Analysis of the lsi region involved in lipooligosaccharide biosynthesis in Neisseria gonorrhoeae

Abstract

The genetic locus (lsi-1) responsible for the transformation of the lipooligosaccharide (LOS)-defective Neisseria gonorrhoeae mutant FA5100 to LOS expression was studied by deletion mutagenesis and sequence analysis. An open reading frame that was preceded by a leader sequence containing regions with the potential to form hairpin loops was identified. A perfect sigma 70 promoter consensus sequence was found upstream from this open reading frame. Promoter function was screened for functionality by using lac fusion cassettes and in vitro transcription-translation analysis. A frameshift mutation in the lsi-1 gene was constructed by site-directed mutagenesis and introduced into the chromosome of FA19, the LOS-expressing isogenic parent strain of FA5100. The mutant was characterized by Southern blotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting (immunoblotting) and found to be phenotypically identical to FA5100.