RESUMEN
Clusters for clavulanic acid (CA) biosynthesis are present in the actinomycetes Streptomyces flavogriseus ATCC 33331 and Saccharomonospora viridis DSM 43017. These clusters, which are silent, contain blocks of conserved genes in the same order as those of the Streptomyces clavuligerus CA cluster but assembled in a different organization. S. flavogriseus was grown in nine different media, but clavulanic acid production was undetectable using bioassays or by high-performance liquid chromatography analyses. Reverse-transcriptase polymerase chain reaction (RT-PCR) of S. flavogriseus CA biosynthesis genes showed that the regulatory genes ccaR and claR and some biosynthetic genes were expressed whereas expression of cyp, orf12, orf13, and oppA2 was undetectable. The ccaR gene of S. clavuligerus was unable to switch on CA production in S. flavogriseus::[Pfur-ccaR C], but insertion of a cosmid carrying the S. clavuligerus CA cluster (not including the ccaR gene) conferred clavulanic acid production on S. flavogriseus::[SCos-CA] particularly in TBO and YEME media; these results suggests that some of the S. flavogriseus CA genes are inactive. The known heptameric sequences recognized by CcaR in S. clavuligerus are poorly or not conserved in S. flavogriseus. Quantitative RT-PCR analysis of the CA gene clusters of S. clavuligerus and S. flavogriseus showed that the average expression value of the expressed genes in the former strain was in the order of 1.68-fold higher than in the later. The absence of CA production by S. flavogriseus can be traced to the lack of expression of the essential genes cyp, orf12, orf13, orf14, and oppA2. Heterologous expression of S. clavuligerus CA gene cluster in S. flavogriseus::[SCos-CA] was 11- to 14-fold lower than in the parental strain, suggesting that the genetic background of the host strain is important for optimal production of CA in Streptomyces.
Asunto(s)
Vías Biosintéticas/genética , Ácido Clavulánico/metabolismo , Regulación Bacteriana de la Expresión Génica , Streptomyces/genética , Streptomyces/metabolismo , Perfilación de la Expresión Génica , Familia de Multigenes , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The dltABCD cluster is involved in the d-alanylation of teichoic acids in gram positive bacteria. In order to determine the role of this alanylation in the physiology and virulence of Lactococcus garvieae, a previously isolated dltA Delta Tn917 signature tagged mutagenesis (STM) clone was analyzed. RT-PCR results revealed that dltABCD genes form an operon. No major differences could be established between the parental and mutant strains with respect to growth rate, autolytic properties, and susceptibility to acid conditions, lysozyme treatment, anionic detergents, or oxidant agents. However, the dltA mutant was more susceptible to nisin than the parental strain, with minimum inhibitory concentration (MIC) values of 8 and 16 microg/ml, respectively. Less proliferation of the mutant was observed in in vivo competence index experiments (CI=0.08). Furthermore, the mutant strain had a 50% lethal dose (LD(50)) 3-fold that of the parental strain. These results, together with the fact that the dltA Delta Tn917 mutant was isolated as a STM clone, reveal that the dltA locus of Lactococcus garvieae is required for full growth and pathogenesis on rainbow trout.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ligasas de Carbono-Oxígeno/metabolismo , Lactococcus/enzimología , Animales , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Ligasas de Carbono-Oxígeno/genética , Proliferación Celular , ADN Bacteriano , Farmacorresistencia Bacteriana , Enfermedades de los Peces/microbiología , Lactococcus/genética , Mutación , Oncorhynchus mykissRESUMEN
Oleandomycin, a macrolide antibiotic produced by Streptomyces antibioticus, contains two sugars attached to the aglycon: L-oleandrose and D-desosamine. oleY codes for a methyltransferase involved in the biosynthesis of L-oleandrose. This gene was overexpressed in Escherichia coli to form inclusion bodies and in Streptomyces lividans, producing a soluble protein. S. lividans overexpressing oleY was used as a biotransformation host, and it converted the precursor L-olivosyl-erythronolide B into its 3-O-methylated derivative, L-oleandrosyl-erythronolide B. Two other monoglycosylated derivatives were also substrates for the OleY methyltransferase: L-rhamnosyl- and L-mycarosyl-erythronolide B. OleY methyltransferase was purified yielding a 43-kDa single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme showed a molecular mass of 87 kDa by gel filtration chromatography, indicating that the enzyme acts as a dimer. It showed a narrow pH range for optimal activity, and its activity was clearly stimulated by the presence of several divalent cations, being maximal with Co(2+). The S. antibioticus OleG2 glycosyltransferase is proposed to transfer L-olivose to the oleandolide aglycon, which is then converted into L-oleandrose by the OleY methyltransferase. This represents an alternative route for L-oleandrose biosynthesis from that in the avermectin producer Streptomyces avermitilis, in which L-oleandrose is transferred to the aglycon by a glycosyltransferase.
Asunto(s)
Antibacterianos/biosíntesis , Desoxiazúcares , Metiltransferasas/metabolismo , Oleandomicina/biosíntesis , Streptomyces antibioticus/enzimología , Escherichia coli/enzimología , Escherichia coli/genética , Hexosas/metabolismo , Metiltransferasas/genética , Metiltransferasas/aislamiento & purificación , Streptomyces/enzimología , Streptomyces/genética , Streptomyces antibioticus/genética , Streptomyces antibioticus/crecimiento & desarrollo , Especificidad por SustratoRESUMEN
The anthracycline-like polyketide drug elloramycin is produced by Streptomyces olivaceus Tü2353. Elloramycin has antibacterial activity against Gram-positive bacteria and also exhibits antitumor activity. From a cosmid clone (cos16F4) containing part of the elloramycin biosynthesis gene cluster, three genes (elmMI, elmMII, and elmMIII) have been cloned. Sequence analysis and data base comparison showed that their deduced products resembled S-adenosylmethionine-dependent O-methyltransferases. The genes were individually expressed in Streptomyces albus and also coexpressed with genes involved in the biosynthesis of l-rhamnose, the 6-deoxysugar attached to the elloramycin aglycon. The resulting recombinant strains were used to biotransform three different elloramycin-type compounds: l-rhamnosyl-tetracenomycin C, l-olivosyl-tetracenomycin C, and l-oleandrosyl-tetracenomycin, which differ in their 2'-, 3'-, and 4'-substituents of the sugar moieties. When only the three methyltransferase-encoding genes elmMI, elmMII, and elmMIII were individually expressed in S. albus, the methylating activity of the three methyltransferases was also assayed in vitro using various externally added glycosylated substrates. From the combined results of all of these experiments, it is proposed that methyltransferases ElmMI, ElmMII, and ElmMIII are involved in the biosynthesis of the permethylated l-rhamnose moiety of elloramycin. ElmMI, ElmMII, and ElmMIII are responsible for the consecutive methylation of the hydroxy groups at the 2'-, 3'-, and 4'-position, respectively, after the sugar moiety has been attached to the aglycon.
Asunto(s)
Antraquinonas/metabolismo , Antibacterianos/metabolismo , Streptomyces/metabolismo , Secuencia de Aminoácidos , Sistema Libre de Células , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Clonación Molecular , Cósmidos/metabolismo , ADN/metabolismo , Glicósidos/química , Glicosilación , Metilación , Metiltransferasas/genética , Modelos Químicos , Modelos Genéticos , Datos de Secuencia Molecular , Naftacenos/química , Proteínas Recombinantes/metabolismo , Ramnosa/metabolismo , Homología de Secuencia de Aminoácido , Factores de TiempoRESUMEN
BACKGROUND: Elloramycin is an anthracycline-like antitumor drug related to tetracenomycin C which is produced by Streptomyces olivaceus Tü2353. Structurally is a tetracyclic aromatic polyketide derived from the condensation of 10 acetate units. Its chromophoric aglycon is glycosylated with a permethylated L-rhamnose moiety at the C-8 hydroxy group. Only limited information is available about the genes involved in the biosynthesis of elloramycin. From a library of chromosomal DNA from S. olivaceus, a cosmid (16F4) was isolated that contains part of the elloramycin gene cluster and when expressed in Streptomyces lividans resulted in the production of a non-glycosylated intermediate in elloramycin biosynthesis, 8-demethyl-tetracenomycin C (8-DMTC). RESULTS: The expression of cosmid 16F4 in several producers of glycosylated antibiotics has been shown to produce tetracenomycin derivatives containing different 6-deoxysugars. Different experimental approaches showed that the glycosyltransferase gene involved in these glycosylation events was located in 16F4. Using degenerated oligoprimers derived from conserved amino acid sequences in glycosyltransferases, the gene encoding this sugar flexible glycosyltransferase (elmGT) has been identified. After expression of elmGT in Streptomyces albus under the control of the erythromycin resistance promoter, ermEp, it was shown that elmG can transfer different monosaccharides (both L- and D-sugars) and a disaccharide to 8-DMTC. Formation of a diolivosyl derivative in the mithramycin producer Streptomyces argillaceus was found to require the cooperative action of two mithramycin glycosyltransferases (MtmGI and MtmGII) responsible for the formation of the diolivosyl disaccharide, which is then transferred by ElmGT to 8-DMTC. CONCLUSIONS: The ElmGT glycosyltransferase from S. olivaceus Tü2353 can transfer different sugars into the aglycon 8-DMTC. In addition to its natural sugar substrate L-rhamnose, ElmGT can transfer several L- and D-sugars and also a diolivosyl disaccharide into the aglycon 8-DMTC. ElmGT is an example of sugar flexible glycosyltransferase and can represent an important tool for combinatorial biosynthesis.
Asunto(s)
Antraquinonas/metabolismo , Antibacterianos/biosíntesis , Glicosiltransferasas/genética , Streptomyces/enzimología , Antibacterianos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , Biblioteca de Genes , Glicosiltransferasas/química , Datos de Secuencia Molecular , Naftacenos/metabolismo , Alineación de Secuencia , Transformación GenéticaRESUMEN
Mithramycin is a glycosylated aromatic polyketide produced by Streptomyces argillaceus, and is used as an antitumor drug. Three genes (mtmV, mtmU and mtmC) from the mithramycin gene cluster have been cloned, and characterized by DNA sequencing and by analysis of the products that accumulate in nonproducing mutants, which were generated by insertional inactivation of these genes. The mtm V gene codes for a 2,3-dehydratase that catalyzes early and common steps in the biosynthesis of the three sugars found in mithramycin (D-olivose, D-oliose and D-mycarose); its inactivation caused the accumulation of the nonglycosylated intermediate premithramycinone. The mtmU gene codes for a 4-ketoreductase involved in D-oliose biosynthesis, and its inactivation resulted in the accumulation of premithramycinone and premithramycin A , the first glycosylated intermediate which contains a D-olivose unit. The third gene, mtmC, is involved in D-mycarose biosynthesis and codes for a C-methyltransferase. Two mutants with lesions in the mtmC gene accumulated mithramycin intermediates lacking the D-mycarose moiety but containing D-olivose units attached to C-12a in which the 4-keto group is unreduced. This suggests that mtmC could code for a second enzyme activity, probably a D-olivose 4-ketoreductase, and that the glycosyltransferase responsible for the incorporation of D-olivose (MtmGIV) shows some degree of flexibility with respect to its sugar co-substrate, since the 4-ketoanalog is also transferred. A pathway is proposed for the biosynthesis of the three sugar moieties in mithramycin.
Asunto(s)
Hexosas/biosíntesis , Familia de Multigenes , Plicamicina/biosíntesis , Streptomyces/genética , Secuencia de Carbohidratos , Mapeo Cromosómico , Clonación Molecular , Desoxiazúcares/biosíntesis , Prueba de Complementación Genética , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Plicamicina/químicaRESUMEN
Elloramycin and oleandomycin are two polyketide compounds produced by Streptomyces olivaceus Tü2353 and Streptomyces antibioticus ATCC11891, respectively. Elloramycin is an anthracycline-like antitumor drug and oleandomycin a macrolide antibiotic. Expression in S. albus of a cosmid (cos16F4) containing part of the elloramycin biosynthetic gene cluster produced the elloramycin non-glycosylated intermediate 8-demethyl-tetracenomycin C. Several plasmid constructs harboring different gene combinations of L-oleandrose (neutral 2,6-dideoxyhexose attached to the macrolide antibiotic oleandomycin) biosynthetic genes of S. antibioticus that direct the biosynthesis of L-olivose, L-oleandrose and L-rhamnose were coexpressed with cos16F4 in S. albus. Three new hybrid elloramycin analogs were produced by these recombinant strains through combinatorial biosynthesis, containing elloramycinone or 12a-demethyl-elloramycinone (= 8-demethyl-tetracenomycin C) as aglycone moiety encoded by S. olivaceus genes and different sugar moieties, coded by the S. antibioticus genes. Among them is L-olivose, which is here described for the first time as a sugar moiety of a natural product.
Asunto(s)
Técnicas Químicas Combinatorias , Desoxiazúcares/metabolismo , Antraciclinas/metabolismo , Antraquinonas/química , Desoxiazúcares/química , Glicosilación , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Macrólidos/metabolismo , Naftacenos/química , Naftacenos/metabolismo , Oleandomicina/metabolismo , Plásmidos/genética , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
A 9.8-kb DNA region from the oleandomycin gene cluster in Streptomyces antibioticus was cloned. Sequence analysis revealed the presence of 8 open reading frames encoding different enzyme activities involved in the biosynthesis of one of the two 2, 6-deoxysugars attached to the oleandomycin aglycone: L-oleandrose (the oleW, oleV, oleL, and oleU genes) and D-desosamine (the oleNI and oleT genes), or of both (the oleS and oleE genes). A Streptomyces albus strain harboring the oleG2 glycosyltransferase gene integrated into the chromosome was constructed. This strain was transformed with two different plasmid constructs (pOLV and pOLE) containing a set of genes proposed to be required for the biosynthesis of dTDP-L-olivose and dTDP-L-oleandrose, respectively. Incubation of these recombinant strains with the erythromycin aglycon (erythronolide B) gave rise to two new glycosylated compounds, identified as L-3-O-olivosyl- and L-3-O-oleandrosyl-erythronolide B, indicating that pOLV and pOLE encode all enzyme activities required for the biosynthesis of these two 2,6-dideoxysugars. A pathway is proposed for the biosynthesis of these two deoxysugars in S. antibioticus.
Asunto(s)
Proteínas Bacterianas/genética , Desoxiazúcares/biosíntesis , Hexosas/biosíntesis , Streptomyces antibioticus/genética , Secuencia de Aminoácidos , Amino Azúcares/biosíntesis , Amino Azúcares/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Regulación Bacteriana de la Expresión Génica , Glicosiltransferasas/biosíntesis , Glicosiltransferasas/genética , Datos de Secuencia Molecular , Familia de Multigenes , Oleandomicina/biosíntesis , Homología de Secuencia de Aminoácido , Streptomyces antibioticus/metabolismoRESUMEN
Mithramycin is an antitumor aromatic polyketide synthesized by Streptomyces argillaceus. Two genes (mtrX and mtrY) of the mithramycin gene cluster were inactivated by gene replacement. Inactivation of mtrX, that encodes an ABC excision nuclease system for DNA repair, produced a mutant that was affected in the normal rate of growth. Expression of mtrX in Streptomyces albus in a multicopy plasmid vector conferred a low increase in resistance to mithramycin. Inactivation of mtrY, that encodes a protein of unknown function, produced a 50% decrease in mithramycin biosynthesis. When mtrY was expressed in the wild-type S. argillaceus in a multicopy plasmid, this caused about 47% increase in the levels of mithramycin production. It is proposed that mtrX and mtrY could code for a secondary defense mechanism and a mithramycin regulatory element, respectively.
Asunto(s)
Antibióticos Antineoplásicos/biosíntesis , Proteínas Bacterianas/genética , Plicamicina/biosíntesis , Streptomyces/crecimiento & desarrollo , Streptomyces/metabolismo , Secuencia de Aminoácidos , Antibióticos Antineoplásicos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Farmacorresistencia Microbiana/genética , Genes Bacterianos , Genes Reguladores , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis Insercional , Plicamicina/farmacología , Alineación de Secuencia , Streptomyces/efectos de los fármacos , Streptomyces/genética , Transformación BacterianaRESUMEN
The oleD gene has been identified in the oleandomycin producer Streptomyces antibioticus and it codes a macrolide glycosyltransferase that is able to transfer a glucose moiety from UDP-glucose (UDP-Glc) to many macrolides. The glycosyltransferase coded by the oleD gene has been purified 371-fold from a Streptomyces lividans clone expressing this protein. The reaction product was isolated, and its structure determined by NMR spectroscopy. The kinetic mechanism of the reaction was analyzed using the macrolide antibiotic lankamycin (LK) as substrate. The reaction operates via a compulsory order mechanism. This has been shown by steady-state kinetic studies and by isotopic exchange reactions at equilibrium. LK binds first to the enzyme, followed by UDP-glucose. A ternary complex is thus formed prior to transfer of glucose. UDP is then released, followed by the glycosylated lankamycin (GS-LK). A pH study of the reaction was performed to determine values for the molecular pK values, suggesting possible amino acid residues involved in the catalytic process.
Asunto(s)
Antibacterianos/metabolismo , Glucosiltransferasas/aislamiento & purificación , Glucosiltransferasas/metabolismo , Streptomyces antibioticus/enzimología , Secuencia de Aminoácidos , Catálisis , Eritromicina/análogos & derivados , Eritromicina/metabolismo , Glicosilación , Concentración de Iones de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Oleandomicina/metabolismo , Pliegue de Proteína , Uridina Difosfato/metabolismo , Uridina Difosfato Glucosa/metabolismoRESUMEN
A 2,580-bp region of the chromosome of Streptomyces argillaceus, the producer of the antitumor polyketide mithramycin, was sequenced. Analysis of the nucleotide sequence revealed the presence of two genes (mtmGIII and mtmGIV) encoding proteins that showed a high degree of similarity to glycosyltransferases involved in the biosynthesis of various antibiotics and antitumor drugs. Independent insertional inactivation of both genes produced mutants that did not synthesize mithramycin but accumulated several mithramycin intermediates. Both mutants accumulated premithramycinone, a non-glycosylated intermediate in mithramycin biosynthesis. The mutant affected in the mtmGIII gene also accumulated premithramycin A1, which contains premithramycinone as the aglycon unit and a D-olivose attached at C-12a-O. These experiments demonstrate that the glycosyltransferases MtmGIV and MtmGIII catalyze the first two glycosylation steps in mithramycin biosynthesis. A model is proposed for the glycosylation steps in mithramycin biosynthesis.
Asunto(s)
Antibióticos Antineoplásicos/biosíntesis , Glicosiltransferasas/metabolismo , Plicamicina/biosíntesis , Streptomyces/metabolismo , Secuencia de Aminoácidos , Antibióticos Antineoplásicos/química , Secuencia de Bases , ADN Bacteriano/genética , Marcación de Gen , Genes Bacterianos , Glicosilación , Glicosiltransferasas/genética , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación , Filogenia , Plicamicina/química , Homología de Secuencia de Aminoácido , Streptomyces/enzimología , Streptomyces/genéticaRESUMEN
A DNA chromosomal region of Streptomyces argillaceus ATCC 12596, the producer organism of the antitumor polyketide drug mithramycin, was cloned. Sequence analysis of this DNA region, located between four mithramycin glycosyltransferase genes, showed the presence of two genes (mtmMI and mtmMII) whose deduced products resembled S-adenosylmethionine-dependent methyltransferases. By independent insertional inactivation of both genes nonproducing mutants were generated that accumulated different mithramycin biosynthetic intermediates. The M3DeltaMI mutant (mtmMI-minus mutant) accumulated 4-demethylpremithramycinone (4-DPMC) which lacks the methyl groups at carbons 4 and 9. The M3DeltaM2 (mtmMII-minus mutant) accumulated 9-demethylpremithramycin A3 (9-DPMA3), premithramycin A1 (PMA1), and 7-demethylmithramycin, all of them containing the O-methyl group at C-4 and C-1', respectively, but lacking the methyl group at the aromatic position. Both genes were expressed in Streptomyces lividans TK21 under the control of the erythromycin resistance promoter (ermEp) of Saccharopolyspora erythraea. Cell-free extracts of these clones were precipitated with ammonium sulfate (90% saturation) and assayed for methylation activity using different mithramycin intermediates as substrates. Extracts of strains MJM1 (expressing the mtmMI gene) and MJM2 (expressing the mtmMII gene) catalyzed efficient transfer of tritium from [(3)H]S-adenosylmethionine into 4-DPMC and 9-DPMA3, respectively, being unable to methylate other intermediates at a detectable level. These results demonstrate that the mtmMI and mtmMII genes code for two S-adenosylmethionine-dependent methyltransferases responsible for the 4-O-methylation and 9-C-methylation steps of the biosynthetic precursors 4-DPMC and 9-DPMA3, respectively, of the antitumor drug mithramycin. A pathway is proposed for the last steps in the biosynthesis of mithramycin involving these methylation events.
Asunto(s)
Metiltransferasas , Plicamicina/biosíntesis , Streptomyces/metabolismo , Secuencia de Aminoácidos , Antibióticos Antineoplásicos/biosíntesis , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Clonación Molecular , Metiltransferasas/análisis , Metiltransferasas/genética , Metiltransferasas/metabolismo , Datos de Secuencia Molecular , Especificidad por SustratoRESUMEN
Mithramycin is an aromatic antitumour polyketide synthesized by Streptomyces argillaceus. Two chromosomal regions located upstream and downstream of the locus for the mithramycin type II polyketide synthase were cloned and sequenced. Analysis of the sequence revealed the presence of eight genes encoding three oxygenases (mtmOI, mtmOII and mtmOIII), three reductases (mtmTI, mtmTII and mtmTIII), a cyclase (mtm Y) and an acyl CoA ligase (mtmL). The three oxygenase genes were each inactivated by gene replacement. Inactivation of one of them (mtmOII) generated a non-producing mutant, while inactivation of the other two (mtmOl and mtmOIII) did not affect the biosynthesis of mithramycin. The mtmOII gene may code for an oxygenase responsible for the introduction of oxygen atoms at early steps in the biosynthesis of mithramycin leading to 4-demethylpremithramycinone. One of the reductases may be responsible for reductive cleavage of an intermediate from an enzyme and another for the reduction of a keto group in the side-chain of the mithramycin aglycon moiety. A hypothetical biosynthetic pathway showing in particular the involvement of oxygenase MtmOII and of various other gene products in mithramycin biosynthesis is proposed.
Asunto(s)
Antibióticos Antineoplásicos/biosíntesis , Cromosomas Bacterianos , Genes Bacterianos , Complejos Multienzimáticos/genética , Familia de Multigenes , Plicamicina/biosíntesis , Streptomyces/genética , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Insercional , Oxigenasas/genética , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Mithramycin is a member of the clinically important aureolic acid group of antitumor drugs that interact with GC-rich regions of DNA nonintercalatively. These drugs contain a chromophore aglycon that is derived from condensation of ten acetate units (catalyzed by a type II polyketide synthase). The aglycones are glycosylated at two positions with different chain length deoxyoligosaccharides, which are essential for the antitumor activity. During the early stages of mithramycin biosynthesis, tetracyclic intermediates of the tetracycline-type occur, which must be converted at later stages into the tricyclic glycosylated molecule, presumably through oxidative breakage of the fourth ring. RESULTS: Two intermediates in the mithramycin biosynthetic pathway, 4-demethyl-premithramycinone and premithramycin B, were identified in a mutant lacking the mithramycin glycosyltransferase and methyltransferase genes and in the same mutant complemented with the deleted genes, respectively. Premithramycin B contains five deoxysugars moieties (like mithramycin), but contains a tetracyclic aglycon moiety instead of a tricyclic aglycon. We hypothesized that transcription of mtmOIV (encoding an oxygenase) was impaired in this strain, preventing oxidative breakage of the fourth ring of premithramycin B. Inactivating mtmOIV generated a mithramycin nonproducing mutant that accumulated premithramycin B instead of mithramycin. In vitro assays demonstrated that MtmOIV converted premithramycin B into a tricyclic compound. CONCLUSIONS: In the late stages of mithramycin biosynthesis by Strepyomyces argillaceus, a fully glycosylated tetracyclic tetracycline-like intermediate (premithramycin B) is converted into a tricyclic compound by the oxygenase MtmOIV. This oxygenase inserts an oxygen (Baeyer-Villiger oxidation) and opens the resulting lactone. The following decarboxylation and ketoreduction steps lead to mithramycin. Opening of the fourth ring represents one of the last steps in mithramycin biosynthesis.
Asunto(s)
Antibióticos Antineoplásicos/biosíntesis , Oxigenasas/genética , Plicamicina/biosíntesis , Streptomyces/metabolismo , Secuencia de Aminoácidos , Antibióticos Antineoplásicos/metabolismo , Secuencia de Carbohidratos , Catálisis , Glicosiltransferasas/metabolismo , Espectroscopía de Resonancia Magnética , Metiltransferasas/metabolismo , Datos de Secuencia Molecular , Complejos Multienzimáticos/metabolismo , Mutagénesis Insercional , Mutación , Oxidación-Reducción , Oxigenasas/metabolismo , Plicamicina/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Streptomyces/genéticaRESUMEN
Sequencing of a 4.3-kb DNA region from the chromosome of Streptomyces argillaceus, a mithramycin producer, revealed the presence of two open reading frames (ORFs). The first one (orfA) codes for a protein that resembles several transport proteins. The second one (mtmR) codes for a protein similar to positive regulators involved in antibiotic biosynthesis (DnrI, SnoA, ActII-orf4, CcaR, and RedD) belonging to the Streptomyces antibiotic regulatory protein (SARP) family. Both ORFs are separated by a 1.9-kb, apparently noncoding region. Replacement of the mtmR region by an antibiotic resistance cassette completely abolished mithramycin biosynthesis. Expression of mtmR in a high-copy-number vector in S. argillaceus caused a 16-fold increase in mithramycin production. The mtmR gene restored actinorhodin production in Streptomyces coelicolor JF1 mutant, in which the actinorhodin-specific activator ActII-orf4 is inactive, and also stimulated actinorhodin production by Streptomyces lividans TK21. A 241-bp region located 1.9 kb upstream of mtmR was found to be repeated approximately 50 kb downstream of mtmR at the other end of the mithramycin gene cluster. A model to explain a possible route for the acquisition of the mithramycin gene cluster by S. argillaceus is proposed.
Asunto(s)
Proteínas Bacterianas/genética , Cromosomas Bacterianos , ADN Bacteriano/genética , Genes Reguladores , Familia de Multigenes , Plicamicina/biosíntesis , Secuencias Repetitivas de Ácidos Nucleicos , Streptomyces/genética , Transactivadores , Secuencia de Aminoácidos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Secuencia de Bases , Secuencia de Carbohidratos , Mapeo Cromosómico , Secuencia de Consenso , ADN Bacteriano/química , Datos de Secuencia Molecular , Estructura Molecular , Sistemas de Lectura Abierta , Plicamicina/química , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Streptomyces/metabolismoRESUMEN
Mithramycin is an antitumor polyketide drug produced by Streptomyces argillaceus that contains two deoxysugar chains, a disaccharide consisting of two D-olivoses and a trisaccharide consisting of a D-olivose, a D-oliose, and a D-mycarose. From a cosmid clone (cosAR3) which confers resistance to mithramycin in streptomycetes, a 3-kb PstI-XhoI fragment was sequenced, and two divergent genes (mtmGI and mtmGII) were identified. Comparison of the deduced products of both genes with proteins in databases showed similarities with glycosyltransferases and glucuronosyltransferases from different sources, including several glycosyltransferases involved in sugar transfer during antibiotic biosynthesis. Both genes were independently inactivated by gene replacement, and the mutants generated (M3G1 and M3G2) did not produce mithramycin. High-performance liquid chromatography analysis of ethyl acetate extracts of culture supernatants of both mutants showed the presence of several peaks with the characteristic spectra of mithramycin biosynthetic intermediates. Four compounds were isolated from both mutants by preparative high-performance liquid chromatography, and their structures were elucidated by physicochemical methods. The structures of these compounds were identical in both mutants, and the compounds are suggested to be glycosylated intermediates of mithramycin biosynthesis with different numbers of sugar moieties attached to C-12a-O of a tetracyclic mithramycin precursor and to C-2-O of mithramycinone: three tetracyclic intermediates containing one sugar (premithramycin A1), two sugars (premithramycin A2), or three sugars (premithramycin A3) and one tricyclic intermediate containing a trisaccharide chain (premithramycin A4). It is proposed that the glycosyltransferases encoded by mtmGI and mtmGII are responsible for forming and transferring the disaccharide during mithramycin biosynthesis. From the structures of the new metabolites, a new biosynthetic sequence regarding late steps of mithramycin biosynthesis can be suggested, a sequence which includes glycosyl transfer steps prior to the final shaping of the aglycone moiety of mithramycin.
Asunto(s)
Antibióticos Antineoplásicos/biosíntesis , Disacáridos/metabolismo , Genes Bacterianos , Glicosiltransferasas/genética , Plicamicina/biosíntesis , Streptomyces/genética , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Plicamicina/químicaRESUMEN
Two genes (mtmD and mtmE) were cloned and sequenced from the mithramycin producer Streptomyces argillaceus. Comparison with proteins in databases and enzymatic assays after expression in Escherichia coli showed that they encode a glucose-1-phosphate:TTP thymidylyl transferase and a TDP-D-glucose 4,6-dehydratase, respectively. The mtmD gene was inactivated by gene replacement, generating a nonproducing mutant that accumulates a tetracyclic compound designated premithramycinone. The identification of premithramycinone reveals new aspects of the mithramycin biosynthetic pathway and suggests that at least some glycosylations occur before breakage of the fourth ring.
Asunto(s)
Antibióticos Antineoplásicos/biosíntesis , Regulación Bacteriana de la Expresión Génica , Hidroliasas/genética , Complejos Multienzimáticos/genética , Mutagénesis Insercional , Nucleotidiltransferasas/genética , Plicamicina/biosíntesis , Streptomyces/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Clonación Molecular , Hidroliasas/fisiología , Datos de Secuencia Molecular , Nucleotidiltransferasas/fisiología , Streptomyces/enzimología , Streptomyces/metabolismoRESUMEN
Production of cephamycin C and clavulanic acid by Streptomyces clavuligerus took place during the exponential phase of growth in a defined medium. Both antibiotic biosynthetic pathways were activated shortly after spore germination, but the timing and kinetics of activation were affected by inoculum density. Rapid activation was favoured by high inoculum density or by growth in medium conditioned by previous incubation of S. clavuligerus spores or mycelium. A heat-resistant conditioning factor able to accelerate the acquisition of antibiotic-biosynthetic capacity when added to low-density cultures was released in suspensions of spores in water. Conditioning factor was also obtained in suspensions of spores from different Streptomyces species or of Bacillus cells, indicating that the signal was not produced specifically by S. clavuligerus. Fractionation of conditioning factor showed that its effect was not due to a single molecule. The fractions contained amino acids (as free amino acids and oligopeptides) in amounts that roughly correlated with their respective conditioning power. Furthermore, the conditioning effect was reproduced by supplementing defined medium with amino acids and peptides in concentration that mimicked those found in conditioning factor. When individually tested at concentrations in the micromolar range, only some amino acids were able to stimulate antibiotic biosynthetic capacity. This stimulation ws also promoted by low concentrations (less than 1 microgram ml-1) of peptide mixtures obtained with different proteolytic enzymes. The results suggest that both amino acids and peptides are responsible for the effects of conditioning factor released by spores. Possible implications of intercellular signalling on activation of secondary metabolism are discussed.
Asunto(s)
Cefamicinas/biosíntesis , Ácidos Clavulánicos/biosíntesis , Streptomyces/metabolismo , Aminoácidos/farmacología , Bacterias/metabolismo , Factores Biológicos/aislamiento & purificación , Factores Biológicos/metabolismo , Factores Biológicos/farmacología , Ácido Clavulánico , Medios de Cultivo/farmacología , Medios de Cultivo Condicionados/farmacología , Calor , Cinética , Péptidos/farmacología , Esporas Bacterianas , Streptomyces/crecimiento & desarrollo , Streptomyces/fisiologíaRESUMEN
Cephalosporin biosynthetic activity and extracellular protease production increased during growth of Streptomyces clavuligerus in defined medium, while the level of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) remained very low and stable. Cephalosporin biosynthesis (measured in resting cell systems) was initiated during early exponential growth in complex media, without appreciable change in the small ppGpp pool. Nutritional shift-down induced by withdrawal of Casamino acids caused a transient increase in ppGpp and a reduction of RNA accumulation. The increase in ppGpp was small in very young cultures, but increased as the culture aged. Twenty-seven spontaneous thiostrepton-resistant mutants were isolated and partially characterized. Most of them had a reduced ppGpp-forming ability and gave normal titres of cephalosporin. However, in complex medium, some mutants did not produce cephalosporins or extracellular protease, whereas others overproduced cephalosporins. The results indicate that, in S. clavuligerus, there is no obligatory relationship between the initiation of secondary metabolism and the stringent response.
Asunto(s)
Cefalosporinas/biosíntesis , Guanosina Tetrafosfato/metabolismo , Streptomyces/metabolismo , Aminoácidos/metabolismo , Amoníaco/metabolismo , Medios de Cultivo , Farmacorresistencia Microbiana/genética , Endopeptidasas/biosíntesis , Glutamina/metabolismo , Cinética , Lisina/metabolismo , Mutación/fisiología , Streptomyces/efectos de los fármacos , Streptomyces/crecimiento & desarrollo , Streptomyces/aislamiento & purificación , Tioestreptona/farmacologíaRESUMEN
Streptomyces clavuligerus was able to accumulate proline intracellularly throughout a wide range of external proline concentrations. Kinetic analysis of proline uptake indicated that this phenomenon is mediated by two saturable systems. One of them was a high-affinity system (Km = 11 microM), with low uptake capacity and specific for proline. The second system had lower affinity for proline (Km = 10.2 mM), higher uptake capacity, and was inhibited by several amino acids. Proline was not required as an inducer of the systems, which were neither repressed nor inhibited by ammonium.