• Almasoud, Nagla
• Rattray, Nicholas
• Muhamadali, Howbeer
• Trivedi, Drupad
• Sayqal, Ali
• Xu, Yun
• Ellis, David
• Goodacre, Royston
• Webb, Carole

Abstract

Introduction<br/><br/>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.<br/><br/>Objective<br/><br/>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.<br/><br/>Methods<br/><br/>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.<br/><br/>Results<br/><br/>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.<br/><br/>Conclusion<br/><br/>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.

Topics

• density
• impedance spectroscopy
• compound
• gas chromatography
• Fourier transform infrared spectroscopy
• spectrometry
• gas chromatography-mass spectrometry