A highly heat-stable amylase gene from an obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, was cloned and expressed in Escherichia coli. The nucleotide sequence of the amylase gene predicts a 686-amino-acid protein of relative molecular mass 81 200, which is consistent with that determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified enzyme. The NH2-terminal sequence determined using the enzyme purified from E. coli cells corresponds precisely to that predicted from the nucleotide sequence, except for the absence of the NH2-terminal methionine in the mature protein. When the amylase gene was expressed in E. coli cells, the enzyme was localized in the cytoplasmic fraction; this is probably explained by the absence of the signal sequence for secretion. By using the amylase purified from the E. coli transformant, some enzymatic properties, such as optimum pH, optimum temperature, pH-stability and heat-stability, were examined. The amylase was found to be a highly liquefying-type.Dictyoglomus thermophilum is a gram-negative, obligately anaerobic and extremely thermophilic bacterium forming a characteristic cell-association structure called 'rotund bodies' and possesses DNA of an extremely low G + C content (29 mol%) [l]. This strain was found to produce more than three different amylases with an extreme heat-stability and acidic optimum pH [2] which will be potentially useful in industrial starch saccharification. We intended to clone the amylase genes and produce the amylases in Escherichia coli which is easy to cultivate. In this paper, we describe the cloning and sequencing of one (tentatively designated as amyA) of the amylase genes along with its expression in E. coli cells. A single species of the amylase purified from the E. coli transformant has enabled us to characterize the enzymatic properties. MATERIALS AND METHODS Bacterial DNA isolationPlasmid DNA from E. coli was prepared by ethidium bromide/CsCl equilibrium centrifugation. For isolation of chromosomal DNA from D. thermophilum, the bacterium was cultured at 73 "C in the anaerobic medium with the gas phase of nitrogen, as previously described [l]. Cells were collected by centifugation and lyzed with lysozyme. DNA was purified by the method of Saito and Miura [4]. Recombinant DNA studiesRestriction endonucleases, T4 DNA ligase and DNAmodifying enzymes were purchased from Takara Shuzo Co. Ltd and New England Biolabs. Transformation of E. coli cells [5], DNA recovery from agarose gel slices [6], and Southern hybridization [7j were performed according to the published procedure. E. coli transformants carrying pBR322-or pYEJOOl -derived plasmids were selected on L agar containing 50 Fg/ml of ampicillin. DNA was sequenced with [IX-~'P]~CTP by the M13-dideoxynucleotide method [8]. Detection of the transformants containing the amylase genePlasmid pYEJOOl was digested with HindIII and the larger fragment was purified by agarose gel electrophoresis. Chromosomal DNA partially digested with Hind...