The capsule of
K. pneumoniae was visualized by transmission electron microscopy. Wild-type NTUHK2044 had a fibrous halo corresponding to the HMV phenotype (
Fig. 1A), similar to that described previously.
8 This halo was absent in the Δ
magA mutant (
Fig. 1B). However, the
magA-complemented strain was similar in appearance to the wild-type strain, with extracellular material visible (
Fig. 1C). After establishing that the wild-type and
magA complement strains did not grow differently in vitro (data not shown), capsule production was assessed qualitatively by sedimentation. When equal CFUs were sedimented at 10,000
g, the Δ
magA mutant cells packed well and formed a solid pellet (
Fig. 1D). However, neither the wild-type nor the
magA-complemented strain formed tight pellets, and the
magA complement had a pellet more than twice the volume of the wild-type (
Fig. 1D). The amount of capsule, as a function of uronic acid content, was quantified. There was no statistical difference in the amount of uronic acid detected in overnight cultures of the wild-type and
magA-complement strains (7.25 ± 0.35 μg/10
9 CFU vs. 6.88 ± 0.68 μg/ 10
9CFU [
P = 0.44]), indicating the complement restored wild-type levels of capsule production. The amount of uronic acid produced by the Δ
magA strain was below the limit of detection for the assay. To verify the deletion and complementation of
magA, we used semiquantitative RT-PCR to detect
magA expression. The presence of the
magA transcript was verified in wild-type and complemented strains but was not detected in the mutant strain (
Fig. 1E). These data suggested that the complemented strain produced capsule equal to that of the wild-type
K. pneumoniae and that the capsule from these strains inhibited tight packing of cells similar to what has been described previously.
19