Cloning and characterization of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferase genes (kdtA) from Acinetobacter baumannii and Acinetobacter haemolyticus.
3-Deoxy-D-manno-oct-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 TTG 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. baumanniii, A. haemnolyticus 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.
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Division of Medical and Biochemical Microbiology, Research Center Borstel, Germany.
Brabetz
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Brade
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D000150:Acinetobacter / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014166:Transferases / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D014166:Transferases; C019917:3-deoxy-D-manno-octulosonate transferase