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PubMed1999

Genes encoding acyl-CoA dehydrogenase (AcdH) homologues from Streptomyces coelicolor and Streptomyces avermitilis provide insights into the metabolism of small branched-chain fatty acids and macrolide antibiotic production.

The cloning, using a PCR approach, of genes from both Streptomyces coelicolor and Streptomyces avermitilis encoding an acyl-CoA dehydrogenase (AcdH), putatively involved in the catabolism of branched-chain amino acids, is reported. The deduced amino acid sequences of both genes have a high similarity to prokaryotic and eukaryotic short-chain acyl-CoA dehydrogenases. When the S. coelicolor and S. avermitilis acyl-CoA dehydrogenase genes (acdH) were expressed in Escherichia coli, each of the AcdH flavoproteins was able to oxidize the branched-chain acyl-CoA derivatives isobutyryl-CoA, isovaleryl-CoA and cyclohexylcarbonyl-CoA, as well as the short straight-chain acyl-CoAs n-butyryl-CoA and n-valeryl-CoA in vitro. NMR spectral data confirmed that the oxidized product of isobutyryl-CoA is methacrylyl-CoA, which is the expected product at the acyl-CoA dehydrogenase step in the catabolism of valine in streptomycetes. Disruption of the S. avermitilis acdH produced a mutant unable to grow on solid minimal medium containing valine, isoleucine or leucine as sole carbon sources. Feeding studies with 13C triple-labelled isobutyrate revealed a significant decrease in the incorporation of label into the methylmalonyl-CoA-derived positions of avermectin in the acdH mutant. In contrast the mutation did not affect incorporation into the malonyl-CoA-derived positions of avermectin. These results are consistent with the acdH gene encoding an acyl-CoA dehydrogenase with a broad substrate specificity that has a role in the catabolism of branched-chain amino acids in S. coelicolor and S. avermitilis.

Acyl-CoA Dehydrogenase↗

PubMed2000

Multiple paralogous genes related to the Streptomyces coelicolor developmental regulatory gene whiB are present in Streptomyces and other actinomycetes.

The whiB sporulation gene of Streptomyces coelicolor was shown [Davis, N. K. & Chater, K. F. (1992). Mol Gen Genet 232, 351-358] to encode a small, cysteine-rich putative transcription factor unlike any that had been described previously. The large database of DNA sequences of mycobacteria (like Streptomyces, members of the Actinomycetales) has revealed a family of genes encoding proteins related to WhiB. Mycobacterium tuberculosis contains at least six such genes (whiB homologues in mycobacteria: whmA-F) and a likely seventh, whmG. Using conserved features of Whm proteins, a PCR-based approach led to the discovery that S. coelicolor A3(2) contains several similar genes. Cloning and sequencing of these whiB-like (wbI) genes revealed likely orthologues of four of the whm genes of M. tuberculosis. In all, S. coelicolor contains at least five wbI genes in addition to whiB itself. All five were shown by RT-PCR to be transcribed. A Southern blotting survey using each wbI gene as a probe showed that nearly all of a series of representatives of ten actinomycete genera (including morphologically simple organisms) contain close homologues of several wbI genes, suggesting that the ancient progenitor of all these organisms already contained a family of such genes, which have not been found in any other organisms.

Actinomycetales↗

PubMed1996

Evidence for a novel class of microbial 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase in Streptomyces coelicolor A3(2), Streptomyces rimosus and Neurospora crassa.

The tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthases from Streptomyces coelicolor A3(2), Streptomyces rimosus and Neurospora crassa have been purified to homogeneity. All three enzymes have a subunit Mr of 54,000. The S. coelicolor DAHP synthase was physically and kinetically characterized and the N-terminal amino acid sequence was obtained. The N-terminal amino acid sequence could not be obtained for the enzymes from S. rimosus and N. crassa, their N-termini apparently being blocked. However, following proteolytic digestion, internal amino acid sequences were obtained from both enzymes. A comparison with the known DAHP synthase sequences indicated that these DAHP synthases are unrelated to other microbial DAHP synthase sequences but are similar to plant DAHP synthases. Up until now, two distinct classes of DAHP synthase have been described, one comprising exclusively enzymes from plants, the other restricted to enzymes from micro-organisms. These studies indicate that the class containing the plant DAHP synthases also contains enzymes from a microbial eukaryote and from several bacteria.

3-Deoxy-7-Phosphoheptulonate Synthase↗

PubMed1999

Regulation of the expression of amy TO1 encoding a thermostable alpha-amylase from Streptomyces sp. TO1, in its original host and in Streptomyces lividans TK24.

In its original host, the thermophilic Streptomyces strain sp. TO1, the amy TO1 gene was expressed during growth but only in the presence of starch in the growth medium. When cloned in Streptomyces lividans, on a low copy number replicative plasmid, amy TO1 expression was detectable in fructose-, mannitol- and galactose-grown cultures but not in glucose- or glycerol-grown cultures. This basal expression could be further induced by maltotriose. In a mutant strain of S. lividans disrupted for the LacI-like negative transcriptional regulator (NTR) Reg1, and when the symmetry of the dyadic symmetry element located in the promoter region of amy TO1 was altered, the basal levels of amy TO1 expression were significantly higher than those of the wild-type strain, and the maltotriose inducibility was abolished. These results suggest that, in S. lividans, amy TO1 expression is under the control of the NTR Reg1 due to its interaction with the dyadic symmetry element.

Base Sequence↗

PubMed1993

Cloning of a Streptomyces clavuligerus DNA fragment encoding the cephalosporin 7 alpha-hydroxylase and its expression in Streptomyces lividans.

A 26-mer DNA probe was designed from N-terminal sequence data for the cephalosporin 7 alpha-hydroxylase (CH) of Streptomyces clavuligerus NRRL 3585 and used to screen a DNA library from this organism. The library was constructed in the lambda GEM-11 phage system. After plaque purification and reprobing, positive recombinant phages were chosen for further analysis. Characterization of the cloned DNA by restriction mapping and Southern hybridization showed that a 1.5-kb SalI fragment hybridized to the probe. Polymerase chain reaction assays using this fragment as a template and the probe as a primer indicated that the fragment carries the entire putative CH gene (cmcI). This was confirmed through the expression of CH enzymatic activity when the fragment was introduced into Streptomyces lividans. A putative beta-lactamase activity was detected in S. lividans.

Amino Acid Sequence↗

PubMed1995

Expression of Streptomyces peucetius genes for doxorubicin resistance and aklavinone 11-hydroxylase in Streptomyces galilaeus ATCC 31133 and production of a hybrid aclacinomycin.

The aklavinone 11-hydroxylase gene and two doxorubicin resistance genes cloned from Streptomyces peucetius subsp. caesius ATCC 27952 were introduced into doxorubicin-sensitive Streptomyces galilaeus ATCC 31133, an aclacinomycin producer. The doxorubicin resistance genes drrA and drrB endowed S. galilaeus with high-level resistance to doxorubicin, indicating that the resistance mechanism for doxorubicin might be different from that for aclacinomycin A. Transformation of S. galilaeus ATCC 31133 with plasmid pMC213 containing the aklavinone 11-hydroxylase gene (dnrF) resulted in the production of many red pigments. A new metabolite was purified, and the position of the newly introduced hydroxyl group was determined. This result indicated that the aklavinone 11-hydroxylase gene was stably expressed in S. galilaeus ATCC 31133 and that it gave rise to a hybrid aclacinomycin A which showed highly specific in vitro cytotoxicity against leukemia and melanoma cell lines.

Aclarubicin↗

PubMed2004

Chalcomycin biosynthesis gene cluster from Streptomyces bikiniensis: novel features of an unusual ketolide produced through expression of the chm polyketide synthase in Streptomyces fradiae.

Chalcomycin, a 16-membered macrolide antibiotic made by the bacterium Streptomyces bikiniensis, contains a 2,3-trans double bond and the neutral sugar D-chalcose in place of the amino sugar mycaminose found in most other 16-membered macrolides. Degenerate polyketide synthase (PKS)-specific primers were used to amplify DNA fragments from S. bikiniensis with very high identity to a unique ketosynthase domain of the tylosin PKS. The resulting amplimers were used to identify two overlapping cosmids encompassing the chm PKS. Sequencing revealed a contiguous segment of >60 kb carrying 25 putative genes for biosynthesis of the polyketide backbone, the two deoxysugars, and enzymes involved in modification of precursors of chalcomycin or resistance to it. The chm PKS lacks the ketoreductase and dehydratase domains in the seventh module expected to produce the 2,3-double bond in chalcomycin. Expression of PKS in the heterologous host Streptomyces fradiae, from which the tyl genes encoding the PKS had been removed, resulted in production of at least one novel compound, characterized as a 3-keto 16-membered macrolactone in equilibrium with its 3-trans enol tautomer and containing the sugar mycaminose at the C-5 position, in agreement with the structure predicted on the basis of the domain organization of the chm PKS. The production of a 3-keto macrolide from the chm PKS indicates that a discrete set of enzymes is responsible for the introduction of the 2,3-trans double bond in chalcomycin. From comparisons of the open reading frames to sequences in databases, a pathway for the synthesis of nucleoside diphosphate-D-chalcose was proposed.

Anti-Bacterial Agents↗

PubMed1986

Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ702.

The cholesterol oxidase gene (cho) of Streptomyces sp. was cloned into Streptomyces lividans with the vector pIJ702. Deletion analysis of the recombinant plasmid showed that entire coding sequence of the cho gene was located within a 2.5-kilobase segment of the chromosomal DNA obtained from the cholesterol oxidase-producing strain. When cloned cells of S. lividans were grown in an appropriate medium, the cells produced severalfold more cholesterol oxidase extracellularly than did the producing strain.

3-Hydroxysteroid Dehydrogenases↗

PubMed2005

Metabolic network analysis of Streptomyces tenebrarius, a Streptomyces species with an active entner-doudoroff pathway.

Streptomyces tenebrarius is an industrially important microorganism, producing an antibiotic complex that mainly consists of the aminoglycosides apramycin, tobramycin carbamate, and kanamycin B carbamate. When S. tenebrarius is used for industrial tobramycin production, kanamycin B carbamate is an unwanted by-product. The two compounds differ only by one hydroxyl group, which is present in kanamycin carbamate but is reduced during biosynthesis of tobramycin. (13)C metabolic flux analysis was used for elucidating connections between the primary carbon metabolism and the composition of the antibiotic complex. Metabolic flux maps were constructed for the cells grown on minimal medium with glucose or with a glucose-glycerol mixture as the carbon source. The addition of glycerol, which is more reduced than glucose, led to a three-times-greater reduction of the kanamycin portion of the antibiotic complex. The labeling indicated an active Entner-Doudoroff (ED) pathway, which was previously considered to be nonfunctional in Streptomyces. The activity of the pentose phosphate (PP) pathway was low (10 to 20% of the glucose uptake rate). The fluxes through Embden-Meyerhof-Parnas (EMP) and ED pathways were almost evenly distributed during the exponential growth on glucose. During the transition from growth phase to production phase, a metabolic shift was observed, characterized by a decreased flux through the ED pathway and increased fluxes through the EMP and PP pathways. Higher specific NADH and NADPH production rates were calculated in the cultivation on glucose-glycerol, which was associated with a lower percentage of nonreduced antibiotic kanamycin B carbamate.

Aminoglycosides↗

PubMed2006

Control of the Streptomyces Subtilisin inhibitor gene by AdpA in the A-factor regulatory cascade in Streptomyces griseus.

AdpA in the A-factor regulatory cascade in Streptomyces griseus activates a number of genes required for secondary metabolism and morphological differentiation, forming an AdpA regulon. The Streptomyces subtilisin inhibitor (SSI) gene, sgiA, in S. griseus was transcribed in response to AdpA, showing that sgiA is a member of the AdpA regulon. AdpA bound a single site upstream of the sgiA promoter at approximately position -70 with respect to its transcriptional start point. Mutational analysis of the AdpA-binding site showed that the AdpA-binding site was essential for transcriptional activation. Mutants in which sgiA was disrupted had higher trypsin, chymotrypsin, metalloendopeptidase, and total protease activities than the wild-type strain, which showed that SgiA modulated the activities of these extracellularly produced proteases. Because a number of genes encoding chymotrypsins, trypsins, and metalloendopeptidases, most of which are SSI-sensitive proteases, are also under the control of AdpA, the A-factor regulatory cascade was thought to play a crucial role in modulating the extracellular protease activities by triggering simultaneous production of the proteases and their inhibitor at a specific timing during growth. Mutants in which sgiA was disrupted grew normally and formed aerial hyphae and spores with the same time course as the wild-type strain. However, exogenous addition of purified SgiA to substrate mycelium grown on agar medium resulted in a delay in aerial mycelium formation, indicating that SgiA is involved in aerial hypha formation in conjunction with proteases.

Amino Acid Sequence↗

PubMed1979

High-frequency fusion of Streptomyces parvulus or Streptomyces antibioticus protoplasts induced by polyethylene glycol.

Conditions were established for the regeneration of protoplasts of Streptomyces parvulus and Streptomyces antibioticus to the mycelial form. Regeneration was accomplished with a hypertonic medium that contained sucrose, CaCl2, MgCl2, and low levels of phosphate. High-frequency fusion of protoplasts derived from auxotrophic strains of S. parvulus or S. antibioticus was induced by polyethylene glycol 4,000 (42%, wt/vol). The frequency of genetic transfer by the fusogenic procedure varied with the auxotrophic strains examined. Fusion with auxotrophic strains of S. parvulus resulted in the formation of true prototrophic recombinants. Similar studies with S. antibioticus revealed that both stable prototrophic recombinants and heterokaryons were formed.

Genotype↗

PubMed1985

Efficient plasmid transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts.

A procedure for efficient transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts with plasmid DNA was developed. Transformation frequencies with S. fradiae protoplasts were strongly influenced by the temperatures for cell growth, protoplast formation, and protoplast regeneration. Transformation frequencies for both species were also influenced by the culture age before protoplast formation, the source and concentration of polyethylene glycol, the transformation-inducing agent, the concentration of protoplasts used in the transformation procedure, and the number of protoplasts added to regeneration plates. Transformation frequencies were substantially higher for both species when calf thymus DNA and protamine sulfate were added to the transformation mix. With S. fradiae, transformation frequencies were much lower with plasmid DNA prepared from other species than with the same plasmids prepared from S. fradiae, suggesting that S. fradiae expresses restriction and modification. With the modified transformation procedures using DNA prepared from homologous hosts, S. ambofaciens and S. fradiae are now transformed routinely at frequencies of 10(6) to 10(7) transformants per micrograms of plasmid DNA.

DNA↗

PubMed1989

Cloning and heterologous expression in Streptomyces lividans of Streptomyces rimosus genes involved in oxytetracycline biosynthesis.

The anhydrotetracycline (ATC) oxygenase enzyme which carries out the conversion of ATC to dehydrotetracycline was purified and the N-terminal amino acid sequence was determined. The sequence displays a significant similarity to that of the p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. This is consistent with the activity of the oxygenase, i.e., addition of a hydroxyl moiety to an aromatic ring structure. Oligonucleotide probes were designed and used to clone the corresponding fragment of chromosomal DNA from Streptomyces rimosus. This DNA fragment was used to screen a cosmid library, allowing the isolation of flanking DNA sequences. Surprisingly, the gene was located within the previously cloned cluster of genes involved in the synthesis of the biosynthetic intermediate ATC and not as had been expected (P. M. Rhodes, N. Winskill, E. J. Friend, and M. Warren, J. Gen. Microbiol. 124:329-338, 1981) at a separate locus on the other side of the chromosome. Subcloning of an appropriate DNA fragment from one of the cosmid clones onto pIJ916 produced Streptomyces lividans transformants which synthesized oxytetracycline.

Amino Acid Sequence↗

PubMed1991

Characterization, expression in Streptomyces lividans, and processing of the amylase of Streptomyces griseus IMRU 3570: two different amylases are derived from the same gene by an intracellular processing mechanism.

Extracellular amylase in Streptomyces lividans was undetectable in starch-supplemented medium. However, S. lividans produced fivefold-higher levels of amylase than Streptomyces griseus IMRU 3570 when transformed with the S. griseus amy gene. Two major proteins of 57 and 50 kDa with amylase activity accumulated in the culture broths of the donor S. griseus and S. lividans transformed with the amy gene. Both proteins were also present in protoplast lysates in the same relative proportion; they gave a positive reaction with antibodies against the 57-kDa amylase. They did not differ in substrate specificity or enzyme kinetics. The two amylases were purified to homogeneity by a two-step procedure. Both proteins showed the same amino-terminal sequence of amino acids, suggesting that both proteins are derived from the same gene. The deduced signal peptide has 28 amino acids with two positively charged arginines near the amino-terminal end. When an internal NcoI fragment was removed from the amy gene, the resulting S. lividans transformants did not synthesize any of the two amylase proteins and showed no reaction in immunoblotting. Formation of the 50-kDa protein was observed when pure 57-kDa amylase was treated with supernatants of protoplast lysates but not when it was treated with membrane preparations, indicating that the native 57-kDa amylase could be processed intracellularly.

Amino Acid Sequence↗

PubMed1992

Heterologous expression and secretion of a Streptomyces scabies esterase in Streptomyces lividans and Escherichia coli.

The esterase gene from Streptomyces scabies FL1 was cloned and expressed in Streptomyces lividans on plasmids pIJ486 and pIJ702. In S. lividans, the esterase gene was expressed during later stages of growth and was regulated by zinc, as is seen with S. scabies. The 36-kDa secreted form of the esterase was purified from S. lividans. N-terminal amino acid sequencing indicated that the processing site utilized in S. lividans for the removal of the signal sequence was the same as that recognized for processing in S. scabies. Western blots (immunoblots) revealed the presence of a 40-kDa precursor form of the esterase in cytoplasmic extracts. A 23-amino-acid deletion was introduced into the putative signal sequence for the esterase. When this deleted form of the esterase was expressed in S. lividans, a cytoplasmic 38-kDa precursor protein was produced but no secreted esterase was detected, suggesting the importance of the deleted sequence for efficient processing and secretion. The esterase gene was also cloned into the pUC119 plasmid in Escherichia coli. By using the lac promoter sequence, the esterase gene was expressed, and the majority of the esterase was localized to the periplasmic space.

Alkaline Phosphatase↗

PubMed1992

Cloning and sequencing of a gene encoding a novel extracellular neutral proteinase from Streptomyces sp. strain C5 and expression of the gene in Streptomyces lividans 1326.

The gene encoding a novel milk protein-hydrolyzing proteinase was cloned on a 6.56-kb SstI fragment from Streptomyces sp. strain C5 genomic DNA into Streptomyces lividans 1326 by using the plasmid vector pIJ702. The gene encoding the small neutral proteinase (snpA) was located within a 2.6-kb BamHI-SstI restriction fragment that was partially sequenced. The molecular mass of the deduced amino acid sequence of the mature protein was determined to be 15,740, which corresponds very closely with the relative molecular mass of the purified protein (15,500) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified neutral proteinase was determined, and the DNA encoding this sequence was found to be located within the sequenced DNA. The deduced amino acid sequence contains a conserved zinc binding site, although secondary ligand binding and active sites typical of thermolysinlike metalloproteinases are absent. The combination of its small size, deduced amino acid sequence, and substrate and inhibition profile indicate that snpA encodes a novel neutral proteinase.

Amino Acid Sequence↗

PubMed1993

Physical map of the Streptomyces lividans 66 genome and comparison with that of the related strain Streptomyces coelicolor A3(2).

A physical map of the chromosome of Streptomyces lividans 66 ZX7 was constructed by ordering the macrorestriction fragments generated from the genomic DNA with the restriction enzymes AseI and DraI. AseI and DraI linking cosmids (i.e., recombinant cosmids including either AseI or DraI sites) were isolated from a gene bank and used as hybridization probes against Southern transfers of pulsed-field gel electrophoresis (PFGE) restriction patterns. The DraI sites were precisely mapped by PFGE analyses of AseI-DraI double digests and hybridization with the AseI junctions. The 16 AseI and 7 DraI fragments were aligned as a single chromosome of about 8,000 kb. The data supported the interpretation that the chromosome is a linear structure. The related strain Streptomyces coelicolor A3(2) M145, recently mapped by H. Kieser, T. Kieser, and D. A. Hopwood (J. Bacteriol. 174:5496-5507, 1992), was compared with S. lividans at the level of the genomic structure by hybridizing the linking cosmids to Southern transfers of PFGE patterns. In spite of little apparent similarity in their restriction patterns, the comparison of the physical maps revealed a common structure with an identical ordering of the cosmid sequences. This conservation of the map order was further confirmed by assigning genetic markers (i.e., cloned genes and DNA elements relevant to the unstable region) to the AseI fragments.

DNA, Bacterial↗

PubMed1995

Nucleotide sequences and expression of genes from Streptomyces purpurascens that cause the production of new anthracyclines in Streptomyces galilaeus.

Six open reading frames, rdmA to rdmF, in a 6,077-bp segment of Streptomyces purpurascens DNA which caused the production of hybrid anthracyclines were identified. The minimal fragment that produced anthracyclines modified at the 10th position contained rdmB to rdmD; rdmE is the gene for aklavinone-11-hydroxylase. RdmC is similar to a putative open reading frame in the daunorubicin biosynthetic cluster of Streptomyces peucetius and is likely to participate in the removal of the side chain at the 10th position.

Amino Acid Sequence↗