Comparative analyses of secondary gene products of 3-deoxy-D-manno-oct-2-ulosonic acid transferases from Chlamydiaceae in Escherichia coli K-12

The waaA gene encoding the essential, lipopolysaccharide (LPS)-specific 3-d eoxy-dmanno-oct-2-ulosonic acid (Kdo) transferase was inactivated in the ch romosome of a heptosyltransferase I and II deficient Escherichia coli K-12 strain by insertion of gene expression cassettes encoding the waaA genes of Chlamydia trachomatis, Chlamydophila pneumoniae or Chlamydophila psittaci. The three chlamydial Kdo transferases were able to complement the knockout mutation without changing the growth or multiplication behaviour. The LPS of the mutants were serologically and structurally characterized in compari son to the LPS of the parent strain using compositional analyses, high perf ormance anion exchange chromatography, matrix-assisted laser desorption/ion ization time-of-flight mass spectrometry and specific monoclonal antibodies . The data show that chlamydial Kdo transferases can replace in E. coli K-1 2 the host's Kdo transferase and retain the product specificities described in their natural background. In addition, we unequivocally proved that Waa A from C. psittaci transfers predominantly four Kdo residues to lipid A, fo rming a branched tetrasaccharide with the structure alpha-Kdo-(2 --> 8)-[al pha-Kdo-(2 --> 4)]-alpha-Kdo-(2 --> 4)-alpha-Kdo.