The gram-negative bacterial pathogen Brucella abortus is a zoonotic pathogen causing brucellosis in a variety of animal species including humans. The loss of the O-side chain in the lipopolysaccharide of the outer membrane decreases Brucella virulence. To understand the genetics of O-side chain biosynthesis and its relationship to virulence, studies were initiated to characterize specific O-side chain mutants. B. abortus rough mutant strain RA2 was derived by transposon (Tn5) mutagenesis of smooth B. abortus 2308. The chromosomal region of strain RA2 with the Tn5 and flanking chromosomal region was cloned into the sequencing vector pGEM-3Z to create a suicide plasmid pNW-2. The plasmid pNW-2, or a derivative of it (pNW-3), in which Tn5 was replaced with a Kank gene, were electroporated into wild type smooth B. abortus 2308 in order to assess the phenotypic conversion from smooth to rough. The electroporation parameters such as cell growth stage, pulse field strength and pulse length were optimized. It was determined that using late log phase cells (approximately 70-77 Klett units), 10 ms and 13 KV/cm were the best conditions for achieving transformation by pNW-2 or pNW3. Kanamycin resistant and ampicillin sensitive Brucella were screened for double reciprocal crossovers between the suicide plasmids (pNW-2 and pNW-3) and Brucella chromosomal DNA. The recombinants were checked for their O-side chain by crystal violet uptake and immunoblotting with monoclonal antibody specific for the O-side chain. The locations of Tn5 and the flanking region in the genome of these recombinants were characterized by Southern blot using either a Tn5 probe or a flanking region probe. An analysis of KanR colonies showed that none of the recombinants were rough. The B. abortus DNA in pNW-2 was sequenced and compared with other genes. No Significant homology was found between the Brucella DNA in pNW-2 and gene sequences in the gene bank. Analysis of the recombinants suggests no linkage between the Tn5 element in strain RA2 and the rough phenotype.