Abstract
Acriflavine resistance regulator (AcrR), a local transcription factor, regulates the expression of the acrRAB genes associated with the AcrAB-TolC multidrug efflux pump. Screening of organic solvent tolerance (OST) with the overexpression of 13 genes in Escherichia coli revealed that the overexpression of acrR improved OST. Overexpression of AcrR in a background strain of wild-type E. coli and in the OST strain LMB015 (ΔfadR ΔmarR; acrR + and ΔfadR ΔmarR acrR + strain, respectively) significantly increased cell growth in the presence of n-hexane/cyclohexane, which attenuated the membrane reduction capacity of the wild-type strain below 50 % of the control level. This was recovered to control levels in the acrR + strain. Quantitative real-time PCR analysis of RNA from the wild-type, ΔacrR, and acrR + strains showed that AcrR represses the transcription of marRAB and soxRS, and its own gene cluster, acrRAB. Electrophoretic mobility shift assay demonstrated that AcrR binds directly to the promoter region of acrRAB, marAB, and soxRS, indicating that AcrR acts on global regulators to affect mar-sox-rob regulon. In the acrR + strain, soxS expression was significantly upregulated compared with the wild-type. The OST of the acrR + strain was completely lost in the ΔsoxS acrR + strain, indicating that SoxS mediated OST improvement in the acrR + strain. The observation that all genes associated with marRAB and soxRS are upregulated in the ΔacrR strain, and that there is only moderate induction of soxS (and marB) in the acrR + strain, provides insight into how acrR overexpression confers bacterial OST and the mar-sox-rob regulon control network.
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References
Alekshun MN, Levy SB (1997) Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon. Antimicrob Agents Chemother 41:2067–2075
Alekshun MN, Levy SB (1999) The mar regulon: multiple resistance to antibiotics and other toxic chemicals. Trends Microbiol 7:410–413
Asako H, Nakajima H, Kobayashi K, Kobayashi M, Aono R (1997) Organic solvent tolerance and antibiotic resistance increased by overexpression of marA in Escherichia coli. Appl Environ Microbiol 63:1428–1433
Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:2006–2008
Berridge MV, Herst PM, Tan AS (2005) Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev 11:127–152
Blanchard JL, Wholey WY, Conlon EM, Pomposiello PJ (2007) Rapid changes in gene expression dynamics in response to superoxide reveal SoxRS-dependent and independent transcriptional networks. PLoS One 2:e1186
Boch J, Kempf B, Schmid R, Bremer E (1996) Synthesis of the osmoprotectant glycine betaine in Bacillus subtilis: characterization of the gbsAB genes. J Bacteriol 178:5121–5129
Campbell JW, Cronan JE Jr (2001) Escherichia coli FadR positively regulates transcription of the fabB fatty acid biosynthetic gene. J Bacteriol 183:5982–5990
Campbell JW, Cronan JE Jr (2002) The enigmatic Escherichia coli fadE gene is yafH. J Bacteriol 184:3759–3764
Cánovas D, Vargas C, Kneip S, Morón MJ, Ventosa A, Bremer E, Nieto JJ (2000) Genes for the synthesis of the osmoprotectant glycine betaine from choline in the moderately halophilic bacterium Halomonas elongata DSM 3043, USA. Microbiology 146:455–463
Choi YJ, Lee SY (2013) Microbial production of short-chain alkanes. Nature 502:571–574
Chubiz LM, Glekas GD, Rao CV (2012) Transcriptional cross talk within the mar-sox-rob regulon in Escherichia coli is limited to the rob and marRAB operons. J Bacteriol 194:4867–4875
Cohen SP, Hächler H, Levy SB (1993) Genetic and functional analysis of the multiple antibiotic resistance (mar) locus in Escherichia coli. J Bacteriol 175:1484–1492
Cronan JE Jr, Subrahmanyam S (1998) FadR, transcriptional co-ordination of metabolic expediency. Mol Microbiol 29:937–943
Demple B (1991) Regulation of bacterial oxidative stress genes. Annu Rev Genet 25:315–337
Demple B (1996) Redox signaling and gene control in the Escherichia coli soxRS oxidative stress regulon—a review. Gene 179:53–57
Doukyu N, Ishikawa K, Watanabe R, Ogino H (2012) Improvement in organic solvent tolerance by double disruptions of proV and marR genes in Escherichia coli. J Appl Micobiol 112:464–474
Foo JL, Leong SS (2013) Directed evolution of an E. coli inner membrane transporter for improved efflux of biofuel molecules. Biotechnol Biofuels 6:81
Goodarzi H, Bennett BD, Amini S, Reaves ML, Hottes AK, Rabinowitz JD, Tavazoie S (2010) Regulatory and metabolic rewiring during laboratory evolution of ethanol tolerance in E. coli. Mol Syst Biol 6:1–12
Greenberg JT, Demple B (1989) A global response induced in Escherichia coli by redox-cycling agents overlaps with that induced by peroxide stress. J Bacteriol 171:3933–3939
Greenberg JT, Monach P, Chou JH, Josephy PD, Demple B (1990) Positive control of a global antioxidant defense regulon activated by superoxide-generating agents in Escherichia coli. Proc Natl Acad Sci 87:6181–6185
Gu M, Imlay JA (2011) The SoxRS response of Escherichia coli is directly activated by redox‐cycling drugs rather than by superoxide. Mol Microbiol 79:1136–1150
Gu R, Li M, Su CC, Long F, Routh MD, Yang F, McDermott G, Yu EW (2008) Conformational change of the AcrR regulator reveals a possible mechanism of induction. Acta Crystallogr Sect F: Struct Biol Cryst Commun 64:584–588
Hatti-Kaul R, Törnvall U, Gustafsson L, Börjesson P (2007) Industrial biotechnology for the production of bio-based chemicals—a cradle-to-grave perspective. Trends Biotechnol 25:119–124
Heipieper HJ, Neumann G, Cornelissen S, Meinhardt F (2007) Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems. Appl Microbiol Biotechnol 74:961–973
Hidalgo E, Demple B (1996) Adaptive responses to oxidative stress: the soxRS and oxyR regulons. In: Regulation of gene expression in Escherichia coli. Springer US, pp 435–452
Howard TP, Middelhaufe S, Moore K, Edner C, Kolak DM, Taylor GN, Parker DA, Lee R, Smirnoff N, Aves SJ, Love J (2013) Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli. Proc Natl Acad Sci 110:7636–7641
Kaprelyants AS, Kell DB (1993) Dormancy in stationary-phase cultures of Micrococcus luteus: flow cytometric analysis of starvation and resuscitation. Appl Environ Microbiol 59:3187–3196
Kwon HJ, Bennik MH, Demple B, Ellenberger T (2000) Crystal structure of the Escherichia coli Rob transcription factor in complex with DNA. Nat Struct Biol 7:424–430
Li Z, Demple B (1994) SoxS, an activator of superoxide stress genes in Escherichia coli. Purification and interaction with DNA. J Biol Chem 269:18371–18377
Luo LH, Seo PS, Seo JW, Heo SY, Kim DH, Kim CH (2009) Improved ethanol tolerance in Escherichia coli by changing the cellular fatty acids composition through genetic manipulation. Biotechnol Lett 31:1867–1871
Ma D, Cook DN, Alberti M, Pon NG, Nikaido H, Hearst JE (1995) Genes acrA and acrB encode a stress‐induced efflux system of Escherichia coli. Mol Microbiol 16:45–55
Ma D, Alberti M, Lynch C, Nikaido H, Hearst JE (1996) The local repressor AcrR plays a modulating role in the regulation of acrAB genes of Escherichia coli by global stress signals. Mol Microbiol 19:101–112
Martin RG, Jair KW, Wolf RE, Rosner JL (1996) Autoactivation of the marRAB multiple antibiotic resistance operon by the MarA transcriptional activator in Escherichia coli. J Bacteriol 178:2216–2223
Oh HY, Lee JO, Kim OB (2012) Increase of organic solvent tolerance of Escherichia coli by the deletion of two regulator genes, fadR and marR. Appl Micobiol Biotechnol 96:1619–1627
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45–e45
Pomposiello PJ, Bennik MH, Demple B (2001) Genome-wide transcriptional profiling of the Escherichia coli responses to superoxide stress and sodium salicylate. J Bacteriol 183:3890–3902
Ramos JL, Duque E, Gallegos MT, Godoy P, Ramos-González MI, Rojas A, Terán W, Segura A (2002) Mechanisms of solvent tolerance in gram-negative bacteria. Annu Rev Microbiol 56:743–768
Ramos JL, Martínez-Bueno M, Molina-Henares AJ, Terán W, Watanabe K, Zhang X, Gallegos MT, Brennan R, Tobes R (2005) The TetR family of transcriptional repressors. Microbiol Mol Biol Rev 69:326–356
Rock K, Korpelshoek M (2007) Bioethers impact on the gasoline pool. Pet Technol Q 12:9
Rodionov DA, Gelfand MS, Mironov AA, Rakhmaninova AB (2001) Comparative approach to analysis of regulation in complete genomes: multidrug resistance systems in gamma-proteobacteria. J Mol Microbiol Biotechnol 3:319–324
Ruiz C, Levy SB (2010) Many chromosomal genes modulate MarA-mediated multidrug resistance in Escherichia coli. Antimicrob Agents Chemother 54:2125–2134
Saka K, Tadenuma M, Nakade S, Tanaka N, Sugawara H, Nishikawa K, Ichiyoshi N, Kitagawa M, Mori H, Ogasawara N, Nishimura A (2005) A complete set of Escherichia coli open reading frames in mobile plasmids facilitating genetic studies. DNA Res 12:63–68
Shah IM, Wolf RE (2006) Inhibition of Lon‐dependent degradation of the Escherichia coli transcription activator SoxS by interaction with ‘soxbox’ DNA or RNA polymerase. Mol Microbiol 60:199–208
Sikkema J, de Bont JA, Poolman B (1995) Mechanisms of membrane toxicity of hydrocarbons. Microbiol Rev 59:201–222
Su CC, Rutherford DJ, Yu EW (2007) Characterization of the multidrug efflux regulator AcrR from Escherichia coli. Biochem Biophys Res Commun 361:85–90
Tsaneva IR, Weiss B (1990) soxR, a locus governing a superoxide response regulon in Escherichia coli K-12. J Bacteriol 172:4197–4205
Tsukagoshi N, Aono R (2000) Entry into and release of solvents by Escherichia coli in an organic-aqueous two-liquid-phase system and substrate specificity of the AcrAB-TolC solvent-extruding pump. J Bacteriol 182:4803–4810
Tsukatani T, Suenaga H, Higuchi T, Akao T, Ishiyama M, Ezoe K, Matsumoto K (2008) Colorimetric cell proliferation assay for microorganisms in microtiter plate using water-soluble tetrazolium salts. J Microbiol Methods 75:109–116
Vinué L, McMurry LM, Levy SB (2013) The 216‐bp marB gene of the marRAB operon in Escherichia coli encodes a periplasmic protein which reduces the transcription rate of marA. FEMS Microbiol Lett 345:49–55
Vollmer W, Bertsche U (2008) Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli. Biochim Biophys Acta 1778:1714–1734
Watanabe R, Doukyu N (2012) Contributions of mutations in acrR and marR genes to organic solvent tolerance in Escherichia coli. AMB Express 2:58
Watanabe R, Doukyu N (2014) Improvement of organic solvent tolerance by disruption of the lon gene in Escherichia coli. J Biosci Bioeng 1–6
White DG, Goldman JD, Demple B, Levy SB (1997) Role of the acrAB locus in organic solvent tolerance mediated by expression of marA, soxS, or robA in Escherichia coli. J Bacteriol 179:6122–6126
Wu J, Weiss B (1991) Two divergently transcribed genes, soxR and soxS, control a superoxide response regulon of Escherichia coli. J Bacteriol 173:2864–2871
Wu J, Weiss B (1992) Two-stage induction of the soxRS (superoxide response) regulon of Escherichia coli. J Bacteriol 174:3915–3920
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This work was supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Education, Science and Technology (ABC-2013-059635).
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Lee, J.O., Cho, KS. & Kim, O.B. Overproduction of AcrR increases organic solvent tolerance mediated by modulation of SoxS regulon in Escherichia coli . Appl Microbiol Biotechnol 98, 8763–8773 (2014). https://doi.org/10.1007/s00253-014-6024-9
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DOI: https://doi.org/10.1007/s00253-014-6024-9