The gene responsible for the transport of lactose into Streptococcus thermophilus (lacS) was cloned in Escherichia coli as a 4.2-kilobase fragment from an EcoRI library of chromosomal DNA by using the vector pKK223-3. From deletion analysis, the gene for lactose transport mapped to two Hindlll fragments with a total size of 2.8 kilobases. The gene was transcribed in E. coli from its own promoter. Functional expression of lactose transport activity was shown by assaying for the uptake and exchange of lactose both in intact cells and in membrane vesicles. The nucleotide sequence of lacS and 200 to 300 bases of 3' and 5' flanking regions were determined. The gene was 1,902 base pairs long, encoding a 69,454-dalton protein with an NH2-terminal hydrophobic region and a COOH-terminal hydrophilic region. The NH2-terminal end was homologous with the melibiose carrier of E. coli (23% similarity overall; >50% similarity for regions with at least 16 amino acids), whereas the COOH-terminal end showed 34 to 41% similarity with the enzyme III (domain) of three different phosphoenolpyruvate-dependent phosphotransferase systems. Among the conserved amino acids were two histidyl residues, of which one has been postulated to be phosphorylated by HPr. Since sugars are not phosphorylated during translocation by the lactose transport system, it is suggested that the enzyme III-like region serves a regulatory function in this protein. The lacS gene also appears similar to the partially sequenced lactose transport gene of LactobaciUlus bulgaricus (lacL; >60% similarity). Furthermore, the 3' flanking sequence of the S. thermophilus lactose transport gene showed approximately 50% similarity with the N-terminal portion of the ,-galactosidase gene of L. bulgaricus. In both organisms, the lactose transport gene and the Il-galactosidase appear to be separated by a 3-base-pair intercistronic region.Lactose transport in bacteria generally proceeds via either the phosphoenolpyruvate-dependent sugar transferase system (PEP-PTS) or a proton motive force (pmf)-linked transport system (W. N. Konings, B. Poolman, and A. J. M. Driessen, Crit. Rev. Microbiol., in press). In the former system, lactose enters the cell as lactose 6-phosphate, which is subsequently cleaved into glucose and galactose 6-phosphate by a ,-D-phosphogalactoside galactohydrolase. Lactose accumulated by the pmf-driven transport system enters the cell as free sugar and is hydrolyzed into galactose and glucose by a 3-D-galactoside galactohydrolase (,B-galactosidase).Dairy lactic acid bacteria, including species of both the lactic streptococci and lactobacilli, utilize lactose as their primary energy source. In a number of species which transport lactose by a PEP-PTS, the lac genes involved, their organization on the chromosome or plasmid, and their regulation are well documented (1, 4, 33). In contrast, very little is known about the lac genes in lactic acid bacteria that encode a pmf-linked lactose transport system in conjunction with a P-galactosidase. Representatives of this gr...
The complete nucleotide sequences of the genes encoding aldose 1-epimerase (mutarotase) (galM) and UDPglucose 4-epimerase (galE) and flanking regions of Streptococcus thermophilus have been determined. Both genes are located immediately upstream of the S. thermophilus lac operon. To facilitate the isolation of galE, a special polymerase chain reaction-based technique was used to amplify the region upstream of galM prior to cloning. The galM protein was homologous to the mutarotase of Acinetobacter calcoaceticus, whereas the galE protein was homologous to UDPglucose 4-epimerase of Escherichia coli and Streptomyces lividans. The amino acid sequences of galM and galE proteins also showed significant similarity with the carboxy-terminal and amino-terminal domains, respectively, of UDPglucose 4-epimerase from Kluyveromyces lactis and Saccharomyces cerevisiae, suggesting that the yeast enzymes contain an additional, yet unidentified (mutarotase) activity. In accordance with the open reading frames of the structural genes, galM and galE were expressed as polypeptides with apparent molecular masses of 39 and 37 kilodaltons, respectively. Significant activities of mutarotase and UDPglucose 4-epimerase were detected in lysates of E. coli cells containing plasmids encoding galM and galE. Expression of galE in E. coli was increased 300-fold when the gene was placed downstream of the tac promoter. The gene order for the gal-lac gene cluster of S. thermophilus is galE-gahM-lacS-lacZ. The flanking regions of these genes were searched for consensus promoter sequences and further characterized by primer extension analysis. Analysis of mRNA levels for the gal and lac genes in S. thermophilus showed a strong reduction upon growth in medium containing glucose instead of lactose. The activities of the lac (lactose transport and Il-galactosidase) and gal (UDPglucose 4-epimerase) proteins of lactose-and glucose-grown S. thermophilus cells matched the mRNA levels.Streptococcus thermophilus transports lactose by means of a proton motive force-linked mechanism (33). Lactose enters the cell as a free sugar, and the disaccharide is hydrolyzed into glucose and galactose by P-galactosidase (20,33). Glucose enters the glycolytic pathway, whereas in the presence of excess lactose, the galactose moiety of lactose is excreted into the medium (39).The lac genes of S. thermophilus have recently been cloned, sequenced, and partially characterized (20, 33; C. J. Schroeder, C. Robert, G. Lenzen, L. L. McKay, and A.Mercenier, submitted for publication). The lactose transport gene (lacS) encodes a 69,454-dalton (Da) protein consisting of an amino-terminal domain with homology to the melibiose carrier of Escherichia coli and a carboxy-terminal domain with homology to enzyme III or enzyme III domains of various phosphoenolpyruvate-dependent phosphotransferase systemns from gram-positive and gram-negative organisms. A similar transport protein has been found in Lactobacillus bulgaricus (33,38), and the function(s) of the different domains of the transport...
Using synthetic oligonucleotide probes, we cloned genomic DNA sequences encoding an alpha-amylase gene from Aspergillus niger var. awamori (A. awamori) on a 5.8 kb EcoRI fragment. Hybridization experiments, using a portion of this cloned fragment to probe DNA from A. awamori, suggested the presence of two alpha-amylase gene copies which were subsequently cloned as 7 kb (designated as amyA) and 4 kb (amyB) HindIII fragments. DNA sequence analysis of the amyA and amyB genes revealed the following: (1) Both genes are arranged as nine exons and eight introns; (2) The nucleotide sequences of amyA and amyB are identical throughout all but the last few nucleotides of their respective coding regions; (3) The amyA and amyB genes from A. awamori share extensive homology (greater than or equal to 98% identity) with the genes encoding Taka-amylase from A. oryzae. In order to test whether both amyA and amyB were functional in the genome, we constructed vectors containing gene fusions of either amyA and amyB to bovine prochymosin cDNA and used these vectors to transform A. awamori. Transformants which contained either the amyA- or amyB-prochymosin gene fusions produced extracellular chymosin, suggesting that both genes are functional.
EMBL accession no. X53576 In several fungi the trpC or equivalent gene codes for a trifunctional polypeptide; glutamine amido transferase, indole-3-glycerol phosphate synthase, and N-(5'-phosphoribosyl) anthranilate isomerase (1). Genomic DNA sequences encoding the trpC genes of Aspergillus nidulans (2), A. niger (3), and Penicillium chrysogenum (4) have been used as selectable markers in DNA mediated transformation studies. A pUC19 library was constructed from BamHI cut Aspergillus niger var. awamori UVK143f (5) genomic DNA and probed with a BgIH fragment of the A. nidulans gene (6). A 4 Kb clone was identified, and the nucleotide sequence of the region encoding the trpC gene is presented below. When compared with several other sequences, the DNA and deduced amino acid sequence are, respectively: 91.5% and 95.8% identical to Aspergillus niger, 66.9% and 78.5% identical to Aspergillus nidulans, 70.5% and 79.3% identical to Penicillium chrysogenum (7). The deduced amino acid sequence is: 62.6% identical to the Neurospora crassa trpF (8).
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