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Lipooligosaccharide biosynthesis in Neiseria gonorrhoeae : cloning, identification and characterization of the α1,5 heptosyltransferase I gene ( rfaC )
Author(s) -
Zhou Daoguo,
Lee NaGyong,
Apicella Michael A.
Publication year - 1994
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1994.tb01300.x
Subject(s) - biology , subcloning , primer extension , complementation , microbiology and biotechnology , neisseria gonorrhoeae , nucleic acid sequence , sequence analysis , gene , mutant , open reading frame , structural gene , genetics , peptide sequence , escherichia coli , rna
Summary The identical partial deep‐core structure of Hepα1–3Hepα1–5KDO 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 α1,5 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 144bp 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 α1,5 LOS heptosyltransferase 1 (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.