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Three efflux pumps are required to provide efficient tolerance to toluene in Pseudomonas putida DOT-T1E.

ABSTRACT: In Pseudomonas putida DOT-T1E multidrug efflux pumps of the resistance-nodulation-division family make a major contribution to solvent resistance. Two pumps have been identified: TtgABC, expressed constitutively, and TtgDEF, induced by aromatic hydrocarbons. A double mutant lacking both efflux pumps was able to survive a sudden toluene shock if and only if preinduced with small amounts of toluene supplied via the gas phase. In this article we report the identification and characterization in this strain of a third efflux pump, named TtgGHI. The ttgGHI genes form an operon that is expressed constitutively at high levels from a single promoter. In the presence of toluene the operon is expressed at an even higher level from two promoters, the constitutive one and a previously unreported one that is inducible and that partially overlaps the constitutive promoter. By site-directed mutagenesis we constructed a single ttgH mutant which was shown to be unable to survive sudden 0.3% (vol/vol) toluene shocks regardless of the preculture conditions. The mutation was transferred to single and double mutants to construct mutant strains in which two or all three pumps are knocked out. Survival analysis of induced and noninduced cells revealed that the TtgABC and TtgGHI pumps extruded toluene, styrene, m-xylene, ethylbenzene, and propylbenzene, whereas the TtgDEF pump removed only toluene and styrene. The triple mutant was hypersensitive to toluene, as shown by its inability to grow with toluene supplied via the vapor phase.

SUBMITTER: Rojas A

PROVIDER: S-EPMC95279 | biostudies-literature | 2001 Jul

REPOSITORIES: biostudies-literature

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Three efflux pumps are required to provide efficient tolerance to toluene in Pseudomonas putida DOT-T1E.

Rojas A A Duque E E Mosqueda G G Golden G G Hurtado A A Ramos J L JL Segura A A

Journal of bacteriology 20010701 13

In Pseudomonas putida DOT-T1E multidrug efflux pumps of the resistance-nodulation-division family make a major contribution to solvent resistance. Two pumps have been identified: TtgABC, expressed constitutively, and TtgDEF, induced by aromatic hydrocarbons. A double mutant lacking both efflux pumps was able to survive a sudden toluene shock if and only if preinduced with small amounts of toluene supplied via the gas phase. In this article we report the identification and characterization in thi ...[more]

PMID: 11395460

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Project description:The basic mechanisms underlying solvent tolerance in Pseudomonas putida DOT-T1E are efflux pumps that remove the solvent from bacterial cell membranes. The solvent-tolerant P. putida DOT-T1E grows in the presence of high concentrations (e.g., 1% [vol/vol]) of toluene and octanol. Growth of P. putida DOT-T1E cells in LB in the presence of toluene supplied via the gas phase has a clear effect on cell survival: the sudden addition of 0.3% (vol/vol) toluene to P. putida DOT-T1E pregrown with toluene in the gas phase resulted in survival of almost 100% of the initial cell number, whereas only 0.01% of cells pregrown in the absence of toluene tolerated exposure to this aromatic hydrocarbon. One class of toluene-sensitive octanol-tolerant mutant was isolated after Tn5-'phoA mutagenesis of wild-type P. putida DOT-T1E cells. The mutant, called P. putida DOT-T1E-18, was extremely sensitive to 0.3% (vol/vol) toluene added when cells were pregrown in the absence of toluene, whereas pregrowth on toluene supplied via the gas phase resulted in survival of about 0.0001% of the initial number. Solvent exclusion was tested with 1,2,4-[14C]trichlorobenzene. The levels of radiochemical accumulated in wild-type cells grown in the absence and in the presence of toluene were not significantly different. In contrast, the mutant was unable to remove 1,2,4-[14C]trichlorobenzene from the cell membranes when grown on Luria-Bertani (LB) medium but was able to remove the aromatic compound when pregrown on LB medium with toluene supplied via the gas phase. The amount of 14C-labeled substrate in whole cells increased in competition assays in which toluene-and xylenes were the unlabeled competitors, whereas this was not the case when benzene was the competitor. This finding suggests that the exclusion system works specifically with certain aromatic substrates. The mutation in P. putida DOT-T1E-18 was cloned, and the knockedout gene was sequenced and found to be homologous to the drug exclusion gene mexB, which belongs to the efflux pump family of the resistant nodulator division type.

Project description:Pseudomonas putida DOT-T1E was used as a model to develop a "phenomics" platform to investigate the ability of P. putida to grow using different carbon, nitrogen, and sulfur sources and in the presence of stress molecules. Results for growth of wild-type DOT-T1E on 90 different carbon sources revealed the existence of a number of previously uncharted catabolic pathways for compounds such as salicylate, quinate, phenylethanol, gallate, and hexanoate, among others. Subsequent screening on the subset of compounds on which wild-type DOT-TIE could grow with four knockout strains in the global regulatory genes Deltacrc, Deltacrp, DeltacyoB, and DeltaptsN allowed analysis of the global response to nutrient supply and stress. The data revealed that most global regulator mutants could grow in a wide variety of substrates, indicating that metabolic fluxes are physiologically balanced. It was found that the Crc mutant did not differ much from the wild-type regarding the use of carbon sources. However, certain pathways are under the preferential control of one global regulator, i.e., metabolism of succinate and d-fructose is influenced by CyoB, and l-arginine is influenced by PtsN. Other pathways can be influenced by more than one global regulator; i.e., l-valine catabolism can be influenced by CyoB and Crp (cyclic AMP receptor protein) while phenylethylamine is affected by Crp, CyoB, and PtsN. These results emphasize the cross talk required in order to ensure proper growth and survival. With respect to N sources, DOT-T1E can use a wide variety of inorganic and organic nitrogen sources. As with the carbon sources, more than one global regulator affected growth with some nitrogen sources; for instance, growth with nucleotides, dipeptides, d-amino acids, and ethanolamine is influenced by Crp, CyoB, and PtsN. A surprising finding was that the Crp mutant was unable to flourish on ammonium. Results for assayed sulfur sources revealed that CyoB controls multiple points in methionine/cysteine catabolism while PtsN and Crc are needed for N-acetyl-l-cysteamine utilization. Growth of global regulator mutants was also influenced by stressors of different types (antibiotics, oxidative agents, and metals). Overall and in combination with results for growth in the presence of various stressors, these phenomics assays provide multifaceted insights into the complex decision-making process involved in nutrient supply, optimization, and survival.

Project description:An exceptionally interesting stress response of Pseudomonas putida strains to toxic substances is the induction of efflux pumps that remove toxic chemical substances from the bacterial cell out to the external environment. To exploit these microorganisms to their full potential a deeper understanding of the interactions between the bacteria and organic solvents is required. Thus, this study focuses on investigation of metabolic changes in P. putida upon exposure to toluene.Investigate observable metabolic alterations during interactions of three strains of P. putida (DOT-T1E, and its mutants DOT-T1E-PS28 and DOT-T1E-18) with the aromatic hydrocarbon toluene.The growth profiles were measured by taking optical density (OD) measurement at 660 nm (OD660) at various time points during incubation. For fingerprinting analysis, Fourier-transform infrared (FT-IR) spectroscopy was used to investigate any phenotypic changes resulting from exposure to toluene. Metabolic profiling analysis was performed using gas chromatography-mass spectrometry (GC-MS). Principal component-discriminant function analysis (PC-DFA) was applied to the FT-IR data while multiblock principal component analysis (MB-PCA) and N-way analysis of variance (N-way ANOVA) were applied to the GC-MS data.The growth profiles demonstrated the effect of toluene on bacterial cultures and the results suggest that the mutant P. putida DOT-T1E-18 was more sensitive (significantly affected) to toluene compared to the other two strains. PC-DFA on FT-IR data demonstrated the differentiation between different conditions of toluene on bacterial cells, which indicated phenotypic changes associated with the presence of the solvent within the cell. Fifteen metabolites associated with this phenotypic change, in P. putida due to exposure to solvent, were from central metabolic pathways. Investigation of MB-PCA loading plots and N-way ANOVA for condition | strain × time blocking (dosage of toluene) suggested ornithine as the most significant compound that increased upon solvent exposure.The combination of metabolic fingerprinting and profiling with suitable multivariate analysis revealed some interesting leads for understanding the mechanism of Pseudomonas strains response to organic solvent exposure.

Dataset Information

Three efflux pumps are required to provide efficient tolerance to toluene in Pseudomonas putida DOT-T1E.

Publications

Three efflux pumps are required to provide efficient tolerance to toluene in Pseudomonas putida DOT-T1E.

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