Effect of static growth and different levels of environmental oxygen on toxA and ptxR expression in the Pseudomonas aeruginosa strain PAO1

Abstract

Within certain infection sites, such as the lung of cystic fibrosis patients,Pseudomonas aeruginosagrows statically under either decreased oxygen tension or anaerobic conditions, a situation that is likely to influence the production of virulence factors. The goal of this study was to determine the effect of static growth under microaerobic (decreased oxygen) and anaerobic conditions on the expression of theP. aeruginosaexotoxin A (ETA) genetoxAand its positive regulatorptxR. UsingtoxA–lacZandptxR–lacZfusion plasmids, the level oftoxAandptxRexpression was measured throughout the growth cycle of strain PAO1, which was grown in either iron-deficient or iron-sufficient medium under four different conditions: 20 %-SH (aerobic, shaking), 20 %-ST (aerobic, static), 10 %-ST (microaerobic, static) and 0 %-ST (anaerobic, static). In iron-deficient medium,toxAexpression was higher under 20 %-ST and 10 %-ST than under 20 %-SH. However, the highest level oftoxAexpression occurred under 0 %-ST. Analysis of ETA protein using sandwich ELISA revealed that at time points between 8 and 24 h of the growth curve, PAO1 produced higher levels of ETA under 0 %-ST than under 20 %-SH. In iron-sufficient medium,toxAexpression was significantly repressed under all conditions. Additional analyses using PAO1 strains that carrylacZfusions with thetoxAregulatory genesregAandpvdSrevealed that the expression ofregAandpvdSis reduced rather than increased at 0 %-ST.ptxRexpression under different conditions paralleled that oftoxAexpression, except that it was repressed by iron under 20 %-SH only. Between 6 and 24 h of growth, and under all conditions, the level of dissolved oxygen (DO) within the PAO1 cultures was sharply reduced. These results suggest that (1) the combined effect of static growth and anaerobic conditions produce a significant increase intoxAandptxRexpression in PAO1; (2) this effect appears to be unique totoxAandptxR, since the level ofregAandpvdSexpression was reduced under the same conditions; (3) neither static growth nor anaerobic conditions interfere with the repression oftoxAexpression by iron, although static growth deregulatesptxRexpression with respect to iron; and (4) the enhanced expression oftoxAandptxRis not related to the reduced levels of DO in PAO1 cultures.