In gram-negative bacteria, galU is typically part of an operon that is involved in galactose
utilization and in the production of various exopolysaccharides [27, 30, 31]. The galU mutant strain characterized here was isolated from a random transposon library of FT LVS and was isolated as a polymyxin B hypersensitive strain (Figure 1A). The increased sensitivity of this galU mutant strain to cationic antimicrobials does not appear to be due to generalized outer envelope disintegrity because the mutant bacterium does not exhibit hypersensitivity to deoxycholate (an anionic bile acid) (Figure 1A) or the antibiotics chloramphenicol or tetracycline (data not shown). Figure 1 Growth kinetics of the galU mutant in vitro. Growth of wild-type, galU mutant, and galU-complemented strains of FT after 48 hrs of culture was measured by Palbociclib purchase the gradient plating technique to determine their sensitivity to polymyxin-B and deoxycholate. All data points represent the
mean (± SEM) of triplicate samples. Statistical analyses were performed via one-way ANOVA with Bonferroni post-tests. Statistically significant differences are indicated as follows: P < 0.01 (**) (Panel A). Growth of each strain cultured in MHB JQ-EZ-05 supplemented with either 0.1% glucose or 2% D-galactose (Panel B) or within macrophage-like murine cell lines (J774 or RAW264.7 at an MOI of 10, Panel C) was monitored over a 24 hour period. All data points represent the mean (± SEM) of triplicate samples. Each panel is representative of at least three experiments of similar design. Statistical analyses were performed via two-way ANOVA with ADP ribosylation factor Bonferroni post-tests. Statistically significant differences are indicated as follows: P < 0.01 (**) and P < 0.001 (***). The galU gene product is also known to be involved (but not required) in the catabolism of glucose and is required
for the catabolism of galactose in bacteria and yeast [31, 33, 34]. Therefore, we predicted that the galU mutant strain would display a mild growth defect in minimal medium containing glucose as a sole sugar source, and would have a more marked growth defect when cultured in medium containing galactose as a sole source of sugar. To determine whether the galU mutant had a galactose utilization phenotype, we characterized its growth in Mueller-Hinton broth (MHB) supplemented with either glucose or galactose as a sole sugar source (it is important to note that our standard medium for culture of FT is MHB supplemented with 0.1% glucose as the sole source of sugar). As predicted, the galU mutant strain of FT displayed a mild growth defect in MHB supplemented with glucose and a severe growth defect in MHB supplemented with galactose. Selleck PND-1186 Complementation of the galU mutation restored WT growth kinetics in MHB supplemented with either glucose or galactose (Figure 1B ).