(6dm) Control of the Interspecies Biofilm Signal Indole in Pathogenic E. Coli O157:H7 and Pseudomonas Aeruginosa and Proteome and Transcriptome Analysis of Engineered E. Coli for Aerobic Mineralization of Cis-1,2-Dichloroethylene

Bacteria exist in biofilms in natural, medical, and engineering environments. Due to their high resistance to antibiotics, biofilms cause serious problems to human health such as lung infections, dental disease, and urinary tract infections. Procaryotes and eucaryotes signal not only themselves but also one another; hence, there is competition and interference of cell signals. As a stationary phase signal, indole is secreted in large quantities (500 µM) into rich medium by Escherichia coli and has been shown to control several genes (e.g., astD, tnaB, gabT), multi-drug exporters, and the pathogenicity island of E. coli; however, its impact on biofilm formation has not been well-studied. Also, hydroxyindoles and indigoid compounds can be easily produced from indole in the presence of toluene o-monooxygenase (TOM) of Burkholderia cepacia G4. Through a series of global transcriptome analyses, confocal microscopy, isogenic mutants, and dual-species biofilms, we found that indole is a non-toxic signal that controls E. coli biofilms by repressing motility, inducing the sensor of the quorum sensing signal autoinducer-1 (SdiA), and influencing acid resistance (e.g., hdeABD, gadABCEX). We have manipulated biofilm formation in a dual-species biofilm of E. coli and Pseudomonas fluorescens by controlling indole concentrations (BMC Microbiol in revision 2007). Since the addition of indole significantly repressed ymgB along with the repression of other acid resistance genes (hdeABD and gadABCEX), we solved the structure of YmgB to 1.8 Å resolution, which is a biological dimer that has a critical role in acid-resistance in E. coli via indole. Also, the exogenous addition of indole, 5-hydroxyindole, and 7-hydroxyindole decreased biofilms of a pathogenic E. coli O157:H7 (EHEC) without cell growth inhibition (which suggests their use as potential therapeutics), while the addition of isatin (indole-2,3-dione) increased biofilm formation of EHEC (Appl Environ Microbiolon-line 2007). Isatin caused increased motility as a result of flagella synthesis, caused a 20-fold reduction in indole concentrations as a result of repression of tnaA, and altered AI-2 transport whereas 7-hydroxyindole inhibited biofilm formation most likely through sulfur metabolism. In the other hand, the addition of indole and 7-hydroxyindole to Pseudomonas aeruginosa PAO1 (an opportunistic human pathogen) increase its biofilm formation without cell growth inhibition although this strain does not synthesize these compounds. We also found that indole (1000 µM) induced three porin import genes of P. aeruginosa, oprH, oprF, and oprD. Indole enhanced sensitivity to the antibiotic imipenem through the amino acid and antibiotic porin protein OprD; therefore, indole increases antibiotic sensitivity by increasing import. 7-hydroxyindole (500 µM) abolished swarming motility and influenced P. aeruginosa biofilm formation by altering increasing synthesis of fimbriae and repressing some aspects of quorum sensing. Additionally, P. aeruginosa PAO1 degrades readily indole and 7HI. Since P. aeruginosa and E. coli are probably present in consortia, this study provides further evidence of the importance of interspecies communication for biofilm formation and the importance of indole.

Proteome and 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) g-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 g-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). Since the effect of adding the evolved epoxide hydrolase was not as definitive, 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. 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. 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. This study shows clearly that cloning eight genes have significantly enhanced the degradation of cis-DCE and that both the whole transcriptome analysis and the proteomic approach have helped to understand the impact of the metabolic engineering.

Publications

1. J. Lee, K. Reardon and T.K. Wood, Transcriptome analysis of Engineered Escherichia coli for Aerobic Mineralization of cis-1,2-Dichloroethylene. in preparation (2007)

2. J. Lee and T.K. Wood, Indole and 7-Hydroxyindole Enhance Pseudomonas aeruginosa Biofilm Formation and Antibiotic Sensitivity. to be submitted (2007)

3. J. Lee, R. Page, R. García-Contreras, J. Palermino X. Zhang, O. Doshi, T. K. Wood, and W, Peti, Structure and Function of the E. coli Protein YmgB: a Protein Critical for Biofilm Formation and Acid-Resistance. J. Mol. Biol. under review (2007)

4. T. Bansal, D. Englert, J. Lee, T. K. Wood, and A. Jayaraman, Epinephrine and Norepinephrine Divergently Regulate Escherichia coli O157:H7 Chemotaxis, Biofilm Formation, and Gene Expression Compared to Indole. Infect. Immun. under review (2007)

5. J. Lee, A. Jayaraman, and T. K. Wood, Indole is an Inter-Species Biofilm Signal Mediated by SdiA. BMC Microbiol. accpeted (2007)

6. J. Lee, T. Bansal, A. Jayaraman, W. Bentley, and T. K. Wood, Enterohemorrhagic Escherichia coli Biofilms Are Inhibited by 7-Hydroxyindole and Stimulated by Isatin. Appl. Environ. Microbiol. on-line (2007)

7. J. Domka, J. Lee, T. Bansal, and T. K. Wood, Temporal Gene-Expression in Escherichia coli K-12 Biofilms Environ. Microbiol. 9: 332-346 (2007).

8. J. Domka, J. Lee and T. K. Wood, YliH and YceP Regulate Escherichia coli K12 Biofilm Formation By Influencing Cell Signaling. Appl. Environ. Microbiol. 72: 2449-2459 (2006).

9. J. Lee, L. Cao, S. Y. Ow, M. E. Barrios-Llerena, W. Chen, T. K. Wood, and P. C. Wright, Proteome Changes after Metabolic Engineering to Enhance Aerobic Mineralization of cis-1,2-Dichloroethylene. J. Proteome Res. 5: 1388-1397 (2006).

10. L. Cao, J. Lee, W. Chen, T. K. Wood, Enantioconvergent Product of (R)-1-phenyl-1,2-Ethanediol From Styrene Oxide by Combining the Solanum tuberosum and an Evolved Agrobacterium radiobactor AD1 Epoxide Hydrolases. Biotechnol. Bioeng. 94: 522-529 (2006).

11. T. K. Wood, A. F. G. Barrios, M. Herzberg, J. Lee, Motility Influences Biofilm Architecture in Escherichia coli. Appl. Microbiol. Biotechnol. 72: 361-367 (2006)

12. G. Vardar, Y. Tao, J. Lee, and T. K. Wood, Alanine 101 and Alanine 110 of the Alpha Subunit of Pseudomonas stutzeri OX1 Toluene-o-Xylene Monooxygenase Influence the Regiospecific Oxidation of Aromatics. Biotechnol. Bioeng. 92: 652-658 (2005)

13. J. Lee and H. Pedersen, Stable Genetic Transformation of Eschscholzia californica Expressing Synthetic Green Fluorescent Proteins. Biotechnol. Prog. 17: 247-251, (2001)

14. J. Lee, M. H. Cho and J. Lee, Characterization of an Oxygen-Dependent Inducible Promoter System, the nar promoter, and Escherichia coli with an Inactivated nar Operon. Biotechnol. Bioeng. 52: 572-578 (1996)

15. J. Lee, M. H. Cho, E.-K. Hong, K.-S. Kim and J. Lee, Characterization of the nar promoter to Use as an Inducible Promoter. Biotechnol. Lett. 18: 129-134, (1996)