Construction and characterization of an acapsular mutant strain of Mannheimia (Pasteurella) haemolytica A1
Two genes, 'nmaA' and 'nmaB', which code for UDP-GlcNAc-2-epimerase and UDP-ManNAc-dehydrogenase, respectively, have been identified in the 'Mannheimia haemolytica' A1 capsular polysaccharide biosynthesis cluster. A chloramphenicol resistance (Cm R) cassette cloned behind a 'M. haemolytica' A1 promoter ('plpCAT') was created and used to interrupt 'nmaA' and 'nmaB' by allelic exchange, resulting in Cm R knockout mutants. The DNA replacement was confirmed by PCR analysis of the 'mnaA, nmaB' loci and Southern hybridization analyses using either an 'nmaA'/'B' or CAT specific probe against genomic DNA from the CmR mutants. Electron microscopy examination of the mutants showed a significant reduction of capsular materials compared to the parent strain. The loss of NmaA and/or NmaB activity was confirmed by analysis of carbohydrate moieties using capillary electrophoresis. Serum sensitivity assays indicated that the acapsular mutant was resistant to complement-mediated killing by naive calf serum, but was more sensitive to killing by adult bovine serum than the encapsulated parent.