Identification of two polyketide synthase gene clusters on the linear plasmid pSLA2-L in Streptomyces rochei.

The 200kb linear plasmid pSLA2-L was suggested to be involved in the production of two macrolide antibiotics, lankamycin (Lm) and lankacidin (Lc), in Streptomyces rochei 7434AN4. Hybridization experiments with the polyketide synthase (PKS) genes for erythromycin and actinorhodin identified two eryAI-homologous regions and an actI-homologous region on pSLA2-L. The nucleotide sequence of a 3.6kb SacI fragment carrying one of the eryAI-homologs revealed that it codes for part of a large protein with four domains for ketoreductase, acyl carrier protein, ketosynthase, and acyltransferase. Gene disruption confirmed that the two eryAI-homologs are parts of a large type-I PKS gene cluster for Lm. A 4.8kb DNA carrying the actI-homologous region contains four open reading frames (ORF1-ORF4) as well as an additional ORF, i.e. ORF5, which might code for a thioesterase. Deletion of the ORF2-ORF4 region showed that it is not involved in the synthesis of Lm or Lc. Thus, it was confirmed that pSLA2-L contains two PKS gene clusters for Lm and an unknown type-II polyketide.

Chromosomal deletions in Streptomyces griseus that remove the afsA locus.

We have recently constructed a physical map of the Streptomyces griseus 2247 genome using the restriction enzymes AseI and DraI, which revealed that this strain carries a 7.8 Mb linear chromosome. Based on this map, precise macrorestriction fragment and cosmid maps were constructed for both ends of the chromosome, which localized the afsA gene 150 Kb from the left end. Two afsA- mutants were found to have suffered chromosomal deletions that removed the afsA locus. The sizes of the deletions were 20 and 130 Kb at the right end and 180 and 350 kb at the left end, respectively. Hybridization experiments using cosmids carrying a deletion endpoint indicated that the ends of the chromosome in the mutants were fused to form a circular chromosome.

Physical map of the linear chromosome of Streptomyces griseus.

The chromosomal DNA of Streptomyces griseus 2247 (a derivative of strain IFO3237) was digested with several restriction endonucleases and analyzed by pulsed-field gel electrophoresis (PFGE). Digestion with AseI and DraI gave 15 and 9 fragments, respectively, the total sizes of which were 7.8 Mb. All the AseI and DraI fragments were aligned on a linear chromosome map by using linking plasmids and cosmids. PFGE analysis of the intact chromosome also showed a linear DNA band of about 8 Mb. Detailed physical maps of both terminal regions were constructed; they revealed the presence of a 24-kb terminal inverted repeat on each end. PFGE analysis with and without proteinase K treatment suggested that each end of the chromosome carries a protein molecule.

Purification and characterization of RNA polymerase holoenzyme (E sigma B) from vegetative-phase mycelia of Streptomyces griseus.

RNA polymerase was purified from vegetative-phase mycelia of Streptomyces griseus by a series of ion-exchange chromatographies. By western blot analysis using antiserum against S. coelicolor HrdB, which is a principal sigma factor (sigma(hrdB)), the purified holoenzyme was found to contain sigmaB (=sigma(hrdB)) of S. griseus. Significant amounts of HrdB protein were, however, eluted from the DEAE column at lower concentrations of KCl than that required for for elution of the holoenzyme containing sigmaB, suggesting that sigmaB is dissociated from the core enzyme, or an excess amount of sigmaB exists in S.griseus cells. The holoenzyme containing sigmaB (EsigmaB) transcribed in vitro the dagA promoter of S. coelicolor, and the hardB and hsp70 promoters of S. griseus, suggesting that it is involved in transcription of the essential genes. EsigmaB may be a major form of RNA polymerase holoenzyme in the growing phase of S. griseus.

Nucleotide sequence of a principal sigma factor gene (hrdB) of Streptomyces griseus.

The hrdB homologue was isolated from a streptomycin-producing Streptomyces griseus 2247 strain, which is independent of A-factor. The nucleotide sequence of the cloned DNA fragment revealed the presence of an open reading frame (ORF) of 1,542bp, which predicted a primary product of 514 amino acids and Mr 56,100. The N-terminal sequence of the purified HrdB protein of S. griseus was identical to the amino acid sequence deduced from the nucleotide sequence. The deduced amino acid sequence contains an "rpoD box" conserved in the principal sigma factors of eubacteria, and shows high similarity to the hrdB products of S. coelicolor A3(2)(89.9%) and S. aureofaciens (88.1%). The cloned gene encodes a principal sigma factor of S. griseus. The promoter region was identified by using a promoter-probe vector and by means of primer extensions experiments. The transcription start point is located 158-bp upstream of the initiation codon.

Isolation and characterization of linear plasmids from lankacidin-producing Streptomyces species.

Streptomyces rochei 7434AN4, a producer of lankacidin and lankamycin contains three large linear plasmids, pSLA2-L (200 kb), M (100 kb), and S (17 kb). Studies on the mutants of 7434AN4 having a different plasmid profile showed a parallel relationship between the presence of pSLA2-L and the production of both lankacidin and lankamycin. When pSLA2-L was transferred by protoplast fusion to S. rochei 2-39, a non-antibiotic-producing mutant of 7434AN4 which contained no detectable plasmid, the fusants gained the capacity to produce both antibiotics. From the physical maps of pSLA2-L and pSLA2-L1, a deletion plasmid (160 kb) of pSLA2-L, the latter plasmid was determined to contain a symmetrical linear repeat composed of the right 80-kb part of pSLA2-L. Four other lankacidin-producing Streptomyces strains were also found to have distinctive large linear plasmids which hybridized with the pSLA2-L probe. These results support the involvement of pSLA2-L in the production of lankacidin and lankamycin in S. rochei 7434AN4.

Use of the tyrosinase gene from Streptomyces to probe promoter sequences for Escherichia coli.

We have constructed a promoter-probe vector utilizing the expression of a promoter-less tyrosinase derived from Streptomyces plasmid pIJ702. The vector, pMX100, has single sites for EcoRI, KpnI, BamHI, XbaI, SalI, and SphI for cloning promoter sequences. When the tac promoter was inserted into pMX100, E. coli harboring the chimeric plasmid produced the melanin pigment.

Nucleotide sequence of the gene for cholesterol oxidase from a Streptomyces sp.

The nucleotide sequence of a 2.1-kilobase-pair fragment containing the Streptomyces choA gene, which codes a secreted cholesterol oxidase, was determined. A single open reading frame encodes a mature cholesterol oxidase of 504 amino acids, with a calculated Mr of 54,913. The leader peptides extend over 42 amino acids and have the characteristics of a signal sequence, including basic amino acids near the amino terminus and a hydrophobic core near the signal cleavage site. Analyses of the total amino acid composition and amino acid sequencing of the first 21 amino acids from the N terminus of the purified extracellular enzyme agree with the values deduced from nucleotide sequencing data.

The nucleotide sequence of a streptomycin streptomycin phosphotransferase (streptomycin kinase) [corrected] gene from a streptomycin producer.

The nucleotide sequence of the DNA fragment containing the streptomycin phosphotransferase (streptomycin kinase) [corrected] gene from the streptomycin-producer Streptomyces griseus strain HUT 6037 was determined. Analysis of the sequence revealed an open reading frame which could encode 325 amino acid residues. A biased codon usage pattern, reflecting the high G + C composition (approximately 74%) of Streptomyces DNA, was observed in the gene.

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.

Acetylation of blasticidin S by its producing actinomycetes.

A blasticidin S-producing actinomycetes, Streptoverticillium sp. JCM 4673 possesses an enzyme activity which acetylates the drug in the presence of acetyl coenzyme A. The modified drug was biologically inactive when tested against protein synthesis in vivo and in vitro. Production of the enzyme which acetylates blasticidin S increases with formation of the antibiotic during cell growth.

Molecular cloning and expression in Streptomyces lividans of a streptomycin 6-phosphotransferase gene from a streptomycin-producing microorganism.

The gene encoding streptomycin 6-kinase involved in the self-resistance of the streptomycin-producing Streptomyces griseus HUT 6037 was cloned in the plasmid vector pIJ703. The resulting plasmid, pSP6, contained 2.5 kb inserts of S. griseus DNA. When streptomycin-susceptible S. lividans 1326 was retransformed with pSP6, all transformants produced streptomycin 6-kinase. Addition of streptomycin to the culture medium of S. lividans carrying pSP6 plasmid brought about a remarkable increase in streptomycin 6-kinase activity in the cell extracts. It is suggested from the results that the production of streptomycin 6-kinase in streptomycin producer was induced by streptomycin accumulated during cultivation.

Purification and characterization of streptomycin 6-kinase, an enzyme implicated in self-protection of a streptomycin-producing micro-organism.

Streptomycin 6-kinase of the streptomycin-producing strain Streptomyces griseus HUT 6037 was purified by fractionation with (NH4)2SO4 and chromatography on DEAE-Sephadex A-25, hydroxyapatite and Sephadex G-100. After PAGE of the final fraction, a protein band corresponding to streptomycin 6-kinase was detected, together with a less intense band having no enzyme activity. Molecular weights determined by SDS-PAGE and by Sephadex G-100 chromatography were about 36000 and 38000, respectively, suggesting that the enzyme was a monomer. The isoelectric point of the enzyme was pH 6.6. Among the nucleoside 5'-triphosphates tested, ATP was the preferred phosphoryl donor. The Km values for streptomycin and ATP were 3.5 mM and 0.4 mM, respectively. The enzyme activity was strongly inhibited by EDTA and AgNO3. It was shown by using an in vitro protein-synthesizing system that purified streptomycin 6-kinase could protect polyphenylalanine synthesis of the streptomycin-susceptible S. griseus strain KSN from inhibition by streptomycin.

Effect of arginine on gramicidin S biosynthesis by Bacillus brevis.

Effects of arginine on gramicidin S (GS) biosynthesis were investigated by growing Bacillus brevis ATCC 9999 in a synthetic medium consisting of 10 g fructose, 0.15 g l-proline, 1.3 g l-histidine, 1.3 g l-glutamine, 0.5 g L-methionine, 1 g L-phenylalanine and six mineral salts per liter. Supplement of 3 g/liter L-arginine to the medium, especially at the logarithmic phase of growth, enhanced the cell growth and GS production. Twice supplement of 3 g/liter arginine at the beginning and middle logarithmic phase of growth gave much more GS production than any once supplement, but the soluble GS synthetase extractable by lysozyme digestion was remarkably decreased. However, the decrease of enzyme by arginine seemed to be merely an apparent phenomenon, because GS-synthesizing ability of the cell was strongly enhanced by arginine and the enzyme which was not extracted by lysozyme digestion could efficiently be solubilized by ultrasonic homogenization. In the soluble fraction of cells grown in an arginine-added synthetic medium, no arginine was detected, but a large amount of ornithine was accumulated. When L-ornithine, instead of L-arginine, was added to the synthetic medium, cell growth and GS production was stimulated with increase of its concentration without decrease in the soluble enzyme activity.

Mechanism of protection of protein synthesis against streptomycin inhibition in a producing strain.

The influence of streptomycin (SM) on protein synthesis in a SM-producing strain was investigated using polyuridylic acid-directed polyphenylalanine synthesis in cell-free extracts. Tolerance of protein synthesis to SM developed with increasing culture age of cells and could be attributed to a decrease in affinity of the ribosomes for SM and in increase in SM 6-kinase activity in cells. SM 6-phosphate produced from SM by SM 6-kinase did not bind to ribosomes and, furthermore, ribosome-bound SM was effectively released on phosphorylation with SM 6-kinase. Also a decrease in cell permeability to SM during the production phase may contribute in protecting protein synthesis from the antibiotic.

Susceptibility of protein synthesis to neomycin in neomycin-producing Streptomyces fradiae.

A cell-free protein-synthesizing system from Streptomyces fradiae was developed by preparing ribosomes and an S-150 fraction with precautions to prevent protease action. Using this system, the ribosomes of this organism were shown to be susceptible to its own product, neomycin.