FIGURE 4.

Capsule polysaccharide production is reduced in the kpfR::kan^R mutant strain. (A) For visualization of capsule production, wild-type (UKP8), mutant (kpfR::kan^R), and complemented (${\mathit{kpfR::kan}}_{\text{comp}}^{\text{R}}$) strains were cultured under the same growth conditions, stained with India ink, and analyzed by optical microscopy. The images are representative of independent experiments and the presence of capsule is indicated by a negative staining area around the bacteria. While capsule is observed in the wild-type and partially in the complemented strains, no visible capsule is observed on kpfR::kan^R, suggesting a reduction in capsule biosynthesis on the hyperfimbriated mutant strain. (B) For quantification of capsule production, glucuronic acid, an important constituent of the K. pneumoniae capsule, was measured from capsular polysaccharides extracted of 0.5 mL cultures of each strain. The results are indicated as percentages of glucuronic acid production by mutant and complemented strain relative to the production by the wild-type UKP8 strain (set at 100%). The mutant strain presents reduced production of capsule compared to the wild-type strain. Although the complemented strain produces more capsules than the mutant strain, this increased production was not significantly different. Data are means of five independent experiments ± SE. ***p ≤ 0.005 vs. wild-type. (C) The less capsule production by the kpfR::kan^R mutant strain correlates with the down-regulation of the capsule-producing gene galF on this strain. ***p ≤ 0.005 vs. wild-type.