Global change of gene expression and cell physiology in YidC-depleted Escherichia coli

Abstract

YidC depletion affects membrane protein insertion and leads to a defect in the growth of the Escherichia coli cell. We analyzed global changes in gene expression upon YidC depletion to determine the importance of YidC for cellular functions using a gene chip method to compare the transcriptomes of JS71 (control) and JS7131 (yidC depletion strain). Of the more than 4,300 genes identified, 163 were upregulated and 99 were downregulated upon YidC depletion, including genes which are responsible for DNA/RNA repair; energy metabolism; various transporters, proteases and chaperones; stress response; and translation and transcription functions. Real-time PCR was performed on selected genes to confirm the results. Specifically, we found upregulation of the genes encoding the energy transduction proteins F(1)F(o) ATP synthase and cytochrome bo(3) oxidase due to perturbation in assembly when YidC was depleted. We also determined that the high-level induction of the PspA stress protein under YidC depletion conditions is roughly 10-fold higher than the activation due to the addition of protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), which dissipates the proton motive force. In addition, the gene chip data reveal the Cpx stress pathway is activated upon YidC depletion. The data show the broad physiological contribution of YidC to the bacterial cell and the considerable ramification to the cell when it is depleted.

FIG. 1.

YidC depletion affects cell growth in liquid medium. Growth of JS71 (control) and JS7131 (YidC depletion) was measured by OD_600. Absorbances were measured from 3 independent cultures of both JS71 and JS7131. A 0.2% concentration of glucose was added at time point 0. Error bars represent standard deviation from the 3 independent culture measurements.

FIG. 2.

Cell morphology is affected by YidC depletion. Images (×100 magnification) were obtained after the cells were grown for 5 h under the YidC depletion condition for JS7131 (right panel) or the normal condition for JS71 (left panel).

FIG. 3.

Selected genes are tested by real-time PCR to confirm the microarray data. The y axis represents the relative expression level of each gene under YidC depletion conditions compared to the normal condition. A value of 1 would represent no change in expression upon YidC depletion. The mRNA levels at both the 3- and 5-h time points were tested. Error bars are calculated from triplicates for each experiment.

FIG. 4.

Gene category summary. All genes showing a different expression pattern (>2 fold difference and P value of <0.05) were grouped according to their functions. The x axis indicates the number of genes within each functional group. Black bars represent those groups of genes which were upregulated by microarray analysis under YidC depletion conditions for 5 h. Gray bars represent those groups of genes which were downregulated by microarray analysis under YidC depletion conditions for 5 h.

FIG. 5.

Regulation of specific Cpx- and sigma E-controlled genes by real-time PCR under YidC depletion conditions. The expected regulation pattern of genes is indicated for each pathway listed in the chart. The y axis represents the relative expression level by real-time PCR of each gene under YidC depletion conditions compared to the normal condition. (A) Relative expression level of selected genes for Cpx pathway at both 3 and 5 h of YidC depletion. (B) Relative expression level of selected genes for sigma E pathway at both 3 and 5 h of YidC depletion.

FIG. 6.

YidC depletion induces a higher level of phage shock response than CCCP treatment. (A) JS7131 was grown in glucose (0.2%) or arabinose (0.2%), treated with various concentrations of CCCP, and then analyzed by Coomassie G-250 staining (top panel) or Western blotting using PspA antibody (lower panel). Soluble proteins were removed before running the gel for Coomassie stain (see Materials and Methods). (B) The growth curve was measured for JS7131 in LB medium containing arabinose (0.2%) with various concentrations of CCCP. (C) The growth curve was determined for JS7131 in LB medium with arabinose (0.2%) or glucose (0.2%). (D) The addition of 40 μM CCCP to the JS71 culture resulted in the accumulation of ³⁵S-labeled pro-OmpA in the cell. The amounts of precursor and mature OmpA in a 1-min pulse-label were determined as described in Materials and Methods.

FIG. 7.

YidC depletion affects cell motility. After growth for 5 h in LB medium supplemented with 0.2% glucose, equal numbers (as determined using a microscope and counting chamber) of JS71 (YidC expressed) and JS7131 (YidC depleted) cells were applied to a motility agar plate containing 0.2% glucose and 0.002% arabinose. This arabinose concentration was tested to be sufficient to keep JS7131 cells alive but did not allow growth within 24 h. Plates were incubated at 37°C for 24 h. The size of the JS7131 colony was 0.6 ± 0.1 cm. The size of the JS71 colony was 3.2 ± 0.3 cm. Sizes were measured independently using 5 plates for each of the strains. Only one representative plate is shown here.

FIG. 8.

Comparison of the protein patterns in the soluble fractions from JS71 (control) and JS7131 (YidC depletion) cells. Two representative 2D gels are shown (JS71 soluble and JS7131 soluble). Gels were stained with SYPRO ruby. The arrow indicates the spots that were cut out for MS analysis.

FIG. 9.

Western blot analysis of selected proteins. To verify the 2D gel results, protein samples were collected from cell lysates derived from JS7131 and JS71 after 5 h of growth in LB medium plus 0.2% glucose and subjected to Western blot analysis using the indicated antibodies. YidC, phage shock protein PspA, MBP, and the chaperones DnaJ and GroEL were analyzed. In the case of MalF and the F_o ATPase subunits, it was necessary to enrich the proteins by isolating the membranes. Inner membrane vesicles from JS7131 and JS71 under the 5-h YidC depletion condition were purified and analyzed by Western blotting using antibodies against F_oa, F_ob, F_oc, and MalF.