesis (29).
It was proposed that all of the intermediates from 2-epi-5-epi-valiolone to valienone in the acarbose biosynthetic pathway are phosphorylated (29), which explains the lack of incorporation of the nonphosphorylated putative intermediates, such as 5-epi-valiolone and valienone. On the contrary, nonphosphorylated 5-epi-valiolone, valienone, and validone were found to be efficiently incorporated into validamycin A (7). If ValC has a role in phosphorylating the intermediates in the validamycin pathway similar to that of AcbM in acarbose biosynthesis, its substrate specificity or catalytic ability to phosphorylate all of the cyclitol intermediates, including 2-epi-5-epi-valiolone, remains to be explored.
Both ValA and AcbC show significant similarities to AroB-related DHQS proteins from diverse organisms (4, 8, 17), whose catalytic function is known to be cyclization of DAHP to dehydroquinate. Based on the analysis of the three-dimensional structure of the DHQS domain of the functional AroM protein of the filamentous fungus Emericella nidulans, a total of 13 amino acid residues were identified to be important for catalysis and as being involved in Zn2+ binding (Co2+ in bacteria instead; indicated as 1 in Fig. 4), heptulose phosphate group binding (indicated as 2 in Fig. 4), C-1 hydroxyl fixation (indicated as 3 in Fig. 4), or C-4 hydroxyl fixation (indicated as 4 in Fig. 4) (4). In ValA, three of the four identified residues for Co2+ binding (1), three of the four for heptulose phosphate group binding (2), two of the three for C-1 hydroxyl fixation (3), and all three for C-4 hydroxyl fixation (4), are conserved. Therefore, ValA seems to be more related to AroB proteins than AcbC.
Preceding valC is a cotranscribed gene, valB, encoding a putative adenyltransferase (ValB). Conceivably, this activity could be involved in the conversion of glucose 1-phosphate, possibly from primary metabolism, into dTDP-glucose. Incorporation of activated glucose has been proven through feeding experiments with validoxylamine A to be the final step in validamycin biosynthesis (14). Alternatively, ValB may be involved in the activation of one of the cyclitol intermediates, e.g., 1-epi-valienol 1-phosphate, to its nucleotide derivative, setting the stage for the coupling reaction that leads to a pseudodisaccharide intermediate.
The combination of the results of the previous feeding experiments (7) with the genetic and biochemical information obtained, especially the results of the in vivo gene inactivation and in vitro enzymatic characterization of ValA, strongly suggests that the identified genes are involved in validamycin biosynthesis. From the overlapping cosmid contig we know that the flanking DNA to the left and right of the 6-kb sequenced region covering the complete valA, valB, and valC genes extends to 29 kb and 35 kb, respectively, likely to cover the complete set of genes necessary for validamycin formation. We can thus expect a detailed understanding of the biosynthesis of validamycin, which is critical for more targeted strain improvement or generating novel validamycin derivatives by combined genetic and biochemical approaches. This will become possible when the entire gene cluster has been completely sequenced and the genes have been individually characterized, work that is now in progress.
|
ACKNOWLEDGMENTS |
|---|
This work received 973 and 863 Funds from the Ministry of Science and Technology, the National Science Foundation of China, the Ph.D. Training Fund from the Ministry of Education, and the Shanghai Municipal Council of Science and Technology. Work at Oregon State University and the University of Washington was supported by NIH grants RAI061528A and AI20264, respectively.
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C31 of Streptomyces coelicolor A3(2). Sov. Genet. 8:629-634.[Medline]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | Microbiol. Mol. Biol. Rev. | Eukaryot. Cell | All ASM Journals |
|---|
