Horinouchi Sueharu

Phosphorylation of the AfsR protein involved in secondary metabolism in Streptomyces species by a eukary otic-type protein kinase

Abstract A global regulatory protein, AfsR, involved in secondary metabolism, was found to be phosphorylated by a membraneassociated phosphokinase, named AfsK, of Streptomyces coelicolor A3(2) and S. lividans . The N-terminal portion of AfsK, deduced from the nucleotide (nt) sequence of the afsK gene, which was located downstream from the afsR gene, showed significant sequence similarity to the catalytic domain of eukaryotic Ser/Thr protein kinases (PKs). Consistent with this, experiments with AfsK produced by use of an Escherichia coli host-vector system revealed a self-catalyzed phosphate incorporation into both Ser and Tyr residues of AfsK. The recombinant AfsK phosphorylated the purified AfsR at both Ser and Thr residues. Disruption of the chromosomal afsK gene with the phage vector KC515 resulted in significant, but not complete, loss of actinorhodin production. This result implies the involvement of afsK in the regulation of secondary metabolism. The presence of an additional PK able to phosphorylate AfsR is predicted, because the afsK -disrupted strain still contained an activity able to phosphorylate Ser and Thr residues of AfsR. Southern hybridization experiments showed that nt sequences homologous to afsK , as well as afsR , were distributed among many Streptomyces spp. It is thus concluded that a signal transduction system similar to that found in higher organisms is involved in the regulation of secondary metabolism in the bacterial genus Streptomyces .

Cloning and nucleotide sequence of a cellulase gene, casA, from an alkalophilic Streptomyces strain

A gene encoding an endo-type semi-alkaline cellulase was cloned from an alkalophilic Streptomyces strain in Streptomyces lividans, and its nucleotide sequence was determined. Downstream from the transcriptional start point, which was determined by high-resolution S1 mapping, an open reading frame of 388 amino acids (aa) was present. The N-terminal amino acid sequence of the mature enzyme determined by an Edman degradation procedure suggested that the cellulase had an extraordinarily long leader sequence of about 70 aa. Comparison with the leader sequences of endoglucosidase H from Streptomyces plicatus and the cellulase from Cellulomonas fimi suggested that the semi-alkaline cellulase was processed in two steps during maturation.

A gene encoding mycinamicin III O-methyltransferase from Micromonospora griseorubida

A DNA fragment of 42 kb encompassing one of the mycinamicin II (Mm)-resistance-encoding genes, myrB, from a Mm-producing strain, Micromonospora griseorubida, was cloned in Escherichia coli using the cosmid vector pJB8. Nucleotide sequencing of the neighboring region of myrB and a computer-aided analysis of the sequence predicted the presence of an open reading frame (ORF) with 254 amino acids which showed great similarity to the macrocin O-methyltransferase (tylF gene product) in tylosin (Ty)-producing Streptomyces fradiae. When a 1.0-kb AluI fragment containing the complete ORF was fused to the lacZ promoter in the correct orientation and expressed in E. coli, a mycinamicin III (MIII) O-methyltransferase (MOMT) activity was detected only upon induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). All these data indicate that this ORF codes for the structural gene of MOMT, and it is designated mycF.

Cloning and sequences of two macrolide-resistance-encoding genes from mycinamicin-producing Micromonospora griseorubida

Abstract Two macrolide-resistance determinants were cloned from a mycinamicin (Mm)-producing Micromonospora griseorubida strain in Streptomyces lividans and Streptomyces parvulus. One of the cloned genes, designated myrA, was cloned as a gene which conferred strong resistance to Mm and tyiosin (Ty), but not to erythromycin (Er) or josamycin (Jm) on S. lividans. Another gene, named myrB, was cloned as an ErRencoding gene which conferred MLS resistance (to macrolide, lincosamide and streptogramine B antibiotics) on S. parvulus. Both myrA and myrB were sequenced and the corresponding ORFs were determined. The deduced amino acid (aa) sequence of myrA showed no similarity to proteins in the available databases, suggesting that an unknown mechanism of macrolide resistance is exerted by the MyrA protein. The deduced aa sequence of myrB exhibited high similarity to 23S rRNA methyltransferases (MTases), such as ErmE and CarB, from a variety of microorganisms.

Cloning of a streptomycin-production gene directing synthesis of N-methyl-L-glucosamine

Abstract A Streptomyces bikiniensis DNA fragment complementing a deficiency in streptomycin (Sm) production was cloned on the plasmid vector pIJ385. Host strain S. bikiniensis SD1 used as the recipient in cloning displayed deficiency in biosynthesis of N -methyl- l -glucosamine, one of the moieties of Sm. The cloned fragment on the multicopy plasmid pIJ385 conferred sevenfold increase in Sm production in comparison with the wild-type parental strain. By subcloning, the region complementing the Sm deficiency of SD1 was narrowed to a 3.0-kb fragment.

Cloning and sequencing the recA+ genes of Acetobacter polyoxogenes and Acetobacter aceti: construction of recA− mutants of by transformation-mediated gene replacement

Abstract The recA + gene of Acetobacter polyoxogenes was cloned as a gene that conferred methyl methanesulfonate resistance (MMS R ) on the RecA − Escherichia coli HB101. The cloned recA + gene also conferred ( i ) resistance to UV irradiation, ( ii ) enhanced intrachromosomal recombination, and ( iii ) permitted prophage φ 80 induction in E. coli recA − lysogens. Nucleotide sequence determination revealed that the recA product consists of 348 amino acids (aa) corresponding to 38 kDa, and shows significant similarity to RecA proteins from other Gram- bacteria. Next, a portion of recA from Acetobacter aceti was cloned by using polymerase chain reaction with oligodeoxyribonucleotide primers design based on the A. polyoxogenes recA sequence. Due to availability of efficient host-vector and transformation systems in A. aceti , recA mutants of A. aceti were obtained by transformation-mediated gene replacement with the cloned A. aceti recA gene which was inactivated by insertion of the kanamycin-resistance-encoding gene from pACYC177. The recA mutants obtained in this way showed similar phenotypes to those of E. coli recA strains, such as increased sensitivity to MMS and to UV irradiation, and decreased homologous recombination.