The Lactobacillus bulgaricus Il-galactosidase gene was cloned on a ca. 7-kilobase-pair HindIII fragment in the vector pKK223-3 and expressed in Escherichia coli by using its own promoter. The nucleotide sequence of the gene and approximately 400 bases of 3'-and 5'-flanking sequences was determined. The amino acid sequence of the 6-galactosidase, deduced from the nucleotide sequence of the gene, yielded a monomeric molecular mass of ca. 114 kilodaltons, slightly smaller than the E. coli lacZ and Klebsiella pneumoniae lacZ enzymes but larger than the E. coli evolved (ebgA) 0-galactosidase. The cloned P-galactosidase was found to be indistinguishable from the native enzyme by several criteria. From amino acid sequence alignments, the L. bulgaricus , -galactosidase has a 30 to 34% similarity to the E. coli lacZ, E. coli ebgA, and K. pneumoniae lacZ enzymes. There are seven regions of high similarity common to all four of these PI-galactosidases. Also, the putative active-site residues (Glu-461 and Tyr-503 in the E. coli lacZ ,I-galactosidase) are conserved in the L. bulgaricus enzyme as well as in the other two ,I-galactosidases mentioned above. The conservation of active-site amino acids and the large regions of similarity suggest that all four of these PI-galactosidases evolved from a common ancestral gene. However, these enzymes are quite different from the thermophilic 0-galactosidase encoded by the Bacillus stearothermophilus bgaB gene.Little is known about the specific amino acids involved in the substrate binding and catalysis of the Escherichia coli lacZ P-galactosidase, even though this enzyme has been studied for many years (35). From iodination (8, 15), fluorotyrosine substitution (27), and active-site-directed inhibitor (11,12,23) experiments, Tyr-503 is thought to be the proton-donating species needed for catalysis. Primarily by the use of active-site-directed reagents (11,13)

Three morphologically similar strains of halophilic, box-shaped procaryotes have been isolated from brines collected in the Sinai, Baja California (Mexico), and southern California (United States). Although the isolates in their morphology resemble Walsby's square bacteria, which are a dominant morphological type in the Red Sea and Baja California brines, they are probably not identical to them. The cells show the general characteristics of extreme halophiles and archaebacteria. They contain pigments similar to bacteriorhodopsin which apparently mediate light-driven ion translocation and photophosphorylation.

The genes encoding the thermostable a-amylases of Bacillus stearothermophilus and Bl. licheniformis were cloned in Escherichia coli, and their DNA sequences were determined. The coding and deduced polypeptide sequences are 59 and 62% homologous to each other, respectively. The B. stearothermophilus protein differs most significantly from that of B. licheniformis in that it possesses a 32-residue COOH-terminal tail. Transformation of E. coli with vectors containing either gene resulted in the synthesis and secretion of active enzymes similar to those produced by the parental organisms. A plasmid was constructed in which the promoter and the NH2-terminal two-thirds of the B. stearothermophilus coding sequence was fused out of frame to the entire mature coding sequence of the B. licheniformis gene. Approximately 1 in 5,000 colonies transformed with this plasmid was found to secrete an active amylase. Hybridization analysis of plasmids isolated from these amylase-positive colonies indicated that the parental coding sequences had recombined by homologous recombination. DNA sequeince analysis of selected hybrid genes revealed symmetrical, nonraudom distribution of loci at which the crossovers had resolved. Several purified hybrid ec-amylases were characterized and found to differ with respect to thermostability and specific activity.The ax-amylases secreted by a variety of Bacillus species have been intensively studied. Ihterest has been focused on their mode of secretion, regulation of synthesis, protein structure, and industrial applications. In recent years, the amylase genes of B. coagulans (4) The mesophile B. licheniformis and the thermophile B. stearothermophilus produce amylases which are active at temperatures in excess of 75°C (7). It is therefore of interest to determine their primary structures and compare them with each other and with those known for other amylases to ascertain which sequences are associated with the unusual thermophilicity of these enzymes. In this study we showed that the B. stearothermophilus and B. licheniformis enzymes differ markedly in their specific activities and thermostabilities. Primary structure analysis might also offer clues to these differences.It has been known for some time that the mesophilic amylase of B. amyloliquefaciens has considerable amino acid homology with the B. licheniformis amylase but no homology to the B. subtilis enzyme (21). Recently, comparison of the B. stearothermophilus and B. amyloliquefaciens primary structures has revealed that these proteins are also evolutionarily related (16). In addition, strong similarities between the restriction endonuclease cleavage maps of the amylase genes of B. coagulans and B. licheniformis indicate that these genes may also show homology (21).Since DNA sequence divergence has led to differing chemical properties of the encoded proteins, it is expected that further diversity in this enzyme family might be found in additional natural Bacillus isolates. Alternatively, methods to generate amylase DNA sequence divergence i...

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