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KMID : 0545119980080030214
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Journal of Microbiology and Biotechnology
1998 Volume.8 No. 3 p.214 ~ p.221
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Cloning, Expression, and Nucleotide Sequencing of the Gene Encoding Glucose Permease of Phosphotransferase System from Brevibacterium ammoniagenes
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Yoon, Ki Hong
Yim, Hyouk/Jung, Kyung Hwa
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Abstract
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A Brevibacterium ammoniagenes gene coding for glucose/mannose-specific enzyme Ⅱ (EⅡ^Glc) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was cloned by complementing an Escherichia coli mutation affecting a ptsG gene, and the complete DNA nucleotide sequence was determined. The cloned gene was identified to be a ptsG, which enables the E. coli transformant to use glucose more efficiently than mannose as the sole carbon source in an M9 minimal medium. The ptsG gene of B. ammonia genes consists of an open reading frame of 1,983 nucleotides putatively encoding a polypeptide of 661 amino acid residues and a TAA stop codon. The deduced amino acid sequence of the B. ammoniagenes EⅡ^Glc shows, at 46%, the highest degree of sequence similarity with the Corynebacterium glutamicum EⅡ specific for both glucose and mannose. In addition, the EⅡ^Glc shares approximately 30% sequence similarities with sucrose-specific and β-glucoside-specific EⅡs of the several bacteria belonging to the glucose-PTS class. The 161-amino-acid C-terminal sequence of EⅡ^Glc is also similar to that of E. coli enzyme ⅡA^Glc, specific for glucose (EⅡA^Glc). The B. ammonia genes EⅡ^Glc consists of three domains; a hydrophobic region (EⅡC) and two hydrophilic regions (EⅡA, EⅡB). The arrangement of structural domains, ⅡBCA, of the EⅡ^Glc is identical to those of EⅡs specific for sucrose or β-glucoside. While the domain ⅡA was removed from the B. ammoniagenes EⅡ^Glc, the remaining domains ⅡBC were found to restore the glucose and mannose-utilizing capacity of E. coli mutant lacking EⅡ^Glc activity with EⅡA^Glc of the E. coli mutant. EⅡ^Glc contains a histidine residue and a cysteine residue which are putative phosphorylation sites for the protein.
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