Cloning and characterization of a new alpha-amylase gene from Streptomyces lividans TK24.
Streptomyces lividans TK24 possesses a very weak amylolytic activity, nevertheless Southern blot analysis carried out at high stringency revealed that this strain does contain a gene strongly related to the well expressed alpha-amylase gene (amlSL) of Streptomyces limosus. To clone this related gene, three genomic banks of S. lividans TK24 were constructed into the multicopy plasmid vector pIJ699 and transformed into the same strain. Two different genes were isolated. One (amlA) has been previously described, whereas the other (amlB) has never been described. Sub-cloning experiments localized amlB to a 3 kb BamHI-NotI fragment that was sequenced. Frame analysis on sequence data revealed the presence of a 1719 bp long open reading frame encoding a 573 amino acid protein of 61214 kDa. Northern blot analysis identified a unique 1.8 kb monocistronic transcript. Primer extension allowed the localization of the transcription start point 108 bp upstream of the translational start codon and demonstrated that the gene was transcribed from a unique typical eubacterial-like promoter. AmlB shares 74.7% amino acid identity with the alpha-amylase of S. limosus and only 27.2% with the amylolytic enzyme encoded by amlA.
Yin
X H
XH
Laboratoire de Biologie et Génétique Moléculaire, Institut de Génétique et Microbiologie, CNRS URA1354, Université Paris-Sud, Orsay, France.
Gagnat
J
J
Gerbaud
C
C
Guérineau
M
M
Virolle
M J
MJ
D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic / Q000235:genetics; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism
D003062:Codon; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger; D000516:alpha-Amylases