Histidine aminotransferase activity in Streptomyces tendae and its correlation with nikkomycin production

Streptomyces tendae Tü901 produces nikkomycins belonging to the nucleoside peptide antibiotics. Mutants defective in histidine catabolism were isolated and characterized with regard to their histidine ammonium-lyase activity and antibiotic synthesis. In the histidine ammonialyase-negative mutant hut-11 which was unimpaired in nikkomycin production histidine aminotransferase activity was detected as an additional histidine metabolizing enzyme. A protein exhibiting histidine aminotransferase activity could be demonstrated on non-denaturing gels of hut-11 crude extracts. Using optimized assay conditions, histidine aminotransferase activity was investigated in the strain hut-11 during growth in nikkomycin production medium. Maximal activity was reached at the end of exponential growth prior to nikkomycin production. In the presence of bromopyruvate, an effective inhibitor of histidine aminotransferase activity in vitro, production of nikkomycin Z and X was markedly reduced in hut-11.     

Cloning and heterologous expression of the entire set of structural genes for nikkomycin synthesis from Streptomyces tendae Tü901 in Streptomyces lividans.

A genomic library from Streptomyces tendae raised in shuttle cosmid vector pKC505 was screened with a previously isolated 8-kb DNA fragment containing the orfP1 gene, which is involved in nikkomycin biosynthesis. The entire set of structural genes for nikkomycin synthesis was heterologously expressed in S. lividans TK23 by introducing recombinant cosmids p24/32 and p9/43-2, carrying inserts of about 31 and 27 kb, respectively, overlapping by 15 kb. S. lividans transformants synthesized nikkomycins X, Z, I, and J, which were identified by high-pressure liquid chromatography analyses of culture filtrates.     

Induction of aerial mycelium formation in a bld mutant of Streptomyces tendae by Borrelidin—a macrolide antibiotic

Abstract In a screening programme for substances with morphogenic effects on the nikkomycin producer strain Streptomyces tendae Tü 901 we identified a metabolite, which induced aerial mycelium formation in the bld mutant Tü 901/S 2566-EM 1. By using a HPLC UV/Vis absorbance spectral library we could confirm that this compound was identical with the macrolide antibiotic borrelidin. 100 ng borrelidin/paperdisc were sufficient to show an evident morphological effect.     

Identification of cellular proteins involved in nikkomycin production in Steptomyces tendae Tü901

Expression of genes involved in nikkomycin production in Streptomyces tendae was investigated by two-dimensional gel electrophoresis of cellular proteins. Ten gene products (P1–P10) were identified that were synthesized when nikkomycin was produced; these proteins were not detected in non-producing mutants. N-terminal sequences of six of the 10 proteins were obtained by microsequencing of protein spots excised from preparative two-dimensional gels. Protein P8 was identified as l -histidine amino-transferase (HisAT), which has been previously correlated with nikkomycin production. By using oligo-nucleotide probes deduced from the N-terminal sequences of protein P2 and P6, we isolated an 8 kb Bam HI fragment and a 6.5 kb Pvu II fragment, respectively, from the genome of Streptomyces tendae Tü901. Restriction analyses revealed that both fragments overlapped within a region of 1.5 kb. Mapping of the oligonucleotide probe hybridizing sites indicated that the genes encoding protein P2 and P6 are closely spaced on the 8 kb Bam HI fragment, and the latter is located on the overlapping region. DNA sequence analysis revealed that proteins P1 and P2 are encoded by a single gene, orfP1, that is translated at two initiation codons. The orfP1 gene was interrupted by homologous recombination using the integrating vector pWHM3. The gene-disrupted transformants did not produce nikkomycin, indicating that proteins P1 and P2 are essential for nikkomycin production. The data presented show that reverse genetics was successfully used to isolate genes Involved in nikkomycin production.     

Molecular cloning and high-level expression of a bromoperoxidase gene from Streptomyces aureofaciens Tü24.

A bromoperoxidase gene was cloned from Streptomyces aureofaciens Tü24 into Streptomyces lividans TK64 by using the promoter-probe vector pIJ486. Subcloning of DNA from the original, unstable clone allowed the gene to be localized to a 1.7-kilobase (kb) fragment of DNA. Southern blotting showed that the cloned 1.7-kb insert hybridized to a 4.3-kb fragment in an SstI digest of S. aureofaciens Tü24 total DNA. The 1.7-kb insert was shown to code for a protein with the electrophoretic properties of the subunits of the nonheme bromoperoxidase isolated from S. aureofaciens Tü24. The protein produced by S. lividans TK64 transformed with pHM621, which contained an 8.0-kb insert, was shown to be identical to the S. aureofaciens Tü24 bromoperoxidase in terms of its electrophoretic mobility on denaturing and nondenaturing polyacrylamide gels and its NH2-terminal amino acid sequence. The bromoperoxidase was overproduced (up to 180 times) by S. lividans TK64 containing pHM621. Based on the heat stability of the S. aureofaciens Tü24 bromoperoxidase, a new and simple purification procedure with very high yields was developed.     

Plasmid transformation of Streptomyces tendae after heat attenuation of restriction

Streptomyces tendae ATCC 31160 produces nikkomycin, a fungicide and insecticide that inhibits chitin synthases. Exposure of S. tendae protoplasts to 50 degrees C for 30 min is required for transformation (10(2) thiostrepton-resistant transformants micrograms of DNA-1) with plasmid pIJ702 or pIJ680 from Streptomyces lividans. pIJ702 and pIJ680 DNA isolated from the S. tendae transformants is efficient (10(6) to 10(7) transformants micrograms of DNA-1) in subsequent transformations of S. tendae protoplasts generated at 30 degrees C. PstI fails to cut the single PstI site in pIJ702 and cuts only one of the two PstI sites in pIJ680 DNA isolated from S. tendae transformants. Digests of plasmid DNA mixtures showed that plasmid DNA from S. tendae does not inhibit PstI activity. pIJ702 and pIJ680 DNA from S. tendae transformants was used to transform S. lividans to show that plasmid DNA remains unchanged, except for modification at some PstI sites in S. tendae, as a consequence of passage through S. tendae. The DNA modification is lost when S. lividans is transformed with plasmid DNA from S. tendae transformants. Since S. tendae modifies only some PstI sites, it appears the modification (presumably restriction activity also) activity in S. tendae recognizes a sequence that includes or overlaps the PstI hexanucleotide recognition sequence.     

Plasmid transformation of Streptomyces tendae after heat attenuation of restriction.

Streptomyces tendae ATCC 31160 produces nikkomycin, a fungicide and insecticide that inhibits chitin synthases. Exposure of S. tendae protoplasts to 50 degrees C for 30 min is required for transformation (10(2) thiostrepton-resistant transformants micrograms of DNA-1) with plasmid pIJ702 or pIJ680 from Streptomyces lividans. pIJ702 and pIJ680 DNA isolated from the S. tendae transformants is efficient (10(6) to 10(7) transformants micrograms of DNA-1) in subsequent transformations of S. tendae protoplasts generated at 30 degrees C. PstI fails to cut the single PstI site in pIJ702 and cuts only one of the two PstI sites in pIJ680 DNA isolated from S. tendae transformants. Digests of plasmid DNA mixtures showed that plasmid DNA from S. tendae does not inhibit PstI activity. pIJ702 and pIJ680 DNA from S. tendae transformants was used to transform S. lividans to show that plasmid DNA remains unchanged, except for modification at some PstI sites in S. tendae, as a consequence of passage through S. tendae. The DNA modification is lost when S. lividans is transformed with plasmid DNA from S. tendae transformants. Since S. tendae modifies only some PstI sites, it appears the modification (presumably restriction activity also) activity in S. tendae recognizes a sequence that includes or overlaps the PstI hexanucleotide recognition sequence.     

Molecular characterization of two genes from Streptomyces tendae Tü901 required for the formation of the 4-formyl-4-imidazolin-2-one-containing nucleoside moiety of the peptidyl nucleoside antibiotic nikkomycin.

The genes nikQ and nikR were identified by sequencing DNA of the nikkomycin biosynthetic gene cluster from Streptomyces tendae Tü901/8c. The nikQ gene encodes a P450 cytochrome, and the predicted NikR gene product shows 48-56% sequence identity with uracil phosphoribosyltransferases from eukaryotic organisms. The nikQ and nikR genes were inactivated separately by insertion of a kanamycin-resistance cassette. Inactivation of the nikQ gene abolished synthesis of nikkomycins containing 4-formyl-4-imidazolin-2-one as the base (nikkomycins X and I), whereas production of nikkomycins containing uracil (nikkomycins Z and J) was not affected. Nikkomycin X and I production could be restored by feeding 4-formyl-4-imidazolin-2-one to the nikQ mutants, indicating that NikQ is responsible for its formation from L-histidine. Disruption of the nikR gene resulted in formation of decreased amounts of nikkomycins X and I, whereas nikkomycins Z and J were synthesized at wild-type levels. A fluorouracil-resistant nikR mutant lacking uracil phosphoribosyltransferase (UPRTase) activity did not synthesize nikkomycins X and I and accumulated 4-formyl-4-imidazolin-2-one in its culture filtrate, whereas formation of nikkomycins Z and J was unimpaired. The mutant was complemented to nikkomycin X and I production by nikR expressed from the mel promoter of plasmid pIJ702. The nikR gene expressed in Escherichia coli led to the production of UPRTase activity. Our results indicate that NikR converts 4-formyl-4-imidazolin-2-one to yield 5'-phosphoribosyl-4-formyl-4-imidazolin-2-one, the precursor of nikkomycins containing this base.     

Disruption of a gene encoding a putative gamma-butyrolactone-binding protein in Streptomyces tendae affects nikkomycin production

A 2.6-kb BamHI fragment from the genome of the wild-type, nikkomycin-producing strain of Streptomyces tendae ATCC 31160 was cloned and sequenced. This 2.6-kb BamHI fragment corresponds to the DNA site where transposon Tn4560 had inserted to create a nikkomycin-nonproducing mutant. A possible ORF of 660 nucleotides was found in this 2.6-kb BamHI fragment, in which the third base of each codon was either G or C in 92% of the codons. The deduced amino acid sequence coded by this ORF (TarA, tendae autoregulator receptor) shows strong homology with several Gamma-butyrolactone-binding proteins that negatively regulate antibiotic production in other streptomycetes and have a helix-turn-helix DNA-binding motif. A portion (179 nucleotides) of tarA that encodes the helix-turn-helix motif was replaced with ermE, and wild-type S. tendae was transformed with this construct borne in pDH5, a gene-disruption vector. Southern hybridization indicated that ermE had inserted in the 2.6-kb BamHI region in one isolate that is erythromycin resistant. Northern hybridization indicated that tarA disruption significantly increased the amount of disrupted-tarA mRNA. This suggests that TarA negatively regulates its own synthesis. Nikkomycin production by the tarA disruptant was delayed but reached the wild-type level after longer incubation in production medium.     

Use of Tn4560 to generate nikkomycin non-producing mutants of Streptomyces tendae

Abstract Transposon Tn 4560 was used to generate three nikkomycin non-producing mutants in Streptomyces tendae ATCC 31160 Southern hybridization confirmed that Tn 4560 was present in 10–12-kb Bam HI fragments of the chromosomes of the mutants. Biologically active nikkomycins were not detected in culture broths of the mutants as determined by bioassays and HPLC. Differences in the HPLC profiles of culture broths suggest that Tn 4560 inserted into different genes in the mutants.     

Restriction enzyme analysis of lactose and bacteriocin plasmids from Streptococcus lactis subsp. diacetylactis WM4 and cloning of BclI fragments coding for bacteriocin production.

The 131.1-kilobase (kb) bacteriocin production (Bac) plasmid pNP2 and the 63.6-kb lactose metabolism (Lac) plasmid pCS26, from Streptococcus lactis subsp. diacetylactis WM4, as well as pWN8, a 116.7-kb recombinant plasmid from a Lac+ transconjugant, were analyzed with restriction enzymes to determine the origin of pWN8. Plasmid pWN8 conferred a Lac+ Bac- phenotype, contained DNA derived from pCS26 and pNP2, and, like pNP2, exhibited self-transmissibility (Tra+). In cloning attempts, Bac+ transformant S. lactis KSH1 was isolated. The recombinant plasmid, pKSH1, contained three BclI fragments from pNP2. Bac- transformants which individually contained each of the three fragments were also identified. Comparison of restriction maps of pKSH1 and pNP2 revealed an 18.4-kb region common to both plasmids, involving two of the three BclI fragments. S. lactis KSH1 also exhibited greater inhibitory activity against the indicator strain S. diacetylactis 18-16 than did a strain containing the 131.1-kb Bac plasmid.     

Restriction enzyme analysis of lactose and bacteriocin plasmids from Streptococcus lactis subsp. diacetylactis WM4 and cloning of BclI fragments coding for bacteriocin production

The 131.1-kilobase (kb) bacteriocin production (Bac) plasmid pNP2 and the 63.6-kb lactose metabolism (Lac) plasmid pCS26, from Streptococcus lactis subsp. diacetylactis WM4, as well as pWN8, a 116.7-kb recombinant plasmid from a Lac+ transconjugant, were analyzed with restriction enzymes to determine the origin of pWN8. Plasmid pWN8 conferred a Lac+ Bac- phenotype, contained DNA derived from pCS26 and pNP2, and, like pNP2, exhibited self-transmissibility (Tra+). In cloning attempts, Bac+ transformant S. lactis KSH1 was isolated. The recombinant plasmid, pKSH1, contained three BclI fragments from pNP2. Bac- transformants which individually contained each of the three fragments were also identified. Comparison of restriction maps of pKSH1 and pNP2 revealed an 18.4-kb region common to both plasmids, involving two of the three BclI fragments. S. lactis KSH1 also exhibited greater inhibitory activity against the indicator strain S. diacetylactis 18-16 than did a strain containing the 131.1-kb Bac plasmid.     

Cloning and analysis of s-triazine catabolic genes from Pseudomonas sp. strain NRRLB-12227.

Pseudomonas sp. strain NRRLB-12227 degrades the s-triazine melamine by a six-step pathway which allows it to use melamine and pathway intermediates as nitrogen sources. With the plasmid pLG221, mutants defective in five of the six steps of the pathway were generated. Tn5-containing-EcoRI fragments from these mutants were cloned and identified by selection for Tn5-encoded kanamycin resistance in transformants. A restriction fragment from ammelide-negative mutant RE411 was used as a probe in colony hybridization experiments to identify cloned wild-type s-triazine catabolic genes encoding ammeline aminohydrolase, ammelide aminohydrolase, and cyanuric acid amidohydrolase. These genes were cloned from total cellular DNA on several similar, but not identical, HindIII fragments, as well as on a PstI fragment and a BglII fragment. Restriction mapping and Southern hybridization analyses of these cloned DNA fragments suggested that these s-triazine catabolic genes may be located on a transposable element, the ends of which are identical 2.2-kb insertion sequences.     

Alterations in the number of rRNA operons within the Bacillus subtilis genome

Deletions and additions of rRNA gene sets in Bacillus subtilis were observed by Southern hybridizations using cloned radiolabeled rDNA sequences. Of the ten rRNA gene sets found in B. subtilis 168M or NCTC3610, one was deleted in strains possessing the leuB1, ilvC1, argA2 and pheA1 mutations. Among EcoRI restriction fragments of genomic DNA products, a 2.9-kb 23S rRNA homolog was missing. In HindIII digest, both 5.5- and 5.1-kb hybrid bands were lost with 16S and 23S probes, respectively. Similarly, genomic DNAs digested with SmaI showed the absence of both 2.1- and 2.0-kb fragments that hybridized to 16S and 5S sequences, respectively, in wild-type genomes. In contrast, B. subtilis strain 166 and its derivatives displayed a gain of a 3.3-kb HindIII fragment homologous to 16S rRNA. Transforming the ilvC1 and leuB1 mutations into new genetic backgrounds revealed in some clones the concomitant introduction of the ribosomal defect. Transformations with the slightly heterologous donor DNA from strain W23 yielded some Leu+ and Arg+ transformants with altered hybridization patterns when probed with cloned sequences. We propose that the deletion of the rRNA operon occurred in the ilv-leu gene cluster of the B. subtilis genome as a result of unequal recombination between redundant sequences.     

Genetic analysis of epidermin biosynthetic genes and epidermin-negative mutants of Staphylococcus epidermidis.

Epidermin is produced by Staphylococcus epidermidis Tü3298 which harbors the 54-kb plasmid, pTü32. The plasmid contains not only the epidermin structural gene epiA, but also a flanking DNA region which is necessary for epidermin biosynthesis. The DNA sequence of this region revealed, in addition to epiA, five additional open reading frames, epiB, C, D, Q and P [Schnell, N., Engelke, G., Augustin J., Rosenstein, R., Ungermann, V., G?tz, F. & Entian, K.-D. (1992) Eur. J. Biochem. 204, 57-68]. We isolated a number of stable mutants from strain Tü3298 which are unable to produce biologically active epidermin. Complementation studies using the newly constructed staphylococcal plasmid vectors pT181mcs and pCU1 led to their classification as epiA, epiB, epiC or epiD mutants. Furthermore, evidence is presented that epiB lacks its own promoter and is co-transcribed from the epiA promoter. There is evidence that epiC and D possess their own promoters. Although epiQ and epiP mutants were not isolated, it could be shown by heterologous gene expression in S. carnosus and S. xylosus that the corresponding DNA region is involved in epidermin biosynthesis. We can not exclude the possibility that, in addition to the four open reading frames, epiA, B, C, D, and the DNA region comprising epiQ and P, host-encoded functions are necessary for epidermin production. Thus, the genetic information for epidermin biosynthesis in S. carnosus and S. xylosus is located on an 8-kb DNA fragment of pTü32. A further characterization of the two epiA mutants revealed that in both mutants, the preepidermin nucleotide sequence was changed. In one mutant, the mutation led to a substitution of Ser3 by Asn; in the other of Gly10 by Glu.     

Cloning, Sequencing, and Function of sanF: A Gene Involved in Nikkomycin Biosynthesis of Streptomyces ansochromogenes

A 111-bp DNA fragment related to nikkomycin biosynthesis of Streptomyces ansochromogenes 7100 was obtained with the method of reverse genetics. Then, a 2.2-kb DNA fragment was cloned from the DNA library of S. ansochromogenes 7100 by using the 111-bp fragment as a probe. Sequence analysis showed that the fragment contains one complete open reading frame (ORF) that encodes a 219-amino acid (aa) protein, and this gene was designated sanF (GenBank Accession No. AF223971). The function of the sanF gene was studied by a strategy of gene disruption, and the resulting sanF mutants lost the ability to synthesize biologically active nikkomycin, indicating that sanF is essential for nikkomycin biosynthesis. Received: 17 April 2000 / Accepted: 23 May 2000     

Tn4560在圈卷产色链霉菌中的转座及其在尼可霉素生物合成相关基因克隆中的应用

采用常规转化方法用来自天蓝色链霉菌J1 5 0 1的质粒pUC1 1 6 9(pMT6 6 0∷Tn45 5 6∷vph)多次转化尼可霉素产生菌圈卷产色链霉菌野生型 71 0 0的原生质体 ,均未得到转化子。采用限制性热衰减法于 5 0℃ ,3 0min溶菌制备 71 0 0的原生质体 ,获得了转化子 ,但转化频率极低 ,只有 0 4个转化子 μgDNA。用来自 71 0 0的pUC1 1 6 9再转化不含pUC1 1 6 9的 71 0 0原生质体 ,转化频率提高 1 0 3 ～ 1 0 4 倍。于 3 9℃ ,MM Vio条件下培养携带有pUC1 1 6 9的 71 0 0孢子 ,Tn45 6 0发生转座 ,筛选到 40 6 8个转座菌落 ,并从中得到 8株尼可霉素阻断突变株 ;对这 8株突变株的总DNA进行Southern杂交分析表明 ,Tn45 6 0至少在 4个不同的位点插入到 71 0 0的染色体上。用实验室已获得的与尼可霉素生物合成有关的 3 0kbDNA片段为探针和经不同酶切的 8株突变株的总DNA进行Southern杂交 ,结果表明 ,除阻断突变株Nik5有杂交信号且杂交信号大小均同野生型…     

Adenylate kinase isoenzymes in Aspergillus nidulans

Multiple HindIII-restriction fragments of Salmonella typhimurium and Salmonella typhi chromosomal DNA exhibited homology with the heat-labile enterotoxin (LT1) gene of Escherichia coli as determined by Southern blot analysis. A 9.4 kb HindIII restriction fragment identified in S. typhimurium and S. typhi chromosomal DNA reacted with both eltA and eltB gene probes. However, the homology of the 9.4 kb DNA fragment from these Salmonella species was greater with eltB than eltA. In addition, a synthetic oligonucleotide probe, made to a portion of the putative GM1-ganglioside binding region of cholera toxin (CT) and LT1, hybridized with the 9.4 kb DNA fragment of S. typhimurium but not with the 9.4 kb fragment found in S. typhi isolates. The hybridization of multiple restriction fragments of Salmonella DNA with eltA and eltB gene sequences further suggests duplication of the stx operon on the chromosome of these bacteria.