The kdsA gene encoding 3-deoxy-~-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-~-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 phosphoennlpyruvate. A chlamydial DNA region upstream of the gene exhibiting similarities to the consensus sequence of a ' " promoters of Escherichia coli was responsible for the heterologous expression of kdsA.Keywords: 3-deoxy-~-manno-2-octulosonate-8-phosphate synthase; lipopolysaccharide biosynthesis; Chlamydia.Lipopolysaccharides (LPS) are amphiphilic molecules anchored in the outer leaflet of the outer membrane of gram-negative bacteria (Rietschel et al., 1994). In general, the architecture of LPS consists of a phosphorylated and acylated p(l+6)-linked glucosainine disaccharide, termed lipid A, to which a variable carbohydrate moiety is covalently linked. For genetic and biosynthetic reasons the sugar moiety may be further divided into a lipid-A proximal core and an outer 0-antigen region (Schnaitman and Klena, 1993). As surface structures, LPS play an important role in the interaction of gram-negative bacteria with higher organisms and are responsible for many immunological and pathophysiological effects which are observed during infectious diseases. On the other hand, many cellular characteristics of gram-negative bacteria like motility or the resistance to antibiotics may be correlated with biophysical and biochemical properities of LPS (Schnaitman and Klena, 1993;Nikaido, 1994). In particular, it is known from the structural analysis of various species, as well as mutants with defects in the biosynthesis of the inner core, that 3-deoxy-D-manno-2-octu~osonic acid (Kdo) linked to lipid A is a basic constituent of all LPS and Abbreviations. Dhd-7-P, 3-deoxy-~-arabino-2-heptulosonate-7-phosphate; Kdo, 3-deoxy-~-manno-2-octulosonate ; Kdo-k-P, 3-deoxy-D-manno-2-octulosonate-8-phosphate; LPS, lipopolysaccharide.Enzymes. 3-Deoxy-~-arabino-2-heptulosonate-7-phosphate synthase (EC 4.1.2.15); 3-deoxy-~-munno-2-octulosonate-8-phosphate synthase (EC 4.1.2.1 6 ) ; CTP: 3-deoxy-D-matmo-2-octu~osonate cytidylyltransferase, CMP-Kdo synthetase (EC 2.7.7.38); 3-deoxy-~-mannu-2-octulosonate transferase (EC