(515bv) Transcriptome Analysis Of Engineered Escherichia Coli For Aerobic Mineralization Of cis-1,2-Dichloroethylene

Metabolically engineered Escherichia coli has previously been used by us to degrade cis-1,2-dichloroethylene (cis-DCE, J Biol Chem 279:46810, 2004; Environ Microbiol 6:491, 2004). The strains express the six genes of an evolved toluene ortho-monooxygenase from Burkholderia cepacia G4 (TOM-Green, which formed a reactive cis-DCE epoxide) and either (1) γ-glutamylcysteine synthetase (which forms glutathione but is not limited by feedback inhibition) and the glutathione S-transferase IsoILR1 from Rhodococcus- AD45 (which adds glutathione to the reactive cis-DCE epoxide) or (2) with an evolved epoxide hydrolase from Agrobacterium radiobacter AD1 (EchA F108L/I219L/C248I which converts the reactive cis-DCE epoxide to a diol). Also, we quantified the impact of this metabolic engineering for bioremediation by measuring the changes in the proteome through a shotgun proteomics technique (iTRAQ) by tracking the changes due to the sequential addition of TOM-Green, the glutathione S-transferase IsoILR1, and γ-glutamylcysteine synthetase and due to adding the evolved EchA vs. the wild-type enzyme to TOM-Green (J Proteome Res 5:1388, 2006). We found that the addition of eight genes involving glutathione induced glutathione synthesis and a stress response (induction of katG, ahpF, and dps) as well as repressed fatty acid synthesis, gluconeogenesis, the tricarboxylic acid cycle, and indole synthesis (down-regulation of tnaA). The effect of adding the evolved epoxide hydrolase was not as definitive. Therefore, in this study, we used DNA microarrays to complement the proteomics study to investigate the impact of the addition of the evolved EchA to TOM-Green; hence, we studied the impact of removing the toxic epoxide from the cell. The whole transcriptome analysis shows that 87 genes are significantly induced or repressed with the addition of the evolved epoxide hydrolase EchA. Among the induced genes, many stress-related genes (yfiD, marA, nemA, ytfE, grxA, marR, ybbT, b3913, b3914, grxD, and ycfR) were most significantly induced. We found recently that YcfR is a membrane protein that regulates E. coli K-12 biofilm formation through hydrophobicity and indole-related stress response (J Bacteriol 189: 3051, 2007). Heat shock proteins (ibpA, ibpB, and htpX), 50S ribosomal proteins (rmpABGF), and several hypothetical genes (yeaR, yogA, and yeaQ) were also induced. Among the repressed genes, genes (tnaA, tnaL, and trpL) involved in the indole biosynthesis were repressed. We have also shown recently that indole is an interspecies biofilm signal (e.g., it can be manipulated by toluene ortho-monooxygenase of Burkholderia cepacia G4 to control dual-species biofilms, BMC Microbiol in revision 2007) and that the exogenous addition of indole, 5-hydroxyindole, and 7-hydroxyindole decreases biofilm formation of a pathogenic E. coli O157:H7 without inhibiting cell growth while the addition of isatin (indole-2,3-dione; i.e., doubly-hydroxylated indole) increases its biofilm formation (Appl Environ Microbiol on-line 2007). Both the whole transcriptome analysis and the proteomic approach identified oxygen-activated genes, 50S ribosomal proteins, indole-biosynthesis genes, soxR, ompX, and cysK; hence, the DNA microarray analysis complements the proteomics approach. In order to enhance cis-DCE degradation, we investigated cis-DCE degradation using an indole-deficient mutant (tnaA); our hypothesis is that the initial cis-DCE oxidation by TOM-Green will be enhanced by removing the competitive substrate indole. In addition, we studied the impact of the newly identified possible stress-related genes, yeaR, yogA, yeaQ, and ymgB on cis-DCE degradation.