Genetic complementation ofStreptomyces tendae deficient in nikkomycin production

Bormann, Christiane; Aberle, Karin; Fiedler, H.-P.; Schrempf, Hildgund

doi:10.1007/bf00903777

Cited by 17 publications

(9 citation statements)

References 22 publications

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“…tendae strains were grown on solid medium HA (Bormann et al 1990) at 37°C, and S. lividans strains were incubated at 30°C. For the selection of plasmid-containing Streptomyces strains, 30 lg thiostrepton/ml or 10 lg kanamycin/ml was added to solid medium, and 10 lg thiostrepton/ml or 10 lg kanamycin/ml was added to liquid medium SP.…”

Section: Methodsmentioning

confidence: 99%

“…Streptomyces protoplasts were transformed as described by Bormann et al (1990). The digoxigenin-11-dUTP labeling kit (Roche Diagnostics GmbH, Mannheim) was used for DNA labeling and detection in Southern experiments according to the recommendations of the manufacturer.…”

Section: Methodsmentioning

confidence: 99%

“…Recombinant pWHM3 plasmids constructed for isolation of nik insertion mutants were passaged through S. lividans TK23 before transformation of S. tendae TuÈ 901/8c protoplasts. To isolate insertion mutations in the nikP1, nikS, or nikV gene, S. tendae carrying pNP1, pNS1, or pNV1 (Table 1), respectively, was cultivated in CRM medium (Bormann et al 1990) containing 5 lg kanamycin/ml for 36 h at 27°C; protoplasts were prepared and regenerated on non-selective regeneration medium. In order to isolate mutants bearing an insertion in the nikP2 or nikT gene, S. tendae carrying pNP2 or pNT1 (Table 1), respectively, was grown as a lawn on non-selective HA medium.…”

Section: Construction Of Insertion Mutantsmentioning

confidence: 99%

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Molecular characterization of co-transcribed genes from Streptomyces tendae Tü901 involved in the biosynthesis of the peptidyl moiety and assembly of the peptidyl nucleoside antibiotic nikkomycin

et al. 2001

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Six genes (nikP1, nikP2, nikS, nikT, nikU, and nikV) from Streptomyces tendae Tu901 were identified by analysis of the nucleotide sequence of the nikkomycin gene cluster. These genes, together with the previously described nikQ and nikR, span 9.39 kb and are transcribed as a polycistronic mRNA in a growth-phase-dependent manner. The nikP1 gene encodes a non-ribosomal peptide synthase consisting of an adenylation domain, a thiolation domain, and an N-terminal 70-residue segment of unknown function. The amino acid sequence encoded by the nikP2 gene displays similarity to the sequences of thioesterases, and the nikS product belongs to a superfamily of proteins characterized by a specific ATP-binding fold. The N-terminal 70 amino acids of the predicted nikT gene product show significant sequence similarity to acyl carrier proteins, and the C-terminal 330 amino acids to aminotransferases. The sequences of the deduced proteins NikU and NikV exhibit similarity to components S and E, respectively, of glutamate mutase from Clostridium. Disruption of the nikP1, nikS, nikT, or nikV gene by insertion of a kanamycin resistance cassette abolished formation of nikkomycins I, J, X, and Z, all of which contain hydroxypyridylhomothreonine as the peptidyl moiety. The nikP1 mutants, and the nikS and nikT mutants accumulated the nucleoside moieties nikkomycin Cz, and nikkomycins Cx and Cz, respectively. The nikV mutants formed nikkomycins Ox and Oz, which contain 2-amino-4-hydroxy-4-(3'-hydroxy-6'-pyridyl) butanoic acid as the peptidyl moiety. The nikP2 mutants synthesized nikkomycins I, J, X, and Z, but amounts of nikkomycins I and X, which contain formylimidazolone as the base, were lower. Feeding formylimidazolone to nikP2 mutants restored the ability to form nikkomycins I and X. Our results indicate that nikU and nikV are required for the synthesis of hydroxypyridylhomothreonine, the genes nikP1, nikP2 and nikS are required for the assembly of nikkomycins, and nikT is required for both pathways. The putative activities of each of their products are discussed.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Construction Of Insertion Mutantsmentioning

confidence: 99%

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Molecular characterization of co-transcribed genes from Streptomyces tendae Tü901 involved in the biosynthesis of the peptidyl moiety and assembly of the peptidyl nucleoside antibiotic nikkomycin

et al. 2001

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“…Streptomyces protoplasts were transformed as dechia coli, and its function in nikkomycin biosynthesis. The rescribed by Bormann et al [31]. The digoxigenin-11-dUTP labelsults identify protein P8 as L-lysine 2-aminotransferase, which ing kit (Boehringer, Mannheim) was used for DNA labeling and catalyzes the initial reaction in the biosynthetic pathway of the detection in Southern blot experiments according to the recomprecursor nikkomycin D.…”

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confidence: 99%

The Streptomyces tendae Tü901 L‐lysine 2‐aminotransferase catalyzes the initial reaction in nikkomycin D biosynthesis

Bruntner

Bormann

1998

European Journal of Biochemistry

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Protein P8 was previously identified as a putative nikkomycin biosynthesis protein. The gene (nikC) encoding protein P8 was cloned from the Streptomyces tendae Tü901 nikkomycin gene cluster and sequenced. The nikC gene was inactivated by inserting a kanamycin resistance cassette; the mutant did not produce the biologically active nikkomycins I, J, X, and Z, but accumulated the nucleoside moieties nikkomycins C X and C Z . The mutant was complemented to nikkomycin production (I, J, X, Z) by nikC expressed from the mel promoter of the vector pIJ702. Furthermore, the nikkomycin-negative phenotype was reversed by the addition of picolinic acid, a precursor of the peptidyl moiety of nikkomycins (nikkomycin D), into the culture medium. The nikC gene was expressed in Escherichia coli and identified and characterized at the enzyme level. NikC encodes an L-lysine 2-aminotransferase, and the activity was exclusively detected in nikkomycin producers and its presence correlated to nikkomycin production. The nikC-inactivated mutant grew with L-lysine as sole source of nitrogen and carbon, indicating that L-lysine 2-aminotransferase is not required for lysine catabolism. Our results identified the nikC-encoded L-lysine 2-aminotransferase as the nikkomycin biosynthetic enzyme that catalyzes the initial reaction in nikkomycin D biosynthesis. The NikC protein belongs to a novel family of pyridoxamine or pyridoxal-phosphate-dependent dehydrases and aminotransferases, some of which are involved in dideoxy-and deoxyaminosugar biosynthesis.Keywords : nikkomycin ; Streptomyces; lysine aminotransferase ; antibiotic production; secondary metabolism.Nikkomycins X and Z exhibit high antifungal, insecticidal, Nikkomycins are peptidyl nucleoside antibiotics that act as and acaricidal activity [7]. Since nikkomycins are nearly nonspecific and competitive inhibitors of chitin synthases from toxic for bees and mammals and are easily degraded in nature, fungi and insects [1Ϫ3]. Streptomyces tendae Tü901 produces they have the potential to be applied as insecticides in agriculvarious types of nikkomycin, and the main components of the ture or as antifungal agents in human therapy [8, 9]. Growth culture filtrate are nikkomycins I, J, X, and Z (Fig. 1). The peptiof Candida albicans is only inhibited at high concentrations of dyl moiety of these nikkomycins is hydroxypyridylhomothreonikkomycin X and Z [10], but combinations of nikkomycin with nine (nikkomycin D), and the nucleoside moiety consists of an antimycotically active azoles, which are directed against the funaminohexuronic acid with N-glycosidically bound 4-formyl-4-gal cytoplasmic membrane, are efficacious in treating mycosis in imidazoline-2-one (nikkomycin C X ) or uracil (nikkomycin C Z ) animals [11]. Nikkomycin Z is very effective against the highly [4]. Both moieties constitute A-amino acid structures that are chitinous, dimorphic fungal pathogens Coccidioides immitis, connected via a peptide bond to yield the dipeptidyl nikkomyBlastomyces dermatitidis, and Histoplasma capsulatum [...

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“…S. tendae Tu901/ hut-ll is a histidine-ammonia-lyase-negative mutant (Roos et al, 1992) and Tug0 1 /NP9, NP47 and NP55 are nikkomycin non-producing mutants (Bormann et For nikkomycin production, mycelia were grown in SM medium consisting of 3 YO (w/v) mannitol, 1 % (w/v) starch, 2 % (w/v) soy bean meal, and 1 YO (w/v) yeast extract, pH 40, and in SP medium (3 YO mannitol, 1 YO soluble starch, 0.5% soy peptone and 0.8 % yeast extract, pH 6.0). S. lividans was grown in CRM medium (Bormann et al, 1990) and YEME (Hopwood et al, 1985).…”

Section: Methodsmentioning

confidence: 99%

Purification and characterization of L-histidine aminotransferase from nikkomycin-producing Streptomyces tendae Tu901

Roos

Bormann

1993

Journal of General Microbiology

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Cell extracts of Streptomyces tendae grown in nikkomycin production media contained an enzyme (HisAT) that transaminated L-histidine as the sole amino substrate with pyruvate as the amino group acceptor. HisAT was purified about 190-fold from the crude extract of S. tendae. The enzyme was determined by gel filtration and SDS-PAGE to be a homodimer with a subunit molecular mass of approximately 45 kDa. The aminotransferase had maximum activity at pH 7.0 and 37 OC. The enzyme was highly specific for L-histidine; pyruvate, 2-oxobutyrate, 2-oxovalerate and Zoxocaproate were used as keto acceptors to about the same extent. The reaction mechanism was ping-pong. The K, values for L-histidine and pyruvate, determined from Lineweaver-Burk plots, were 25 mM and 10 mM, respectively. Neither cell extracts of non-producing S. tendae mutants nor extracts of Streptomyces liuidans, a species that does not synthesize nikkomycins, showed transaminating activity with a narrow substrate specificity for L-histidine as the amino donor. This strongly suggests that the formation of HisAT is essential for nikkomycin production.

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Genetic complementation ofStreptomyces tendae deficient in nikkomycin production

Cited by 17 publications

References 22 publications

Molecular characterization of co-transcribed genes from Streptomyces tendae Tü901 involved in the biosynthesis of the peptidyl moiety and assembly of the peptidyl nucleoside antibiotic nikkomycin

Molecular characterization of co-transcribed genes from Streptomyces tendae Tü901 involved in the biosynthesis of the peptidyl moiety and assembly of the peptidyl nucleoside antibiotic nikkomycin

The Streptomyces tendae Tü901 L‐lysine 2‐aminotransferase catalyzes the initial reaction in nikkomycin D biosynthesis

Purification and characterization of L-histidine aminotransferase from nikkomycin-producing Streptomyces tendae Tu901

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