The impact of ColRS two-component system and TtgABC efflux pump on phenol tolerance of Pseudomonas putida becomes evident only in growing bacteria
Tóm tắt
We have recently found that Pseudomonas putida deficient in ColRS two-component system is sensitive to phenol and displays a serious defect on solid glucose medium where subpopulation of bacteria lyses. The latter phenotype is significantly enhanced by the presence of phenol in growth medium. Here, we focused on identification of factors affecting phenol tolerance of the colR-deficient P. putida. By using transposon mutagenesis approach we identified a set of phenol-tolerant derivatives of colR-deficient strain. Surprisingly, half of independent phenol tolerant clones possessed miniTn5 insertion in the ttgABC operon. However, though inactivation of TtgABC efflux pump significantly enhanced phenol tolerance, it did not affect phenol-enhanced autolysis of the colR mutant on glucose medium indicating that phenol- and glucose-caused stresses experienced by the colR-deficient P. putida are not coupled. Inactivation of TtgABC pump significantly increased the phenol tolerance of the wild-type P. putida as well. Comparison of phenol tolerance of growing versus starving bacteria revealed that both ColRS and TtgABC systems affect phenol tolerance only under growth conditions and not under starvation. Flow cytometry analysis showed that phenol strongly inhibited cell division and to some extent also caused cell membrane permeabilization to propidium iodide. Single cell analysis of populations of the ttgC- and colRttgC- deficient strains revealed that their membrane permeabilization by phenol resembles that of the wild-type and the colR mutant, respectively. However, cell division of P. putida with inactivated TtgABC pump seemed to be less sensitive to phenol than that of the parental strain. At the same time, cell division appeared to be more inhibited in the colR-mutant strain than in the wild-type P. putida. ColRS signal system and TtgABC efflux pump are involved in the phenol tolerance of P. putida. However, as they affect phenol tolerance of growing bacteria only, this indicates that they participate in the regulation of processes which are active during the growth and/or cell division. Single cell analysis data indicated that the cell division step of cell cycle is particularly sensitive to the toxic effect of phenol and its inhibition can be considered as an adaptive response under conditions of phenol stress.
Tài liệu tham khảo
Dominguez-Cuevas P, Gonzalez-Pastor JE, Marques S, Ramos JL, de Lorenzo V: Transcriptional Tradeoff between Metabolic and Stress-response Programs in Pseudomonas putida KT2440 Cells Exposed to Toluene. J Biol Chem. 2006, 281 (17): 11981-11991. 10.1074/jbc.M509848200.
Ramos JL, Duque E, Gallegos MT, Godoy P, Ramos-Gonzalez MI, Rojas A, Teran W, Segura A: Mechanisms of solvent tolerance in gram-negative bacteria. Annu Rev Microbiol. 2002, 56: 743-768. 10.1146/annurev.micro.56.012302.161038.
Sikkema J, de Bont JA, Poolman B: Mechanisms of membrane toxicity of hydrocarbons. Microbiol Rev. 1995, 59 (2): 201-222.
Hallsworth JE, Heim S, Timmis KN: Chaotropic solutes cause water stress in Pseudomonas putida. Environ Microbiol. 2003, 5 (12): 1270-1280. 10.1111/j.1462-2920.2003.00478.x.
Wery J, de Bont JAM: Solvent-tolerance of Pseudomonads: a new degree of freedom in biocatalysis. Pseudomonas: Biosynthesis of macromolecules and molecular metabolism. Edited by: Ramos JL. 2004, New York: Kluwer Academic/Plenum Publishers, 3: 609-634.
Hoch JA, Varughese KI: Keeping signals straight in phosphorelay signal transduction. J Bacteriol. 2001, 183 (17): 4941-4949. 10.1128/JB.183.17.4941-4949.2001.
Dekkers LC, Bloemendaal CJ, de Weger LA, Wijffelman CA, Spaink HP, Lugtenberg BJ: A two-component system plays an important role in the root-colonizing ability of Pseudomonas fluorescens strain WCS365. Mol Plant Microbe Interact. 1998, 11 (1): 45-56. 10.1094/MPMI.1998.11.1.45.
Kivistik PA, Putrinš M, Püvi K, Ilves H, Kivisaar M, Hõrak R: The ColRS two-component system regulates membrane functions and protects Pseudomonas putida against phenol. J Bacteriol. 2006, 188 (23): 8109-8117. 10.1128/JB.01262-06.
Hõrak R, Ilves H, Pruunsild P, Kuljus M, Kivisaar M: The ColR-ColS two-component signal transduction system is involved in regulation of Tn4652 transposition in Pseudomonas putida under starvation conditions. Mol Microbiol. 2004, 54 (3): 795-807. 10.1111/j.1365-2958.2004.04311.x.
Putrinš M, Ilves H, Kivisaar M, Hõrak R: ColRS two-component system prevents lysis of subpopulation of glucose-grown Pseudomonas putida. Environ Microbiol. 2008, 10 (10): 2886-2893. 10.1111/j.1462-2920.2008.01705.x.
de Weert S, Dekkers LC, Bitter W, Tuinman S, Wijfjes AH, van Boxtel R, Lugtenberg BJ: The two-component colR/S system of Pseudomonas fluorescens WCS365 plays a role in rhizosphere competence through maintaining the structure and function of the outer membrane. FEMS Microbiol Ecol. 2006, 58 (2): 205-213. 10.1111/j.1574-6941.2006.00158.x.
Kivistik PA, Kivi R, Kivisaar M, Hõrak R: Identification of ColR binding consensus and prediction of regulon of ColRS two-component system. BMC molecular biology. 2009, 10: 46-10.1186/1471-2199-10-46.
Bayley SA, Duggleby CJ, Worsey MJ, Williams PA, Hardy KG, Broda P: Two modes of loss of the Tol function from Pseudomonas putida mt-2. Mol Gen Genet. 1977, 154 (2): 203-204. 10.1007/BF00330838.
Nelson KE, Weinel C, Paulsen IT, Dodson RJ, Hilbert H, Martins dos Santos VA, Fouts DE, Gill SR, Pop M, Holmes M: Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol. 2002, 4 (12): 799-808. 10.1046/j.1462-2920.2002.00366.x.
Carter P, Bedouelle H, Winter G: Improved oligonucleotide site-directed mutagenesis using M13 vectors. Nucleic Acids Res. 1985, 13 (12): 4431-4443. 10.1093/nar/13.12.4431.
Herrero M, de Lorenzo V, Timmis KN: Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol. 1990, 172 (11): 6557-6567.
Boyer HW, Roulland-Dussoix D: A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969, 41 (3): 459-472. 10.1016/0022-2836(69)90288-5.
Figurski DH, Helinski DR: Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA. 1979, 76 (4): 1648-1652. 10.1073/pnas.76.4.1648.
Miller JH: A short course in bacterial genetics: a laboratory manual and handbook for Echerichia coli and related bacteria. 1992, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY
Adams MH: Bacteriophages. 1959, Intersciensce Publishers Inc., NY
Wilson KJ, Sessitsch A, Corbo JC, Giller KE, Akkermans AD, Jefferson RA: beta-Glucuronidase (GUS) transposons for ecological and genetic studies of rhizobia and other gram-negative bacteria. Microbiology. 1995, 141 (Pt 7): 1691-1705. 10.1099/13500872-141-7-1691.
O'Toole GA, Kolter R: Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol Microbiol. 1998, 28 (3): 449-461. 10.1046/j.1365-2958.1998.00797.x.
de Lorenzo V, Herrero M, Jakubzik U, Timmis KN: Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol. 1990, 172 (11): 6568-6572.
Pavel H, Forsman M, Shingler V: An aromatic effector specificity mutant of the transcriptional regulator DmpR overcomes the growth constraints of Pseudomonas sp. strain CF600 on para-substituted methylphenols. J Bacteriol. 1994, 176 (24): 7550-7557.
Hõrak R, Kivisaar M: Expression of the transposase gene tnpA of Tn4652 is positively affected by integration host factor. J Bacteriol. 1998, 180 (11): 2822-2829.
Tover A, Ojangu EL, Kivisaar M: Growth medium composition-determined regulatory mechanisms are superimposed on CatR-mediated transcription from the pheBA and catBCA promoters in Pseudomonas putida. Microbiology. 2001, 147 (Pt 8): 2149-2156.
Stocks SM: Mechanism and use of the commercially available viability stain, BacLight. Cytometry A. 2004, 61 (2): 189-195. 10.1002/cyto.a.20069.
Rojas A, Duque E, Mosqueda G, Golden G, Hurtado A, Ramos JL, Segura A: Three efflux pumps are required to provide efficient tolerance to toluene in Pseudomonas putida DOT-T1E. J Bacteriol. 2001, 183 (13): 3967-3973. 10.1128/JB.183.13.3967-3973.2001.
Duque E, Segura A, Mosqueda G, Ramos JL: Global and cognate regulators control the expression of the organic solvent efflux pumps TtgABC and TtgDEF of Pseudomonas putida. Mol Microbiol. 2001, 39 (4): 1100-1106. 10.1046/j.1365-2958.2001.02310.x.
Teran W, Felipe A, Segura A, Rojas A, Ramos JL, Gallegos MT: Antibiotic-dependent induction of Pseudomonas putida DOT-T1E TtgABC efflux pump is mediated by the drug binding repressor TtgR. Antimicrob Agents Chemother. 2003, 47 (10): 3067-3072. 10.1128/AAC.47.10.3067-3072.2003.
Teran W, Krell T, Ramos JL, Gallegos MT: Effector-Repressor Interactions, Binding of a Single Effector Molecule to the Operator-bound TtgR Homodimer Mediates Derepression. J Biol Chem. 2006, 281 (11): 7102-7109. 10.1074/jbc.M511095200.
Santos PM, Benndorf D, Sa-Correia I: Insights into Pseudomonas putida KT2440 response to phenol-induced stress by quantitative proteomics. Proteomics. 2004, 4 (9): 2640-2652. 10.1002/pmic.200300793.
Santos PM, Roma V, Benndorf D, von Bergen M, Harms H, Sa-Correia I: Mechanistic insights into the global response to phenol in the phenol-biodegrading strain Pseudomonas sp. M1 revealed by quantitative proteomics. Omics. 2007, 11 (3): 233-251. 10.1089/omi.2007.0009.
Heipieper HJ, de Bont JA: Adaptation of Pseudomonas putida S12 to ethanol and toluene at the level of fatty acid composition of membranes. Appl Environ Microbiol. 1994, 60 (12): 4440-4444.
Denich TJ, Beaudette LA, Lee H, Trevors JT: Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes. Journal of microbiological methods. 2003, 52 (2): 149-182. 10.1016/S0167-7012(02)00155-0.
Neumann G, Veeranagouda Y, Karegoudar TB, Sahin O, Mausezahl I, Kabelitz N, Kappelmeyer U, Heipieper HJ: Cells of Pseudomonas putida and Enterobacter sp. adapt to toxic organic compounds by increasing their size. Extremophiles. 2005, 9 (2): 163-168. 10.1007/s00792-005-0431-x.
Ramos JL, Duque E, Godoy P, Segura A: Efflux pumps involved in toluene tolerance in Pseudomonas putida DOT-T1E. J Bacteriol. 1998, 180 (13): 3323-3329.
Pearson JP, Van Delden C, Iglewski BH: Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals. J Bacteriol. 1999, 181 (4): 1203-1210.
Yang S, Lopez CR, Zechiedrich EL: Quorum sensing and multidrug transporters in Escherichia coli. Proc Natl Acad Sci USA. 2006, 103 (7): 2386-2391. 10.1073/pnas.0502890102.
Heipieper HJ, Keweloh H, Rehm HJ: Influence of phenols on growth and membrane permeability of free and immobilized Escherichia coli. Appl Environ Microbiol. 1991, 57 (4): 1213-1217.