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 e nabled us to isolate a DNA fragment from N. gonorrhoeae that was able to complement the alpha 1,5 LOS heptosyltransferase defect in the S. t yphimurium rfaC630 (SA1377) mutant. SDS-PAGE analysis confirmed the pr oduction of wild-type LPS in the transformant. Subcloning revealed tha t complementation was due to a 1.2 kb fragment. Sequence analysis reve aled a complete open reading frame capable of encoding a 36-37 kDa pep tide. 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 (RfaC). Primer extension analysis ind icated that transcription of the cloned gene in N. gonorrhoeae strain 1291 begins 144 bp upstream of the start codon at a G nucleotide. An i sogenic mutant of N. gonorrhoeae strain 1291 with an m-Tn3 insertion i nside the coding sequence expressed a single truncated LOS with a simi lar molecular mass to S. typhimurium rfaC LPS. We conclude that the 1. 2 kb fragment encodes the alpha 1,5 LOS heptosyltransferase I (RfaC) i n N. gonorrhoeae. Our studies also provide further evidence that the t hird KDO residue in S. typhimurium LPS is added after the core synthes is is completed.