The Organophosphate Degradation (opd) Island-borne Esterase-induced Metabolic Diversion in Escherichia coli and Its Influence on p-Nitrophenol Degradation

Abstract

In previous studies of the organophosphate degradation gene cluster, we showed that expression of an open reading frame (orf306) present within the cluster in Escherichia coli allowed growth on p-nitrophenol (PNP) as sole carbon source. We have now shown that expression of orf306 in E. coli causes a dramatic up-regulation in genes coding for alternative carbon catabolism. The propionate, glyoxylate, and methylcitrate cycle pathway-specific enzymes are up-regulated along with hca (phenylpropionate) and mhp (hydroxyphenylpropionate) degradation operons. These hca and mhp operons play a key role in degradation of PNP, enabling E. coli to grow using it as sole carbon source. Supporting growth experiments, PNP degradation products entered central metabolic pathways and were incorporated into the carbon backbone. The protein and RNA samples isolated from E. coli (pSDP10) cells grown in (14)C-labeled PNP indicated incorporation of (14)C carbon, suggesting Orf306-dependent assimilation of PNP in E. coli cells.

Keywords: Escherichia coli (E. coli); biodegradation; gene expression; gene knock-out; metabolic regulation; microarray.

FIGURE 1.

PNP supported growth of E. coli MG1655 (pSDP10). A indicates growth of MG1655 (pSDP10), MG1655 (pMMB206), and MG1655 cells in PNP-containing minimal medium. Closed (growth) and open (PNP) circles represent growth and PNP concentration in culture medium containing MG1655 (pSDP10). Similar parameters are indicated with closed (growth) and open (PNP) squares for MG1655 (pMMB206) and closed (growth) and open (PNP) rhombuses for MG1655 cultures. Proteins extracted from MG1655 (pSDP10) cells grown in the presence of normal and ¹⁴C-labeled PNP were analyzed by SDS-PAGE, and incorporation of ¹⁴C into proteins is shown in the corresponding autoradiogram (B). RNA extracted from similarly grown cultures and the corresponding autoradiogram are shown in C. The intense sharp signal shown by the arrow indicates incorporation of ¹⁴C into 5S rRNA. D indicates a decrease in concentration of PNP and concomitant release of stoichiometric amounts of nitrite in resting cells of E. coli (pSDP10). HPLC profiles showing the time-dependent decrease of PNP in the culture medium and the appearance of nitrocatechol (4.5 min) and benzenetriol (8.3 min) are shown in E and F. AU, absorbance units; ND, not detected.

FIGURE 2.

The Orf306-dependent induction of the hca and mhp operons. A, heat map showing expression of the hca and mhp operons at 0, 1.5, and 3.0 h after induction of orf306. The portion of the two-dimensional gel showing Orf306-specific induction of HcaR and MhpA proteins is shown in B. The quantification (qPCR) of mhpA-, mhpR-, hcaE-, and hcaR-specific transcripts under similar growth conditions is shown in C with the error bars representing the S.D. Growth of the MG1655 (pDS10) (●) and hcaE (■), mhpA (▴), and mhpR (♦) mutants in PNP-containing minimal medium is shown in D. E represents agarose gel pictures indicating deletion of hcaE, mhpA, and mhpR genes. The PNP-dependent growth of MG1655 (pDS10) (●) and paaA (■) and maoA (▴) mutants (F) and an agarose gel showing the deletion of the paaA and maoA genes (G) are shown.

FIGURE 3.

Orf306-induced metabolic diversion in MG1655 (pSDP10). Heat maps represent differential expression of genes encoding glycolysis, glyoxylate, and TCA cycle enzymes at 0, 1.5, and 3.0 h. The green arrows represent down-regulated pathways. The up-regulated pathways are shown with red arrows. Up-regulation of the phenylpropionate pathway and down-regulation of glycolysis are shown with dotted green and red arrows, respectively. Quantitative PCR results showing either an Orf306-dependent decrease (pfkA, eno, and acnA) or increase (glcB, glcC, and prpB) in the concentration of specific mRNAs are inserted at places showing the corresponding enzyme reactions with the error bars representing the S.D. The two-dimensional gel portions indicate Orf306-dependent induction of MhpA, HcaR, GlcC, PrpB, and SdhA. Eno, enolase; PEP, phosphoenolpyruvate.

FIGURE 4.

Propionate-dependent induction of hcaR and mhpR genes. The lacZ-negative strains of MG1655 containing promoter test vector pMP220 and hcaR-, mhpR-, and mhpA-lacZ fusions were grown either on a propionate + X-gal (A) or a glucose + X-gal plate (B). The quantification of promoter activity for propionate- and glucose-grown cultures is shown in C and D, respectively, with the error bars representing the S.D. The Orf306-dependent induction of the hcaR, mhpR, and mhpA genes in MG1655 (pSDP10) cells is shown in E and F, respectively.

FIGURE 5.

Expression and subcellular localization of Orf306 in S. fuliginis ATCC 27551. The proteins extracted from whole cells (W), membrane (M), and cytoplasm (C) were analyzed by SDS-PAGE (A). PM, protein molecular weight marker. The corresponding Western blot developed using Orf306-specific antibodies is shown in B. Arrows indicate the existence of Orf306 in two different forms. The asterisk (*) indicates the exclusive presence of posttranslationally modified Orf306 in the membrane fraction. Reciprocal pulldown assays performed to show OPH and Orf306 interactions are shown in C–F. C indicates proteins extracted from BL21 (pSM5) and BL21 (pSM5 + pSDP5) loaded in lanes 1 and 2. The corresponding pulldown samples are loaded in lanes 3 and 4. Co-elution of Orf306 along with OPH^His6 is shown with an arrow (lane 4). D, lanes 1 and 3 represent protein extracts prepared either from BL21 (pSM5 + pSDP4) or BL21 (pGEX4T1 + pSM5) as input. Lane 3 and 4 represent proteins in pulldown samples. The Western blots developed with either OPH-specific antibodies or GST-specific antibodies are shown in E and F, respectively.