Proteomic and transcriptomic analyses reveal genes upregulated by cis-dichloroethene in Polaromonas sp. strain JS666

Abstract

Polaromonas sp. strain JS666 is the only bacterial isolate capable of using cis-dichloroethene (cDCE) as a sole carbon and energy source. Studies of cDCE degradation in this novel organism are of interest because of potential bioremediation and biocatalysis applications. The primary cellular responses of JS666 to growth on cDCE were explored using proteomics and transcriptomics to identify the genes upregulated by cDCE. Two-dimensional gel electrophoresis revealed upregulation of genes annotated as encoding glutathione S-transferase, cyclohexanone monooxygenase, and haloacid dehalogenase. DNA microarray experiments confirmed the proteomics findings that the genes indicated above were among the most highly upregulated by cDCE. The upregulation of genes with antioxidant functions and the inhibition of cDCE degradation by elevated oxygen levels suggest that cDCE induces an oxidative stress response. Furthermore, the upregulation of a predicted ABC transporter and two sodium/solute symporters suggests that transport is important in cDCE degradation. The omics data were integrated with data from compound-specific isotope analysis (CSIA) and biochemical experiments to develop a hypothesis for cDCE degradation pathways in JS666. The CSIA results indicate that the measured isotope enrichment factors for aerobic cDCE degradation ranged from -17.4 to -22.4 per thousand. Evidence suggests that cDCE degradation via monooxygenase-catalyzed epoxidation (C C cleavage) may be only a minor degradation pathway under the conditions of these experiments and that the major degradation pathway involves carbon-chloride cleavage as the initial step, a novel mechanism. The results provide a significant step toward elucidation of cDCE degradation pathways and enhanced understanding of cDCE degradation in JS666.

FIG. 1.

Effects of growth substrate on subsequent cDCE degradation. Ethanol-grown cultures were washed and suspended in medium containing cDCE (A) or ethanol (B), and acetate-grown cultures were washed and suspended in medium containing cDCE (C) or acetate (D). Ethanol and acetate concentrations were monitored indirectly by determining the decrease in the partial pressure of oxygen (p_O2). All cultures had approximately the same initial level of biomass (i.e., an optical density at 600 nm of 0.05). The data are the results of duplicate experiments (filled and open symbols).

FIG. 2.

Overlays of 2D gel images comparing proteins extracted from cDCE-grown (blue) cells to proteins extracted from acetate-grown (red) cells (A) and proteins extracted from cDCE-grown (blue) cells to proteins extracted from glycolate-grown (red) cells (B). Differentially expressed spots in cDCE gels that were subsequently analyzed by MS are circled.

FIG. 3.

Distribution of genes upregulated by cDCE based on COG functional categories. Statistically enriched COGs (P < 0.10, Fisher's exact test) are indicated by light gray bars.

FIG. 4.

Hierarchical clustering of transcripts. The rows represent selected transcripts that were upregulated by growth on cDCE (>1.5-fold change with an FDR of 5%). The columns represent individual microarrays, and the samples in columns 1 and 3 are technical replicates that were extracted separately from aliquots of the same biological culture. The samples in columns 1 and 2 and the samples in columns 2 and 3 are biological replicate pairs. Darker gray indicates high levels of expression, while lighter gray indicates low levels. Clusters of genes with expression similar to that of the GST or CMO gene are indicated by a gray background. GLY, glycolate.

FIG. 5.

Expression of selected transcripts in cDCE-grown cells, expressed in arbitrary fluorescence units. The levels of expression of corresponding transcripts in glycolate-grown cells were low (data not shown). The following enzymes were examined: HAD 1 (Bpro5186), HAD 2 (Bpro0530), GST (Bpro0645), CMO (Bpro5565), CO DHase (Bpro0577), and Hlase (Bpro5566).

FIG. 6.

Proposed cDCE degradation pathways.