The effect of growth temperature on the biosynthesis of Yersinia enterocolitica O:3 lipopolysaccharide: temperature regulates the transcription of the rfb but not of the rfa region.

The rfb region of Yersinia enterocolitica O:3 (YeO3) that determines the synthesis of the O-side chain of the lipopolysaccharide was cloned and expressed in Escherichia coli K12 previously. The clone did not show the in vitro temperature variation in O-side chain expression known for YeO3, instead analogous O-side-chain was produced at 25 degrees C and at 37 degrees C and both were similar to that produced by YeO3 at 25 degrees C. In Northern blot analysis marked reduction in the amount of rfb-specific mRNA was observed from YeO3 grown at 37 degrees C when compared with those grown at 25 degrees C. On the other hand, equal amounts of rfb-specific mRNA were detected in the E. coli clone at both growth temperatures. This indicates that the transcription of YeO3 rfb region is dramatically repressed at 37 degrees C. The repressor gene is located outside the rfb region with no analogous locus in E. coli chromosome. We cloned 18.2 kilobase pairs of YeO3 chromosomal DNA that rendered E. coli K12 reactive with 2B5, a YeO3 core-specific monoclonal antibody, in colony blotting, indirect immunofluorescence and immunoblotting. Hence this clone contains the YeO3 rfa region, encompassing the genes involved in the core oligosaccharide biosynthesis. No apparent difference, in the aforementioned tests, was noticed in the expression of the 2B5 epitope at different growth temperatures either in YeO3 or in E. coli. In Northern blot analysis comparable amounts of rfa-specific mRNA were detected at 25 degrees and 37 degrees C. This argues that in YeO3 the core oligosaccharide biosynthesis is not temperature-regulated.