Hidenori Shinkawa

MSG5, a novel protein phosphatase promotes adaptation to pheromone response in S. cerevisiae.

Pheromone‐stimulated yeast cells and haploid gpa1 deletion mutants arrest their cell cycle in G1. Overexpression of a novel gene called MSG5 suppresses this inhibition of cell division. Loss of MSG5 function leads to a diminished adaptive response to pheromone. Genetic analysis indicates that MSG5 acts at a stage where the protein kinases STE7 and FUS3 function to transmit the pheromone‐induced signal. Since loss of MSG5 function causes an increase in FUS3 enzyme activity but not STE7 activity, we propose that MSG5 impinges on the pathway at FUS3. Sequence analysis suggests that MSG5 encodes a protein tyrosine phosphatase. This is supported by the finding that recombinant MSG5 has phosphatase activity in vitro and is able to inactivate autophosphorylated FUS3. Thus MSG5 might stimulate recovery from pheromone by regulating the phosphorylation state of FUS3.

Cloning and Analysis of the Telomere and Terminal Inverted Repeat of the Linear Chromosome of Streptomyces griseus

Cloning and sequencing of the telomere of Streptomyces griseus revealed five palindromic sequences in the terminal 116 nucleotides, all of which can make a hairpin loop structure. However, the end sequence cannot form the foldback secondary structure that is common in Streptomyces telomeres and is suggested to be necessary for terminal replication. Both inside ends of the terminal inverted repeat (TIR) were also cloned and sequenced. The results confirmed the size of the TIR to be 24 kb and identified two almost identical open reading frames that might have been involved in the formation of the TIR.

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

Abstract 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.

Improvement of transformation system in Streptomyces using a modified regeneration medium

Abstract For improvement of the regeneration frequency of Streptomyces griseus 2247 protoplasts, the effects of growth phase and components of the regeneration medium on the frequency were investigated. Protoplasts prepared from transition-phase mycelia of S. griseus 2247 exhibited the highest regeneration frequency. An improved regeneration medium designated R1M was established by supplementing Polypepton and yeast extract which were shown to increase the S. griseus 2247 growth rate to R1 previously developed for S. griseus. The regeneration frequencies of S. griseus 2247 and its pleiotropic mutant HT-3 were greatly increased to 87.5% and 92.5%, respectively, on R1M medium. Using this protoplast-regeneration system, the transformation efficiency for the plasmid pRES18 was increased to 106 transformants per μg DNA. R1M medium was also effective for the protoplast regeneration of some other antibiotic-producing streptomycete species.

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.

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 .

Silence of streptomycin 6-phosphotransferase gene derived by incubation at a high temperature inStreptomyces griseus

SummaryThe bald mutants from streptomycin (SM)-producingStreptomyces griseus 2247 obtained by incubation at high temperature (36° C), designated as HT strains, lost resistance to their own antibiotic and scarcely produced the antibiotic. Although SM susceptibility in the mutant was due to loss of SM 6-phosphotransferase activity produced in the cell, the gene coding for the enzyme cloned from an HT strain was surely expressed inS. lividans 1326 as a host. Northern blot analysis showed that the corresponding RNA is not detected in the mutant, indicating that though the gene encoding SM 6-phosphotransferase, at least, the structural gene is not deleted in the cell, the expression is silent.

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.