Differential Impact of Hexuronate Regulators ExuR and UxuR on the Escherichia coli Proteome

Abstract

ExuR and UxuR are paralogous proteins belonging to the GntR family of transcriptional regulators. Both are known to control hexuronic acid metabolism in a variety of Gammaproteobacteria but the relative impact of each of them is still unclear. Here, we apply 2D difference electrophoresis followed by mass-spectrometry to characterise the changes in the Escherichia coli proteome in response to a uxuR or exuR deletion. Our data clearly show that the effects are different: deletion of uxuR resulted in strongly enhanced expression of D-mannonate dehydratase UxuA and flagellar protein FliC, and in a reduced amount of outer membrane porin OmpF, while the absence of ExuR did not significantly alter the spectrum of detected proteins. Consequently, the physiological roles of proteins predicted as homologs seem to be far from identical. Effects of uxuR deletion were largely dependent on the cultivation conditions: during growth with glucose, UxuA and FliC were dramatically altered, while during growth with glucuronate, activation of both was not so prominent. During the growth with glucose, maximal activation was detected for FliC. This was further confirmed by expression analysis and physiological tests, thus suggesting the involvement of UxuR in the regulation of bacterial motility and biofilm formation.

Keywords: Ashwell pathway; ExuR; UxuR; biofilm formation; fliC; motility; proteome; uxuAB.

Figure 1

Proteomic maps of the wild type E. coli K-12 MG1655 and E. coli K-12 MG1655 ΔexuR during growth with D-glucose (A) and D-glucuronate (B). For each condition, 218 (A) and 160 (B) proteins were detected in both strains.

Figure 2

Proteomic maps of the wild type E. coli K-12 MG1655 and E. coli K-12 MG1655 ΔuxuR during growth with D-glucose. For D-glucuronate, gels are shown in Figure S2. Two hundred and eighteen proteins were detected in both strains.

Figure 3

(A) Bacterial motility assay in 0.3% agar in the presence of 0.2% D-glucose or D-glucuronate. (B) Effects of UxuR and ExuR on fliC transcription. A panel represents qRT-PCR data for the E. coli K-12 MG1655 strain and its uxuR or exuR deletion mutants. No changes were detected in the hns-mRNA levels that were used as controls. mRNA levels are expressed relative to the parent strain during the growth on glucose. Error bars represent the standard deviations from four biological replicates. Significant differences were defined by p < 0.01 (**) and p < 0.001 (***) compared to the wild type strain during growth with glucose.

Figure 4

(A) Relative efficiency of biofilm formation of the wild type E. coli K-12 MG1655 strain and its uxuR deletion mutant after 48 h of microaerobic growth on LB or M9 medium. (B) Example of the staining intensity after 48 h of microaerobic growth on LB. Error bars represent the standard deviations from six biological replicates. Significant differences were defined by p < 0.05 (*) and p < 0.001 (***) compared to the wild type strain.

Figure 5

Effects of UxuR on different processes in E. coli during growth with D-glucose, based on the proteomic analysis. Activation is shown in green, inhibition—in red.