Cloning and expression in Escherichia coli of two additional amylase genes of a strictly anaerobic thermophile, Dictyoglomus thermophilum, and their nucleotide sequences with extremely low guanine-plus-cytosine contents.
An obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, produces multiple extracellular amylases. In addition to one of the amylase genes, amyA, which we previously cloned and characterized, we have cloned two additional genes, amyB and amyC, coding for amylases of this thermophile, into Escherichia coli and determined their nucleotide sequences. The two amylase genes were expressed under the control of E. coli promoters. Almost all activity was detected in the intracellular fraction in the E. coli cells. The molecular mass and NH2-terminal amino acid sequence of the AmyB enzyme, which was purified from an E. coli transformant containing the amyB gene, confirmed that the reading frame of amyB consisted of 562 amino acids (Mr 67,000). The molecular mass of the AmyC enzyme, estimated by activity staining of a crude extract of E. coli containing amyC, confirmed that AmyC consisted of 498 amino acids (Mr 59,000). The optimal temperatures for AmyB and AmyC activities on soluble starch were 80 degrees C and 70 degrees C, respectively. Both AmyB and AmyC showed a pH optimum of 5.5. AmyB and AmyC showed a different pattern of starch hydrolysis when examined by thin-layer chromatography. Some homology in the amino acid sequences with the functional regions of Taka-amylase A was found in both AmyB and AmyC. The codon usage in the amyA, amyB and amyC genes was highly biased, which reflects the fact that the guanine-plus-cytosine (G + C) content of DNA of D. thermophilum is 29 mol%. The distribution of G and C at each position of the codons was non-random; the G + C content of the first position of codons is significantly high, whereas that of the third position is somewhat low. In addition, codons consisting only of A and T were preferentially used in this thermophile.
Horinouchi
S
S
Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Japan.
Fukusumi
S
S
Ohshima
T
T
Beppu
T
T
D000595:Amino Acid Sequence; D000681:Amylases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology / Q000235:genetics*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010957:Plasmids; D013213:Starch / Q000378:metabolism
D003062:Codon; D004269:DNA, Bacterial; D013213:Starch; D004262:DNA Restriction Enzymes; D000681:Amylases