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Infection and Immunity, August 1999, p. 3830-3835, Vol. 67, No. 8
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Genetic Characterization of a Tn5-Disrupted Glycosyltransferase Gene Homolog in Brucella abortus and Its Effect on Lipopolysaccharide Composition and Virulence

J. R. McQuiston,1 R. Vemulapalli,1 T. J. Inzana,1 G. G. Schurig,1 N. Sriranganathan,1 D. Fritzinger,2 T. L. Hadfield,3 R. A. Warren,4 N. Snellings,4 D. Hoover,4 S. M. Halling,5 and S. M. Boyle1,*

Department of Biomedical Sciences and Pathobiology, Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-03421; SRA Technologies, Life Science, Rockville, Maryland 208052; Department of Infectious and Parasitic Diseases, Armed Forces Institute of Pathology, Washington, D.C. 20306-60003; Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, D.C. 20307-51004; and National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Ames, Iowa 500115

Received 7 December 1998/Returned for modification 3 March 1999/Accepted 4 May 1999

We constructed a rough mutant of Brucella abortus 2308 by transposon (Tn5) mutagenesis. Neither whole cells nor extracted lipopolysaccharide (LPS) from this mutant, designated RA1, reacted with a Brucella O-side-chain-specific monoclonal antibody (MAb), Bru-38, indicating the absence of O-side-chain synthesis. Compositional analyses of LPS from strain RA1 showed reduced levels of quinovosamine and mannose relative to the levels in the parental, wild-type strain, 2308. We isolated DNA flanking the Tn5 insertion in strain RA1 by cloning a 25-kb XbaI genomic fragment into pGEM-3Z to create plasmid pJM6. Allelic exchange of genomic DNA in B. abortus 2308 mediated by electroporation of pJM6 produced kanamycin-resistant clones that were not reactive with MAb Bru-38. Southern blot analysis of genomic DNA from these rough clones revealed Tn5 in a 25-kb XbaI genomic fragment. A homology search with the deduced amino acid sequence of the open reading frame disrupted by Tn5 revealed limited homology with various glycosyltransferases. This B. abortus gene has been named wboA. Transformation of strain RA1 with a broad-host-range plasmid bearing the wild-type B. abortus wboA gene resulted in the restoration of O-side-chain synthesis and the smooth phenotype. B. abortus RA1 was attenuated for survival in mice. However, strain RA1 persisted in mice spleens for a longer time than the B. abortus vaccine strain RB51, but as expected, neither strain induced antibodies specific for the O side chain.

* Corresponding author. Mailing address: Department of Biomedical Sciences and Pathobiology, Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0342. Phone: (540) 231-4641. Fax: (540) 231-3426. E-mail: smboyle{at}vt.edu.

Infection and Immunity, August 1999, p. 3830-3835, Vol. 67, No. 8
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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