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The fact that adpA promoter activity is enhanced by S-adenosylmethionine without the involvement of the A-factor/ArpA regulatory cascade suggests the existence of additional transcriptional regulators for adpA expression in Streptomyces griseus. In this study, an additional adpA promoter regulatory protein, named ArfA, that is conserved among many bacteria was identified using DNA affinity purification from the cell extracts of Streptomyces lividans. The interactions of ArfA with the adpA promoter from S. griseus and with the bldH promoter from S. lividans were specific and both adpA and bldH promoters required ArfA for the wild-type level of their expressions in S. lividans. bldH of S. lividans is a homolog of adpA of S. lividans. ArfA-deletion mutant had only 70% of the normal undecylprodigiosin production. This result was confirmed by reduced redD promoter activity in the ArfA-deletion mutant. These results suggest that ArfA is a new type of DNA-binding regulator.

The biosynthesis of cyanophycin granule polypeptides is catalyzed by cyanophycin synthetase, CphA. In this study, the role of the C-terminal region of CphA from Nostoc ellipsosporum NE1, CphA(NE1), was analyzed using a tailor-made C-terminus truncated library. The expression level of truncated CphA(NE1) in E. coli depended on the stop codons that were used. The expression vector that had the amber stop codon TAG produced more than twice amount of CphA(NE1) as a vector that contained the ochre codon TAA. CphA(NE1DeltaC45), which was truncated up to 45 amino acids at its C-terminus, retained full enzymatic activity and produced polymers. However, the removal of one additional amino acid, Glu(856), resulted in complete inactivation of CphA(NE1DeltaC46). Replacement of Glu(856) by valine or alanine confirmed the importance of this residue for the activity of CphA(NE1), as it resulted in the complete inactivation of the enzyme. In addition, thermostability analysis revealed a dramatic decrease in the thermostability of CphA(NE1) after removal of the region from Leu(867) to Leu(870). The gel filtration analysis showed that CphA(NE1Delta46C) still formed a dimer form even its enzyme activity was lost completely. These results suggest that Glu(856) is critical for CphA(NE1) catalytic activity and that the predicted alpha-helical region that ranges from Val(858) to Leu(870) is important for the thermostability of the enzyme.

In this study, three trehalose gene clusters, treX-Y-Z, tpS1, and treS, of the acarbose-producing strain, Actinoplanes sp. SN223/29, have been identified. In particular, five trehalose synthetic genes were sequenced and characterized in detail. They were cloned and expressed in Escherichia coli BL21(DE3)pLysS using the His-tag vector pET19b. The recombinant proteins were purified by Ni(2+)-nitrilotriacetic acid agarose affinity chromatography, and their functions were characterized biochemically. Both the maltooligosyltrehalose synthase (TreY-TreZ) pathway and the trehalose synthase (TreS) pathway have maximum activity at 40 degrees C and at pH 7.5 and 7.0, respectively, in 100-mM phosphate buffer. Meanwhile, the trehalose-6-phosphate synthase (TpS1) showed maximum activity at 35 degrees C and at pH 7.5 in 100 mM Tris-HCl. As a cofactor candidate, Mg(2+) enhanced the activities of all three trehalose synthetic reactions significantly. TreY produced component C from acarbose by its proposed isomerase activity, but TreS did not. This study suggests that the mutation of treY can improve acarbose production by repressing component C production. Based on the data obtained in this study, a model for component C production in Actinoplanes sp. SN 223/29 is proposed.

The gene cluster for spectinomycin biosynthesis from Streptomyces spectabilis was analyzed completely and registered under the accession number EU255259 at the National Center for Biotechnology Information. Based on sequence analysis, spcM of the S. spectabilis cluster is the only methyltransferase candidate required for methylation in spectinomycin biosynthesis. It has high similarity with the conserved domain of DNA methylase, which contains both N-4 cytosine-specific DNA methylases and N-6 adenine-specific DNA methylases. Nucleotide methylation can provide antibiotic resistance, such as 16S rRNA methyltransferase, to Enterobacteriaceae. We therefore tested a hypothesis that SpcM offers aminoglycoside resistance to bacteria. The heterologous expression of spcM in Escherichia coli and S. lividans enhanced resistance against spectinomycin and its relative aminoglycoside antibiotics. We therefore propose that one of the functions of SpcM may be conferring aminoglycoside antibiotic resistance to cells.

Because ATP is an extracellular effector in animal and plant systems and derivatives of ATP, such as S-adenosylmethionine and cAMP, can control antibiotic production and morphological differentiation in Streptomyces, we hypothesized that extracellular ATP (exATP) can also affect physiologies of Streptomyces. We found that the addition of 10 microM exATP to Streptomyces coelicolor A3(2) cultures resulted in enhanced actinorhodin and undecylprodigiosin production and morphological differentiation on solid medium. However, these phenotypes were reduced by the addition of a 10-fold higher concentration of exATP (100 microM). Intracellular ATP concentrations were also modulated in response to changes in exATP. ATP analogs, added at a 100-fold lower concentration, affected Streptomyces similarly to that seen for 10 microM exATP. The enhanced promoter activity of actII-orf4 indicated that 10 microM exATP affect the transcriptional level for actinorhodin production. Results from this study suggest that exATP is an effector for the physiology of S. coelicolor and careful manipulation of exATP may significantly enhance the high-yield production of antibiotics by S. coelicolor.

A gene inactivation study was performed on gntE, a member of the gentamicin biosynthetic gene cluster in Micromonospora echinospora. Computer-aided homology analysis predicts a methyltransferase-related cobalamin-binding domain and a radical S-adenosylmethionine domain in GntE. It is also found that there is no gntE homolog within other aminoglycoside biosynthetic gene clusters. Inactivation of gntE was achieved in both M. echinospora ATCC 15835 and a gentamicin high-producer GMC106. High-performance liquid chromatographic analysis, coupled with mass spectrometry, revealed that gntE mutants accumulated gentamicin A2 and its derivative with a methyl group installed on the glucoamine moiety. This result substantiated that GntE participates in the first step of pseudotrisaccharide modifications in gentamicin biosynthesis, though the catalytic nature of this unusual oxidoreductase/methyltransferase candidate is not resolved. The present gene inactivation study also demonstrates that targeted genetic engineering can be applied to produce specific gentamicin structures and potentially new gentamicin derivatives in M. echinospora.

Three new polyketides, cyathuscavins A (1), B (2), and C (3) were isolated from the mycelium culture of Cyathus stercoreus. The structures of the compounds were elucidated on the basis of NMR and mass spectroscopic data. Antioxidant activities of the compounds were evaluated by the scavenging ability against ABTS(+), DPPH, and superoxide anion radicals. Cyathuscavins A-C showed significant antioxidant activity comparable to those of reference antioxidants, BHA and Trolox. Cyathuscavins A-C protected supercoiled plasmid DNA from Fe(2+)/H(2)O(2)-induced breakage.

In the present study, a mechanism for S-adenosylmethionine (SAM) to promote secondary metabolism was characterized in terms of bldH sl) expression in Streptomyces lividans. A previous study demonstrated that SAM, on application at 2 microM, induces the transcription of the strR promoter (strRp), which originates from Streptomyces griseus, in S. lividans. An inactivation study verified that bldH sl is essential to strRp transcription in S. lividans and it was demonstrated that the effects of SAM on the induction of strRp activity, on the transcription of redZ and actII-orf4, and on antibiotic production were compromised when the unique leucine TTA-codon of bldH sl was changed to TTG. Western blot analysis revealed that SAM supplementation enhances the expression of bldH sl when the TTA-codon was intact but not when the TTG replacement was provided. This study validates the involvement of BldH sl in the potentiating effect of SAM on the antibiotic production and substantiates that SAM controls the expression of bldH sl through the TTA-codon control in translating bldH mRNA. It is argued here that the intracellular SAM-level modulates the maturation of bldA, which encodes the UUA-codon tRNA and controls secondary metabolism in S. lividans.

The Mycobacterium tuberculosis genome contains 11 serine/threonine kinase genes, and the products of two of these, PknA and PknB, are key components of a signal transduction pathway that regulates cell division and/or morphology. Previously, we have shown that one substrate of these kinases is Wag31, a homologue of the cell division protein DivIVA that is present, but not known to be phosphorylated, in other Gram-positive bacteria. Here, we investigate the localization and function of Wag31 and its phosphorylation. We demonstrate that Wag31 is localized to the cell poles. We further show that wag31 is an essential gene and that depletion of its product causes a dramatic morphological change in which one end of the cell becomes round rather than rod-shaped. This abnormal morphology appears to be caused by a defect in polar peptidoglycan synthesis. Finally, expression of M. tuberculosis wag31 in the wag31 conditional mutant of Mycobacterium smegmatis altered the growth rate in a manner that depended on the phospho-acceptor residue encoded by the allele being expressed. Taken together, these results indicate that Wag31 regulates cell shape and cell wall synthesis in M. tuberculosis through a molecular mechanism by which the activity of Wag31 can be modulated in response to environmental signals.

Adenosine kinase (ADK) catalyses phosphorylation of adenosine (Ado) and generates adenosine monophosphate (AMP). ADK gene (adk(Sli), an ortholog of SCO2158) was disrupted in Streptomyces lividans by single crossover-mediated vector integration. The adk(Sli) disruption mutant (Deltaadk(Sli)) was devoid of sporulation and a plasmid copy of adk(Sli) restored sporulation ability in Deltaadk(Sli), thus indicating that loss of adk(Sli) abolishes sporulation in S. lividans. Ado supplementation strongly suppressed sporulation ability in S. lividans wild-type (wt), supporting that disruption of adk(Sli) resulted in Ado accumulation, which in turn suppressed sporulation. Cell-free experiments demonstrated that Deltaadk(Sli) lacked ADK activity and in vitro characterization confirms that adk(Sli) encodes ADK. The intracellular level of Ado was highly elevated while the AMP level was significantly reduced after loss of adk(Sli) while Deltaadk(Sli) displayed no significant derivation from wt in the levels of S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM). Notably, Ado supplementation to wt lowered AMP content, albeit not to the level of Deltaadk(Sli), implying that the reduction of AMP level is partially forced by Ado accumulation in Deltaadk(Sli). In Deltaadk(Sli), actinorhodin (ACT) production was suppressed and undecylprodigiosin (RED) production was dramatically enhanced; however, Ado supplementation failed to exert this differential control. A promoter-probe assay verified repression of actII-orf4 and induction of redD in Deltaadk(Sli), substantiating that unknown metabolic shift(s) of ADK-deficiency evokes differential genetic control on secondary metabolism in S. lividans. The present study is the first report revealing the suppressive role of Ado in Streptomyces development and the differential regulatory function of ADK activity in Streptomyces secondary metabolism, although the underlying mechanism has yet to be elucidated.

S-Adenosylmethionine (SAM) was previously documented to activate secondary metabolism in a variety of Streptomyces spp. and to promote actinorhodin (ACT) and undecylprodigiosin (RED) in Streptomyces coelicolor. The SAM-induced proteins in S. coelicolor include several ABC transporter components (SCO5260 and SCO5477) including BldKB, the component of a well-known regulatory factor for differentiations. In order to assess the role of these ABC transporter complexes in differentiation of Streptomyces, SCO5260 and SCO5476, the first genes from the cognate complex clusters, were individually inactivated by gene replacement. Inactivation of either SCO5260 or SCO5476 led to impaired sporulation on agar medium, with the more drastic defect in the SCO5260 null mutant (ASCO5260). ASCO5260 displayed growth retardation and reduced yields of ACT and RED in liquid cultures. In addition, SAM supplementation failed in promoting the production of ACT and RED in ASCO5260. Inactivation of SCO5476 gave no significant change in growth and production of ACT and RED, but impaired the promoting effect of SAM on ACT production without interfering with the effect on RED production. The present study suggests that SAM induces several ABC transporters to modulate secondary metabolism and morphological development in S. coelicolor.

AfsKav is a eukaryotic-type serine/threonine protein kinase, required for sporulation and avermectin production in Streptomyces avermitilis. In terms of their ability to complement SJW4001 (DeltaafsK-av), afsK-av mutants T165A and T168A were not functional, whereas mutants T165D and T168D retained their ability, indicating that Thr-165 and Thr-168 are the phosphorylation sites required for the role of AfsKav. Expression of the S-adenosylmethione synthetase gene promoted avermectin production in the wild-type S. avermitilis, yet not in the mutant harboring T168D or T165D, demonstrating that tandem phosphorylation on Thr-165 and Thr-168 in AfsKav is the mechanism modulating avermectin production in response to S-adenosylmethione accumulation in S. avermitilis.

The validamycin biosynthetic gene cluster in Streptomyces hygroscopicus var. limoneus contains vldI, the gene encoding a glucoamylase (1,4-alpha-D-glucan glucohydrolase). The knock-out of vldI (vldI::neo) reduced the yield of validamycin-A, thus indicating that VldI contributes to validamycin-A productivity by supplying glucose with the hydrolysis of 1,4-alpha-D-glucan(s). Promoter-probe assays employing xylE fusions indicated that the transcription of vldI correlates to those of other biosynthetic genes, which are organized with two divergently arranged vldABC and vldDEFGH sets. These results reveal that the contiguous region covering nine genes of vldABCDEFGHI represents the core of the validamycin biosynthetic cluster. After confirming that genes vldABCDEFGH confer validamycin production ability to Streptomyces lividans, genes vldBCHI were eliminated from the expression construct and the remaining genes, vldADEFG, were tested for the ability to confer validamycin-A production to S. lividans. Ion-exchange chromatographic purification and a subsequent HPLC analysis confirmed that S. lividans/vldADEFG yielded a 75 microg/l of validamycin-A, showing that the validamycin pathway involves a single NDP-sugar glycosyltransferase reaction. It was also demonstrated that VldE is capable of coupling validoxylamine-A and UDP-glucose to generate validamycin-A. The proposal that VldADEFG catalyze the biosynthesis of validamycin-A from sedo-heptulose 7-phosphate and UDP-glucose and include a N-bridge-forming catalyst will serve as a guideline for future biochemical studies and a platform to explore this m-C7N cyclitol pathway for biotechnological applications.

Streptomyces coelicolor A3(2) produces an antibiotic, actinorhodin, which belongs to the aromatic polyketides and which can function as an acid/base indicator. Its production results in the death of microorganisms in the vicinity of S. coelicolor A3(2), and this phenomenon can be used in concert with biopesticides. The exogenous addition of S-adenosyl-L-methionine (SAM) to S. coelicolor A3(2) enhances its actinorhodin production and may initiate actinorhodin biosynthesis, with at least four genes being involved. Of these (because afsK initiates the others), AfsK, the protein expressed from afsK, may be interacting with SAM. Although the three-dimensional structure of AfsK has not been determined, the differences between nuclear magnetic resonance (NMR) signals obtained from the free form of SAM and those from a SAM-protein complex can help us to determine whether SAM binds to the C-terminal of AfsK or not. In the present study, NMR data analysis strongly supported the idea that SAM binds to AfsK.

In our screening program for new antifungal agents from microbial secondary metabolites, two new isoflavonol glycosides, with potent antifungal activity, talosins A and B, were isolated from the culture broth of Kitasatospora kifunensis MJM341. Talosins A and B were determined to be genistein 7-alpha-L-6-deoxy-talopyranoside and genistein 4',7-di-alpha-L-6-deoxy-talopyranoside, respectively, by spectroscopic studies. They are the first flavonoid glycosides incorporating 6-deoxy-talose as a sugar component.

In our screening program for new antifungal agents from microbial secondary metabolites, we isolated two new isoflavonol glycosides, genistein 7-alpha-L-6-deoxy-talopyranoside (talosin A) and genistein 4',7-di-alpha-L-6-deoxy-talopyranoside (talosin B), from the culture broth of Kitasatospora kifunensis MJM341. The talosins exhibited strong antifungal activity against Candida albicans, Aspergillus niger and Cryptococcus neoformans with minimal inhibitory concentrations (MIC) in the range of 3- 15 microg/ml while genistein and genistein-7-glucopyranoside did not show antifungal activity at 100 microg/ml. These talosins are the first isoflavonol glycosides with a 6-deoxy-talose sugar component and they may be useful as antifungal agents with low toxicity because of no visible cytotoxicity against the human hepatic HepG2 cell.

The validamycin biosynthetic gene cluster was isolated from Streptomyces hygroscopicus var. limoneus KTCC 1715 (IFO 12704) using a pair of degenerated PCR primers designed from the sequence of AcbC, 2-epi-5-epi-valiolone synthase in the acarbose biosynthesis. The nucleotide sequence analysis of the 37-kb DNA region revealed 22 complete ORFs including vldA, the acbC ortholog. Located around vldA, vldB to K were predicted to encode adenyltransferase, kinase, ketoreductase (or epimerase/dehydratase), glycosyltransferase, aminotransferase, dehydrogenase, phosphatase/phosphomutase, glycosyl hydrolase, transport protein, and glycosyltransferase, respectively. Apparently absent were any regulatory components within the sequenced region. The disruption of vldA abolished the validamycin biosynthesis and the plasmid-based complementation with vldABC restored production to the vldA-mutant; this substantiated that vldABC are essential to validamycin biosynthesis. This finding enabled us to discover the complete validamycin biosynthetic cluster. The cosmid clone of pJWS3001 harboring the 37-kb DNA region conferred validamycin-accumulation to Streptomyces lividans, indicating that the entire gene cluster of validamycin biosynthesis had been isolated. Additionally, Streptomyces albus, transformed with pJWS3001, produced a high level of alpha-glucosidase inhibitory activity in a R2YE liquid culture, which highlights the portability of the cluster within Streptomyces. The product of vldI was characterized as a glucoamylase (kcat, 32 s(-1); K(m), 5 mg/ml of starch) that does not play any apparent role in the validamycin biosynthesis. In order to characterize the upstream region, a vldW knockout was achieved via gene-replacement. A phenotypic study of the resulting mutant revealed that vldW is not essential for the host's ability to control Pellicularia filamentosa growth. The current information suggests that vldA to vldH is the genetic region essential to validamycin biosynthesis. This promises excellent opportunities to elucidate biosynthetic route(s) to the validamycin complex and to engineer the pathway for industrial application.

S-adenosylmethionine (SAM), the major methyl donor in diverse biological processes, was previously documented as a regulator for secondary metabolism in Streptomyces. The present study demonstrates that exogenous SAM, in a quantity as low as 10 muM, enhanced streptomycin production and adpA transcription in both Streptomyces griseus wild-type strain and mutant HO1, which displays no ArpA repression on the adpA promoter. SAM also enhanced xylE expression driven by the promoter of adpA or strR in a heterologous host, S. lividans. This report substantiates that exogenous SAM promotes adpA transcription in S. griseus, which is involved in the SAM-mediated promotion of streptomycin, and that the mechanism underlying this event is shared in S. lividans.

Cys-29 and Cys-251 of Streptomyces albus valine dehydrogenase (ValDH) were highly conserved in the corresponding region of NAD(P)(+)-dependent amino acid dehydroganase sequences. To ascertain the functional role of these cysteine residues in S. albus ValDH, site-directed mutagenesis was performed to change each of the two residues to serine. Kinetic analyses of the enzymes mutated at Cys-29 and Cys-251 revealed that these residues are involved in catalysis. We also constructed mutant ValDH by substituting valine for leucine at 305 by site-directed mutagenesis. This residue was chosen, because it has been proposed to be important for substrate discrimination by phenylalanine dehydrogenase (PheDH) and leucine dehydrogenase (LeuDH). Kinetic analysis of the V305L mutant enzyme revealed that it is involved in the substrate binding site. However it displayed less activity than the wild type enzyme toward all aliphatic and aromatic amino acids tested.

S-Adenosylmethionine (SAM), the major methyl donor in diverse biological processes, was recently found to be involved in the regulation of differentiation in streptomycetes. Exogenous SAM, in a quantity as low as 2muM, enhanced antibiotic production and inhibited morphological development of Streptomyces coelicolor M145. Total protein profiling of S. coelicolor M145 revealed that SAM enhanced the expression of oligopeptide-binding components related to ABC transporters that included BldK, a well-known regulatory factor in S. coelicolor differentiation. A radiolabeled SAM feeding experiment verified that exogenous SAM can be imported into the cell, which is under the control of the bld cascade. This study substantiated that BldK serves as a transducer of the SAM signal and uncovered the possible role of oligopeptide import in the regulation of Streptomyces differentiation, particularly in relation to SAM.

In this study, we demonstrate the use of a PCR-based method for the detection of the specific genes involved in natural-product biosynthesis. This method was applied, using specifically designed PCR primers, to the amplification of a gene segment encoding for sedo-heptulose 7-phosphate cyclase, which appears to be involved in the biosynthetic pathways of C7N aminoacyclitol or its keto analogue-containing metabolites, in a variety of actinomycetes species. The sequences of DNA fragments (about 540 bp) obtained from three out of 39 actinomycete strains exhibited a high degree of homology with the sedo-heptulose 7-phosphate cyclase gene, which has been implicated in acarbose biosynthesis. The selective cultivation conditions of this experiment induced the expression of these loci, indicating that the range of C7 aminoacyclitol or its keto analogue-group natural products might be far greater than was previously imagined. Considering that a total of approximately 20 C7 aminoacyclitol metabolites, or its keto analogue-containing metabolites, have been described to date, it appears likely that some of the unknown loci described herein might constitute new classes of C7 aminoacyclitol, or of its keto analogue-containing metabolites. As these metabolites, some of which contain valienamine, are among the most potent antidiabetic agents thus far discovered, the molecular detection of specific metabolite-producing actinomycetes may prove a crucial step in current attempts to expand the scope and diversity of natural-product discovery.

An actinomycetes expression vector (pIBR25) was constructed and applied to express a gene from the kanamycin biosynthetic gene cluster encoding 2-deoxy-scyllo-inosose synthase (kanA) in Streptomyces lividans TK24. The expression of kanA in pIBR25 transformants reached a maximum after 72 h of culture. The plasmid pIBR25 showed better expression than pSET152, and resulted in the formation of insoluble KanA when it was expressed in Escherichia coli. This strategy thus provides a valuable tool for expressing aminoglycoside-aminocyclitols (AmAcs) biosynthetic genes in Streptomyces spp.

The effect of S-adenosylmethionine (SAM) on the production of various antibiotics was investigated to determine whether SAM-dependent methylation is required in biosynthetic pathways of antibiotics. Pristinamycin II(B) and granaticin do not require SAM-dependent methylation in their biosynthesis pathways, and production of these two antibiotics was increased about 2-fold when a low concentration (50 and 10 microM, respectively) of SAM was treated; in contrast, oleandomycin and avermectin B1a require SAM as a methyl donor in their biosynthesis, and production of these two antibiotics was increased 5-fold and 6-fold, depending on the SAM concentration within a certain range. We also found that the transcription of a pathway-specific regulator, gra-ORF9, was activated by exogenous SAM treatment. Production of oleandomycin and avermectin B1a was decreased by using a methyltransferase inhibitor, sinefungin, but the production levels of these antibiotics were restored to the control level by simultaneously adding SAM and sinefungin. Interestingly, we have found a similar stimulatory effect of S-adenosylhomocysteine (SAH), the methylation product of SAM, on antibiotic production in the four strains. Our results clearly demonstrate the widespread activation of antibiotic production using SAM in streptomycetes.

Two actinomycete strains, DSA1T and HD9T, were isolated from a potato tuber and soil from a potato-cultivating field in Jeju, Korea, respectively. A comprehensive 16S rDNA analysis revealed that the isolates belong to the genus Kribbella and share 97.7-98.6% sequence similarity to Kribbella species. The strains also contained typical chemotaxonomic markers of the genus Kribbella: ll-diaminopimelic acid, alanine, glycine and glutamic acid in the cell wall peptidoglycan; mannose, glucose, galactose and ribose as whole cell sugars; diphosphatidylglycerol, phosphatidylcholine and phosphatidylinositol as characteristic phospholipids; and the major menaquinone MK-9 (H4). DNA-DNA hybridization experiments showed that the isolates represented two distinct genomic species. A number of phenotypic properties can be used to differentiate the two isolates from Kribbella species. On the basis of polyphasic evidence, two novel species are proposed: Kribbella solani sp. nov. for strain DSA1T (=KACC 20196T=JCM 12205T) and Kribbella jejuensis sp. nov. for strain HD9T (=KACC 20266T=JCM 12204T).

The 16S rRNA gene sequences for 34 strains, including 11 isolates, were determined to classify scab-causing Streptomyces spp. and relatives isolated from potato scab lesions collected in Jeju, Korea. The 16S-23S rDNA internally transcribed spacer (ITS) sequences were determined to investigate whether the 16S-23S ITS region is useful for analysing intra- and interspecific relationships in these bacteria. On the basis of phylogenetic analysis of 16S rRNA gene sequences, most of the isolates were classified as Streptomyces scabiei and Streptomyces acidiscabies. Isolate KJO61 was placed in an ambiguous taxonomic position between Streptomyces reticuliscabiei and Streptomyces turgidiscabies. 16S-23S ITS region sequence analysis showed that tRNA genes were not found in this region of Streptomyces spp. The 16S-23S ITS regions of Streptomyces spp. exhibited various lengths and highly variable sequence similarities (35-100%) within strains as well as intra- and interspecies. It was revealed that Streptomyces europaeiscabiei could be clearly differentiated from Streptomyces scabiei. However, it was clarified that ITS regions are not useful in phylogenetic analysis of Streptomyces spp.

The biosynthetic gene cluster for bluensomycin, a member of the aminoglycoside family of antibiotics, was isolated and characterized from the bluensomycin producing strain, Streptomyces bluensis ATCC27420. PCR primers were designed specifically to amplify a segment of the dTDP-glucose synthase gene based on its conserved sequences among several actinomycete strains. By screening a cosmid library using amplified PCR fragments, a 30-kb DNA fragment was isolated. Sequence analysis identified 15 open reading frames (ORFs), eight of which had previously been identified by Piepersberg et al. But seven are novel to this study. We demonstrated that one of these ORFs, blmA, confers resistance against the antibiotic dihydrostreptomycin, and another, blmD, encodes a dTDP-glucose synthase. These findings suggest that the isolated gene cluster is very likely to be responsible for the biosynthesis of bluensomycin.

S-Adenosyl-L-methionine synthetase (SAM-s) catalyzes the biosynthesis of SAM from ATP and L-methionine. Despite extensive research with many organisms, its role in Streptomyces sp. remains unclear. In the present study, the putative SAM-s gene was isolated from a spectinomycin producer, Streptomyces spectabilis. The purified protein from the transformed Escherichia coli with the isolated gene synthesized SAM from L-methionine and ATP in vitro, strongly indicating that the isolated gene indeed encoded the SAM-s protein. The overexpression of the SAM-s gene in Streptomyces lividans TK23 inhibited sporulation and aerial mycelium formation but enhanced the production of actinorhodin in both agar plates and liquid media. Surprisingly, the overexpressed SAM was proven by Northern analysis to increase the production of actinorhodin through the induction of actII-ORF4, a transcription activator of actinorhodin biosynthetic gene clusters. In addition, we found that a certain level of intracellular SAM is critical for the induction of antibiotic biosynthetic genes, since the control strain harboring only the plasmid DNA did not show any induction of actII-ORF4 until it reached a certain level of SAM in the cell. From these results, we concluded that the SAM plays important roles as an intracellular factor in both cellular differentiation and antibiotic production in Streptomyces sp.