title: Cloning, characterisation and regulation of an alpha-amylase gene from Streptomyces venezuelae.
abstract: The alpha-amylase gene (aml) of Streptomyces venezuelae ATCC15068 was cloned in Streptomyces lividans TK24 using the plasmid vector pIJ702. Sub-cloning and exonuclease III deletion experiments localised the sequences required for alpha-amylase production to a segment of 2.05 kb. Low-resolution nuclease S1 mapping revealed a aml transcript of approx. 1.7 kb, and the extracellular form of alpha-amylase was estimated by SDS-polyacrylamide gel electrophoresis to be 59 kDa, suggesting that aml mRNA is monocistronic. The nucleotide sequence of aml was determined and high-resolution nuclease S1 mapping experiments identified transcripts that appeared to initiate at a promoter identical to that of the alpha-amylase gene of Streptomyces limosus [Long et al., J. Bacteriol. 169 (1987) 5745-5754]. Transcription of aml in S. venezuelae, and of the cloned gene in Streptomyces coelicolor A3(2), was induced by maltose and repressed by glucose. Glucose repression in S. coelicolor A3(2) depended on a functional glucose kinase gene. The predicted amino acid sequence of the extracellular enzyme was very similar (75% identity) to the alpha-amylase of S. limosus and shared with this enzyme a strong susceptibility to tendamistat, a potent inhibitor of mammalian alpha-amylases. Sequence inspection revealed a putative signal sequence of 28 amino acids that preceded the probable signal peptidase cleavage site.
authors.affiliation: John Innes Institute, Norwich, U.K.
authors.forename: M J
authors.initials: MJ
authors.lastname: Virolle
authors.forename: C M
authors.initials: CM
authors.lastname: Long
authors.forename: S
authors.initials: S
authors.lastname: Chang
authors.forename: M J
authors.initials: MJ
authors.lastname: Bibb
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D013302:Streptomyces / Q000235:genetics*; D014158:Transcription, Genetic; D014169:Transformation, Bacterial; D000516:alpha-Amylases / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D000516:alpha-Amylases