Cloning of a region in the Brucella abortus chromosome necessary for o-side chain biosynthesis

As a first step in characterizing the genes involved in O-side chain synthesis in Brucella abortus strain 2308, a portion of the genomic DNA was cloned from a rough mutant created by Tn5 (KnR) mutagenesis. This mutant was rough based on the lack of reactivity by either whole cells or extracted LPS to an O-side chain monoclonal antibody (BRU-38). A 30 kb XbaI genomic fragment (including Tn5) from the rough strain was subcloned into a sequencing vector to create pJM6. When B. abortus 2308 was electroporated with pJM6, KnR clones were unable to react with BRU-38; a Southern analysis of these clones revealed Tn5 in the 30 kb XbaI genomic fragment. Various regions of the 30kb fragment were subcloned and tested for their ability to complement specific rfa and rfb mutants of Escherichia coli and Salmonella typhimurium. One particular DNA fragment complemented an rfbD mutation in E. coli as judged by agglutination with E. coli anti-O (0:85) serum. The same DNA fragment failed to cause E. coli rfbD to react with either BRU-38 or B. abortus anti-O polyclonal antisera. The B. abortus 30 kb XbaI fragment contains a gene which has been identified by comple-mentation as containing the equivalent of the rfbD gene encoding dTDP-rhamnose synthetase in E. coli. Since Brucella is not known to have rhamnose in its core this enzyme may have a different function in Brucella LPS synthesis.