Characterization of UDP-glucose dehydrogenase and UDP-glucose pyrophosphorylase mutants of Proteus mirabilis: defectiveness in polymyxin B resistance, swarming, and virulence

Abstract

Proteus mirabilis is known to be highly resistant to the action of polymyxin B (PB). However, the mechanism underlying PB resistance is not clear. In this study, we used Tn5 transposon mutagenesis to identify genes that may affect PB resistance in P. mirabilis. Two genes, ugd and galU, which may encode UDP-glucose dehydrogenase (Ugd) and UDP-glucose pyrophosphorylase (GalU), respectively, were identified. Knockout mutants of ugd and galU were found to be extremely sensitive to PB, presumably because of alterations in lipopolysaccharide (LPS) structure and cell surface architecture in these mutants. These mutants were defective in swarming, expressed lower levels of virulence factor hemolysin, and had lower cell invasion ability. Complementation of the ugd or galU mutant with the full-length ugd or galU gene, respectively, led to the restoration of wild-type phenotypic traits. Interestingly, we found that the expression of Ugd and GalU was induced by PB through RppA, a putative response regulator of the bacterial two-component system that we identified previously. Mutation in either ugd or galU led to activation of RpoE, an extracytoplasmic function sigma factor that has been shown to be activated by protein misfolding and alterations in cell surface structure in other bacteria. Activation of RpoE or RpoE overexpression was found to cause inhibition of FlhDC and hemolysin expression. To our knowledge, this is the first report describing the roles and regulation of Ugd and GalU in P. mirabilis.

FIG. 1.

LPS profiles of wild-type P. mirabilis (N2), the galU knockout mutant (dG1), the GalU-complemented strain (dG1c), the ugd knockout mutant (dU2), and the Ugd-complemented strain (dU2c). Six microliters of LPS purified from the same number of cells (OD₆₀₀ × volume [in ml] = 100) of the respective strains was subjected to SDS-PAGE analysis.

FIG. 2.

(A) Swarming migration of wild-type P. mirabilis (N2), the ugd knockout mutant (dU2), and the galU knockout mutant (dG1) on LB swarming plates. Aliquots (5 μl) of overnight culture were inoculated centrally onto the plates. The plates were incubated at 37°C, and the representative pictures were taken at 18 h after incubation. (B) The swarming migration of wild-type P. mirabilis (N2), the ugd knockout mutant (dU2), the Ugd-complemented strain (dU2c), the galU knockout mutant (dG1), and the GalU-complemented strain (dG1c). Aliquots (5 μl) of overnight culture were inoculated centrally onto the LB swarming plates. The plates were incubated at 37°C, and the migration distance was measured hourly after inoculation. The data represent the averages of three results of independent experiments with standard deviations.

FIG. 3.

(A) Flagellin level of wild-type P. mirabilis (N2), the ugd knockout mutant (dU2), and the galU knockout mutant (dG1). The flagellin level was determined at different time points after seeding the strains on LB agar plates. The value obtained with the wild-type cells at 4 h postseeding was set at 100%, and all other values were expressed relative to this value. The data represent the averages of results of three independent experiments with standard deviations. (B) Microscopic observation of cell differentiation of wild-type P. mirabilis (N2), the ugd knockout mutant (dU2), and the galU knockout mutant (dG1). Cells were Gram-stained and viewed under oil (magnification, ×1,000). Three independent experiments were performed, and the representative pictures showing cell differentiation at 4, 5, and 6 h postseeding on LB agar plates are shown. The increase in cell length was taken as a sign of cell differentiation. (C) Expression of flhDC mRNA in wild-type P. mirabilis (N2), the ugd knockout mutant (dU2), and the galU knockout mutant (dG1). Total RNA was isolated from respective cells at 4 h postseeding on LB agar plates and was then subjected to real-time RT-PCR for the measurement of flhDC mRNA. The value obtained with the wild-type cells was set at 1. The data represent the averages of results of three independent experiments with standard deviations.

FIG. 4.

Hemolysin activities of wild-type P. mirabilis (N2), the ugd knockout mutant (dU2), the Ugd-complemented strain (dU2c), the galU knockout mutant (dG1), and the GalU-complemented strain (dG1c). Hemolysin activity was determined at different time points after seeding the strains on LB agar plates. The value obtained with the wild-type cells at 4 h postseeding was set at 100%, and all other values were expressed relative to this value. The data represent the averages of results of three independent experiments with standard deviations.

FIG. 5.

(A) Effect of rppA mutation on the expression of ugd and galU mRNA in the presence or absence of PB (1 μg/ml). The mRNA amounts of ugd and galU in the wild type, the rppA knockout mutant, and the RppA-complemented strain were quantified by real-time RT-PCR at 4 h postseeding on LB agar plates as described in Materials and Methods. The value obtained with the wild-type cells in the absence of PB was set at 1. The data represent the averages of results of four independent experiments with standard deviations. (B) XylE activities of ugd-xylE and galU-xylE reporters in the wild type and the rppA knockout mutant in the presence or absence of 1 μg/ml PB. Representative data of activities for cells grown on the LB agar plates for 4 h are shown. The data represent the averages of results of three independent experiments with standard deviations. *, P < 0.01, for comparison of the value in the absence of PB with that in the presence of PB; N2, wild type; dA10, rppA knockout mutant; dA10c, RppA-complemented strain.

FIG. 6.

(A) Effect of ugd and galU mutation on the promoter activity of rpoE. XylE activities of rpoE-xylE reporter were determined for the wild type, the ugd knockout mutant (dU2), and the galU knockout mutant (dG1). Representative data of activities for cells grown on the LB agar plates for 4 h are shown. (B) Effect of RpoE on the expression of flhDC mRNA. The flhDC mRNA amounts in the wild type (N2), the rseA mutant (ΔrseA), and the RpoE-overexpressed strain (rpoE-over) were quantified by real-time RT-PCR at 4 h postseeding on LB agar plates, as described in Materials and Methods. The value obtained with the wild-type cells was set at 1. The data represent the averages of results of three independent experiments with standard deviations. (C) Effect of RpoE on the hemolysin activities of the wild type (N2), the rseA mutant (ΔrseA), and the RpoE-overexpressed strain (rpoE-over). Hemolysin activity was determined at 4 h after seeding the strains on LB agar plates. The value obtained with the wild-type cells was set at 1, and all other values were expressed relative to this value. The data represent the averages of results of three independent experiments with standard deviation.