Molecular cloning, sequence analysis and functional characterization of the gene kdsA, encoding...

The kdsA gene encoding 3-deoxy-D-manno-2-octulosonate-8-phosphate (Kdo-8-P) synthase of Chlamydia psittaci 6BC was cloned by complementing the temperature-sensitive kdsA mutant Salmonella enterica serovar Typhimurium AG701i50. The sequence analysis of a recombinant DNA fragment revealed an open reading frame of 807 nucleotides which codes for a polypeptide of 269 amino acids with a high degree of similarity to known KdsA proteins. In addition, alignments of Kdo-8-P synthases with bacterial and fungal 3-deoxy-D-arabino-2-heptulosonate-7-phosphate (Dha-7-P) synthases suggested that both classes of enzymes are structurally related and may belong to a family of 2-keto-3-deoxy-aldonic acid synthases. The chlamydial protein was overexpressed and functionally characterized in vitro to synthesize Kdo-8-P from D-arabinose 5-phosphate and phosphoenolpyruvate. A chlamydial DNA region upstream of the gene exhibiting similarities to the consensus sequence of sigma 70 promoters of Escherichia coli was responsible for the heterologous expression of kdsA.