Cloning of cellobiose phosphoenolpyruvate-dependent phosphotransferase genes: Functional expression in recombinant Escherichia coli and identification of a putative binding region for disaccharides?

被引:43
作者
Lai, XK
Davis, FC
Hespell, RB
Ingram, LO
机构
[1] UNIV FLORIDA,DEPT MICROBIOL & CELL SCI,GAINESVILLE,FL 32611
[2] USDA ARS,NATL CTR AGR UTILIZAT RES,FERMENTAT BIOCHEM UNIT,PEORIA,IL 61604
关键词
D O I
10.1128/AEM.63.2.355-363.1997
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Genomic libraries from nine cellobiose-metabolizing bacteria were screened for cellobiose utilization, Positive clones were recovered from six libraries, all of which encode, phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) proteins. Clones from Bacillus subtilis, Butyrivibrio fibrisolvens, and Klebsiella oxytoca allowed the growth of recombinant Escherichia coli in cellobiose-M9 minimal medium. The K. oxytoca clone, pLOI1906, exhibited an unusually broad substrate range (cellobiose, arbutin, salicin, and methylumbelliferyl derivatives of glucose, cellobiose, mannose, and xylose) and was sequenced. The insert in this plasmid encoded the carboxy-terminal region of a putative regulatory protein, cellobiose permease (single polypeptide), and phospho-beta-glucosidase, which appear to form an operon (casRAB). Subclones allowed both casA and casB to be expressed independently, as evidenced by in vitro complementation. An analysis of the translated Sequences from the EIIC domains of cellobiose, aryl-beta-glucoside, and other disaccharide permeases allowed the identification of a 50-amino-acid conserved region. A disaccharide consensus sequence is proposed for the most conserved segment (13 amino acids), which may represent part of the EIIC active site for binding and phosphorylation.
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页码:355 / 363
页数:9
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