The production of a capsular polysaccharide (CPS; K antigen) is characteristic of KiebsieUla pneumoniae, but CPS structure varies among strains, and many different serotypes are now known. In this study, cps gene clusters encoding the elements of capsular polysaccharide biosynthesis were exchanged by homologous recombination between strains expressing different serotypes. The wild-type K. pneumoniae strains used for genetic exchange were KPA1 (cpsK2), expressing K2 CPS, and KPB1 (cpsK2la), expressing K21a CPS. Plasmid R68.45 was used to mobilize fragments of chromosomal DNA from auxotrophic derivatives of donor strains. Auxotrophic his alleles introduced into recipient strains provided selectable markers to coinherit the adjacent cps gene clusters from donors expressing a heterologous CPS. Each of the capsule-switched recombinants, KPA5 (cpsK21a) and KPB20 (cpsK2), was shown to have a CPS that was immunologically identical to the serotype of the respective donor. The recombinants retained their respective recipient strain background, as evidenced by a genetic marker and demonstration of a distinctive restriction fragment length polymorphism in genomic DNA. KPB1 CPS contained a sequence (mannose-ca-2-mannose) that binds to a macrophage lectin and may be responsible for their higher susceptibility to macrophage binding and phagocytosis compared with KPA1, whose CPS lacked such sequences. The recombinant strains expressing heterologous cps genes inherited the macrophage-binding phenotype of the donor, thus confirming that relative susceptibility to phagocytosis was determined by the capsule type expressed. KPA1 was highly virulent in a mouse lethality assay, which is a feature typical of K2 strains, whereas KPB1 was not virulent in mice. Recombinant KPA5 retained relatively high virulence in mice, even though it produced the heterologous K21a CPS, which suggests that a virulence factor other than capsule biosynthesis is encoded by the KPA genomic strain background. In contrast, KPB20 gained marginal virulence in the mouse lethality assay through the inheritance and expression of the K2 CPS from the virulent strain. Thus, pathogenesis in K. pneumoniae may be multifactorial. Specific antibody was used to stabilize the CPS on the surface of K. pneumonwae, and the structural organization of the homologous and heterologous capsules was examined by electron microscopy. Recombinant KPB20, expressing heterologous K2 CPS, had a uniform layer of capsule surrounding the organism that was similar to that seen on the surfaces of the parental strains. However, KPA5, expressing the heterologous K21a CPS, was unusual in that the uniform capsular layer was physically separated from the cell wall by-50 nm. Within this zone of separation, numerous and regularly spaced filaments of CPS were * Corresponding author. isolated. This analysis showed that the genes (cps) encoding CPS map close to his and are probably clustered. Recently, the genes determining K pneumoniae K2 CPS were cloned as a cps gene cluster occupying more than 15 kb of ...
