Carbohydrate utilization in Streptococcus thermophilus: characterization of the genes for aldose 1-epimerase (mutarotase) and UDPglucose 4-epimerase.
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-galM-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 beta-galactosidase) and gal (UDPglucose 4-epimerase) proteins of lactose- and glucose-grown S. thermophilus cells matched the mRNA levels.
Poolman
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GENENCOR Inc., South San Francisco, California 94080.
Royer
T J
TJ
Mainzer
S E
SE
Schmidt
B F
BF
D000595:Amino Acid Sequence; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics*; D050260:Carbohydrate Metabolism; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013291:Streptococcus / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic; D014534:UDPglucose 4-Epimerase / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011994:Recombinant Proteins; D002238:Carbohydrate Epimerases; D014534:UDPglucose 4-Epimerase; C019646:aldose 1-epimerase