Construction and characterization of an acapsular mutant of Mannheimia haemolytica A1

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 (Cm(r)) 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 Cm(r) 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.