Cloning and characterization of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferase genes (kdtA) from Acinetobacter baumannii and Acinetobacter haemolyticus

被引:27
作者
Bode, CE [1 ]
Brabetz, W [1 ]
Brade, H [1 ]
机构
[1] Res Ctr Borstel, Ctr Med & Biosci, Div Med & Biochem Microbiol, D-23845 Borstel, Germany
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1998年 / 254卷 / 02期
关键词
3-deoxy-D-manno-oct-2-ulosonic acid transferase; Acinetobacter; lipopolysaccharide biosynthesis; shuttle plasmid;
D O I
10.1046/j.1432-1327.1998.2540404.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
3-Deoxy-D-manno-act-2-ulosonic acid (Kdo) transferases (KdtA) are multifunctional glycosyltransferases with primary structures of low similarity. Totally degenerated primers were deduced from two stretches of identical amino acids between known KdtA sequences and used to amplify by PCR a kdtA-specific fragment from Acinetobacter baumannii ATCC 15308 DNA which was then applied as a probe for the cloning and sequencing of the complete Kdo transferase gene. With conserved PCR primers for this structural gene from A. baumannii ATCC 15308, also kdtA genes of A. baumannii ATCC 19606 and A. haemolyticus ATCC 17906 were obtained, cloned from the chromosome and sequenced. The genes coded for proteins with similarities to known Kdo transferases. Within the genus Acinetobacter, the identity and similarity of the deduced amino acid sequences were 71% and 84.5%, respectively. The kdtA sequences of both A. baumannii strains were identical and possessed a TIG start codon, whereas ATG was found in the case of A. haemolyticus. The genes from Acinetobacter, and kdtA from Escherichia coli K-12 were expressed in the Gram-positive bacterium Corynebacterium glutamicum. In vitro tests confirmed the function of the gene products as Kdo transferases, which transferred mainly two Kdo residues to a synthetic lipid A precursor of E. coli. Also, no differences between the cloned kdtA genes from A. baumannii, A. haemolyticus and E. coli were observed when tetraacyl or hexaacyl lipid A were tested, since all transferases acted more efficiently on the former With limiting amounts of acceptor, all Kdo transferases were able to transfer a third Kdo residue with varying efficiency.
引用
收藏
页码:404 / 412
页数:9
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