Osamu Nimi

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.

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.

Susceptibility of protein synthesis to streptomycin in streptomycin-producing Streptomyces griseus.

Streptomycin inhibits protein synthesis in the streptomycin-sensitive bacteria by binding to their ribosomes. Since a streptomycin producer must be resistant to streptomycin, it is of interest to know if the protein synthesis of the streptomycin producer is inhibited by its own product. Cella and Vining [l] could not demonstrate the effect of streptomycin on protein synthesis in an in vitro system prepared from Sfreptomyces griseus, because the cell extract contained some factor inhibitory to polyuridylatedependent polyphenylalanine synthesis. Teraoka and Tanaka [2] reported that the binding of dihydrostreptomycin to ribosomes of S. griseus was very low. On the other hand, Piwowarski and Shaw [3] showed that the crude ribosomes of S. bikiniensis (streptomycin producer) bound 0.3 dihydrostreptomycin molecules/ 30 S ribosomal subunit. This paper describes the establishment of an in vitro polyphenylalanine-synthesizing system in S. griseus. Using this system, investigations were carried out on the inhibitory effect of streptomycin on polyphenylalanine synthesis as well as on the binding affinity of streptomycin to the ribosomal subunits.

Structural analysis of the giant linear plasmid SCP1 in various Streptomyces coelicolor strains

Summary: The methylenomycin biosynthetic gene cluster was previously shown to be present on the 350 kb giant linear plasmid SCP1. The structures of SCP1 in various Streptomyces coelicolor A3(2) strains were analysed by pulsed field gel electrophoresis (PFGE). S. coelicolor A3(2) strains 1147, M138 and M146, previously genetically characterized as SCP1+, contained the free form of SCP1. S. coelicolor 1984 and 2106, earlier characterized as SCP1′-cysB and a stable cysD donor respectively, were found to contain very large linear plasmids, 550 kb and 1700 kb in size. Strain JCM4979, another SCP1+ derivative of A3(2), had a series of SCP1-related linear plasmids of 390–630 kb. The integrated states of SCP1 in S. coelicolor SCP1-NF strains, an NF-like strain and a stable pabA donor were confirmed by PFGE, which revealed a lack of the intact SCP1 termini. In addition, a 70 kb linear plasmid was found in Streptomyces violaceoruber SANK95570, another methylenomycin producer. However, the plasmid was shown by hybridization to a methylenomycin resistance gene not to carry the methylenomycin gene cluster of this strain.

Cloning and nucleotide sequence of a gene conferring ability to grow at a low temperature on Saccharomyces cerevisiae tryptophan auxotrophs

Abstract A gene conferring on yeast YNN140 the ability to grow at a low temperature was cloned and designated LTG3 (low temperature growth gene). Disruption of the LTG3 gene resulted in no growth at a low temperature. It was shown that the effect of the presence of the gene in multicopy was dependent on the strain. DNA sequencing analysis of the LTG3 gene revealed an open reading frame which can encode 592 amino acids. The deduced amino acid sequence of the protein was homologous with that of some amino acid permeases. Since the addition of a large amount of l -tryptophan to YPAD medium enabled the YNN140 strain to grow at a low temperature, it is possible that a limiting step for YNN140 to grow at a low temperature is to take up tryptophan from the medium. A part of the nucleotide sequence of the cloned DNA containing the LTG3 gene coincided with that of the SUP3 gene, indicating that the LTG3 gene exists on chromosome XV.

The nucleotide sequence of a streptomycin 6-phosphotransferase gene from a streptomycin producer

SUMMARY: The nucleotide sequence of the DNA fragment containing the streptomycin 6-phosphotransferase (streptomycin 6-kinase) 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.

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.

Roles of streptomycin 6 -kinase and ribosomal affinity to streptomycin in self-protection of streptomycin producer

SummaryIn streptomycin (SM)-producing organisms, the lower affinity of ribosomes for SM gives rise to a lower susceptibility of protein synthesis to SM. But, even in a strain with considerably low affinity of ribosomes for SM, phosphorylation of SM in the cells by SM 6-kinase is necessary for the protein synthesis to be fully tolerant to SM.

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.

Cloning and nucleotide sequence of a hsp70 gene from Streptomyces griseus

Abstract The cultivation of Streptomyces griseus 2247 at the growth-limited temperature (37°C) or in liquid medium containing 5% ethanol (toxic for growth) revealed the presence of heat-induced proteins in the total cellular proteins. Among them, a 70 kDal protein was isolated and its N-terminal amino acid sequence was determined. The 70 kDal protein possessed a possible ATP-binding site in the N-terminus, which was conserved among the HSP70 family. A DNA fragment encoding the HSP70 homologue was isolated from a genomic library of S. griseus 2247 strain using an oligonucleotide probe based on the N-terminal amino acid sequence of the 70 kDal protein. DNA sequence analysis of the cloned gene revealed an open reading frame consisting of 618 amino acid residues. The deduced amino acid sequence is highly homologous to the HSP70 family proteins; it is 59.8 % identical to Clostridium perfringens HSP70, 59.7% to the Bacillus megaterium DnaK protein, 58.4% to the Methanosarcina mazei DnaK protein, 58.1% to Synechocystis HSP70, 52.8% to the DnaK protein of Escherichia coli , and about 50% to some of the mitochondrial heat shock proteins. The cloned gene could encode the HSP70 of S. griseus .