Lipooligosaccharide biosynthesis in Neisseria gonorrhoeae: cloning, identification and characterization of the alpha 1,5 heptosyltransferase I gene (rfaC)

The identical partial deep-core structure of Hep alpha 1-3Hep alpha 1-5KDO in Salmonella typhimurium LT2 LPS and Neisseria gonorrhoeae LOS enabled us to isolate a DNA fragment from N. gonorrhoeae that was able to complement the alpha 1,5 LOS heptosyltransferase defect in the S. typhimurium rfaC630 (SA1377) mutant. SDS-PAGE analysis confirmed the production of wild-type LPS in the transformant. Subcloning revealed that complementation was due to a 1.2 kb fragment. Sequence analysis revealed a complete open reading frame capable of encoding a 36-37 kDa peptide. In vitro transcription-translation analysis of the 1.2 kb clone confirmed that a 37 kDa protein was encoded by this DNA fragment. The DNA sequence-deduced protein had 36% identity and 58% similarity to S. typhimurium heptosyltransferase I (RfaC). Primer extension analysis indicated that transcription of the cloned gene in N. gonorrhoeae strain 1291 begins 144 bp upstream of the start codon at a G nucleotide. An isogenic mutant of N. gonorrhoeae strain 1291 with an m-Tn3 insertion inside the coding sequence expressed a single truncated LOS with a similar molecular mass to S. typhimurium rfaC LPS. We conclude that the 1.2 kb fragment encodes the alpha 1,5 LOS heptosyltransferase I (RfaC) in N. gonorrhoeae. Our studies also provide further evidence that the third KDO residue in S. typhimurium LPS is added after the core synthesis is completed.