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Infection and Immunity, May 2002, p. 2622-2629, Vol. 70, No. 5
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.5.2622-2629.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Construction and Characterization of an Acapsular Mutant of Mannheimia haemolytica A1

Linda J. McKerral and Reggie Y. C. Lo*

Department of Microbiology, University of Guelph, Guelph, Ontario, Canada, N1G 2W1

Received 29 October 2001/ Returned for modification 3 January 2002/ Accepted 31 January 2002

The nmaA and nmaB genes, which code for UDP-GlcNAc-2-epimerase and UDP-ManNAc-dehydrogenase, respectively, are involved in capsular polysaccharide biosynthesis in Mannheimia haemolytica A1. A chloramphenicol resistance (Cmr) cassette cloned behind an M. haemolytica A1 promoter, plpcat, was created and used to interrupt nmaA and nmaB. A 1.3-kbp DNA fragment that encompasses part of nmaA and nmaB was replaced by the 1.0-kbp plpcat, resulting in a knockout mutant which is Cmr and unable to synthesize N-acetylmannosamine (ManNAc) and N-acetylmannosaminuronic acid (ManNAcA). The DNA replacement was confirmed by Southern hybridization and PCR analyses of the nmaA and nmaB loci. Electron microscopy examination of the mutant showed the absence of capsular materials compared to the parent strain. The loss of NmaA and NmaB activity was confirmed by analysis of carbohydrate moieties using capillary electrophoresis. Serum sensitivity assays indicated that the acapsular mutant is as resistant as the encapsulated parent to complement-mediated killing by colostrum-deprived calf serum but is more sensitive to killing by immune bovine serum. Analysis of lipopolysaccharide prepared from the acapsular mutant and encapsulated parent confirmed that these strains have long O-polysaccharide chains, possibly conferring resistance to serum-mediated killing.

* Corresponding author. Mailing address: Department of Microbiology, University of Guelph, Guelph, Ontario, Canada, N1G 2W1. Phone: (519) 824-4120, ext. 3363. Fax: (519) 837-1802. E-mail: RLO{at}micro.uoguelph.ca.

Editor: B. B. Finlay

Infection and Immunity, May 2002, p. 2622-2629, Vol. 70, No. 5
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.5.2622-2629.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.





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