Mechanisms for Solvent Tolerance in Bacteria

Abstract

The development of tolerance in Pseudomonas putida DOT-T1 to toluene and related highly toxic compounds involves short-and long-term responses. The short-term response is based on an increase in the rigidity of the cell membrane by rapid transformation of the fatty acid cis-9,10-methylene hexadecanoic acid (C17:cyclopropane) to unsaturated 9-cis-hexadecenoic acid (C16:1,9 cis) and subsequent transformation to the trans isomer. The long-term response involves in addition to the changes in fatty acids, alterations in the level of the phospholipid polar head groups: cardiolipin increases and phosphatidylethanolamine decreases. The two alterations lead to increased cell membrane rigidity and should be regarded as physical mechanisms that prevent solvent penetrance. Biochemical mechanisms that decrease the concentration of toluene in the cell membrane also take place and involve: (i) a solvent exclusion system and (ii) metabolic removal of toluene via oxidation. Mutants unable to carry out cis 3 trans isomerization of unsaturated lipids, that exhibit altered cell envelopes because of the lack of the OprL protein, or that are unable to exclude toluene from cell membranes are hypersensitive to toluene.Organic solvents with a logP OW value (logarithm of the partition coefficient of the target compound in a mixture of octanol/ water) between 1.5 and 3 are extremely toxic to microorganisms, a characteristic that has been well documented for toluene (logP OW 2.5) (1-4). De Smet et al. (2) demonstrated that toluene destabilizes the inner membrane of Gram-negative bacteria, causing a transition from a lamellar bilayer state to a hexagonal state, which results in the leakage of proteins, lipids, and ions and disruption of the cell membrane potential (1, 2). The consequent collapse of ATP synthesis together with other lesions lead to cell death.Inoue and Horikoshi (5) isolated a Pseudomonas putida strain able to grow in a double phase system that contained up to 50% (v/v) toluene, despite the fact that this microorganism was not able to use this aromatic as a carbon source. This report was followed by three independent studies that described the isolation of three different P. putida strains that tolerated related organic solvents, e.g. styrene (6), xylenes (7), and toluene (8). The toluene-tolerant isolate, called P. putida DOT-T1, metabolized toluene via the p-cresol pathway (8). The "unexpected" ability of these Pseudomonas strains to tolerate toxic solvents opens new avenues of research into cellular metabolism. In this study, we have explored the molecular basis for solvent tolerance by P. putida DOT-T1. EXPERIMENTAL PROCEDURESBacterial Strains and Culture Conditions-P. putida DOT-T1 is a solvent-tolerant strain (8), whereas P. putida mt-2 is a toluene-sensitive strain (9).Isolation of Toluene-sensitive Tn5 Mutants of P. putida DOT-T1-About 2000 Tn5 transconjugants of P. putida DOT-T1 were obtained after mating this strain with Escherichia coli (pGS9). The suicide plasmid pGS9 bears Tn5, and mutagenesis was carried out a...

TrgI, toluene repressed gene I, a novel gene involved in toluene-tolerance in Pseudomonas putida S12

“…In each case, different flagella genes were found to influence solvent tolerance. Our findings confirm the existence of a link between flagella and solvent tolerance, but its nature remains to be elucidated and may be found at the regulatory level Ramos et al 2002). However, a direct relationship between flagellar systems and toluene tolerance could exist if the flagellar export system is able to export toluene molecules as well.…”

“…Three types of membrane-associated solvent tolerance mechanisms have been elucidated (Heipieper and de Bont 1994;Weber and de Bont 1996;Ramos et al 1997;Isken and de Bont 1998;Kieboom and de Bont 2000;Ramos et al 2002): (1) cytoplasmic membrane changes, (2) outer membrane changes, and (3) active efflux of organic solvents. All three modes of solvent tolerance have been found in solvent-tolerant Pseudomonads, but it is likely that additional mechanisms exist.…”

“…The solvent-tolerant bacterium Pseudomonas putida S12 is quite exceptional in its ability to withstand a wide range of toxic organic solvents such as toluene and benzene, in concentrations that are lethal to most microorganisms (Isken and de Bont 1998;Kieboom and de Bont 2000;Ramos et al 2002). Organic solvents accumulate in lipid membranes of living cells, causing an increase of the membrane fluidity and a decrease of bilayer stability (Sikkema et al 1995;Weber and de Bont 1996).…”

YLR099C (ICT1) Encodes a Soluble Acyl-CoA-dependent Lysophosphatidic Acid Acyltransferase Responsible for Enhanced Phospholipid Synthesis on Organic Solvent Stress in Saccharomyces cerevisiae

“…For example in Pseudomonas putida DOT-T1, the conversion of cis-9,10-methylene hexadecanoic acid to unsaturated cis-9-hexadecenoic acid was observed on exposure to toluene. However, the mechanism for the conversion is unknown (5). Change in the phospholipid content was shown to be an important factor in providing tolerance against the organic solvents.…”

Engineering Complex Phenotypes in Industrial Strains

“…Recently, a similar approach was used to identify solvent-tolerant genes in Clostridium acetobutylicum. Mechanisms for solvent tolerance include alteration of membrane-lipid headgroups, overexpression of stress proteins such as groESL, and active pumping out of the solvent molecules (84)(85)(86)(87). In the case of butanol toxicity in C. acetobutylicum, overexpression of groESL and knocking out of butyrate kinase resulted in increased butanol production, suggesting the complexity of the phenotype (88,89).…”