Multiple responses of Gram‐negative bacteria to organic solvents

Since the Industrial Revolution, the production and use of chemicals has increased immensely. As a consequence, all kinds of wastes are produced, which are released into the environment. Many products such as herbicides or insecticides will be released into the environment to control weeds and pests; other products such as organic solvents or fuels will reach the biosphere as a result of production or storage losses, accidents and solvent evaporation. Nowadays, there is a growing awareness concerning the possible toxic or even carcinogenic effects of chemicals. Although the release of many of them is restricted by legislation, a number of pollutants have already reached the biosphere and need to be eliminated. The use of biological treatments for the removal of toxic chemicals seems to be promising (Ramos et al., 1994). However, chemical toxicity can hamper the application of microorganisms in the removal of pollutants from waste streams and dump sites. This is a serious problem when dealing with microbial bioremediation in reactors, biofilters and soils. The main function of the cell membrane of microorganisms is to form a permeability barrier, regulating the passage of solutes between the cell and the external environment (Nikaido, 1999). The barrier properties of the cytoplasmic membrane are of special importance for the energy transduction of the cell (Sikkema et al., 1995). The major damage caused by organic solvents on the cell membrane is the impairment of vital functions, e.g. loss of ions, metabolites, lipids and proteins, the dissipation of the pH gradient and electrical potential or the inhibition of membrane protein functions. This is often followed, in turn, by cell lysis and death (de Smet et al., 1978; Sikkema et al., 1995). The logarithm of the partitioning coefficient of a solvent in a defined octanol–water mixture (log Pow) is commonly used as a measure of the lipophilicity of a solvent (Rekker and de Kort, 1979). Aromatic solvents with a log Pow below 4.0, e.g. benzene (log Pow 2.0), styrene (log Pow 3.6), xylene (log Pow 3.2) and toluene (log Pow 2.5), accumulate in the cytoplasmic membrane of bacteria, causing disorganization of the cell membrane structure and impairment of the above-cited vital membrane functions (Sikkema et al., 1992; 1994). Nevertheless, several Pseudomonas species have been isolated that are able to grow on rich and minimal medium in the presence of high concentrations of toxic organic solvents, such as toluene, styrene and p-xylene (Inoue and Horikoshi, 1989; Cruden et al., 1992; Weber et al., 1994; Ramos et al., 1995). Recently, it has been shown that P. putida Idaho and P. putida DOT-T1 are not only resistant to toluene in a two-phase system, but can even use toluene at these high concentrations as a carbon and energy source (Cruden et al., 1992; Ramos et al., 1995). Regarding tolerance to aromatic hydrocarbons, a number of elements have been suggested as being involved in the response to these toxic chemicals: (i) metabolism of the toxic hydrocarbons, which can contribute to their transformation into non-toxic compounds; (ii) rigidification of the cell membrane via alteration in the composition of phospholipids; and (iii) efflux of the toxic compound in an energy-dependent process. Although the metabolism of the toxic chemicals can help to reduce their toxicity, two lines of evidence suggest that it is of minor importance. (i) A number of microorganisms tolerant to different organic solvents cannot metabolize the toxic compound, e.g. Escherichia coli strains tolerant to 1% (v/v) hexane do not use (or biotransform) this compound at all (Aono et al., 1991). Furthermore, a number of Pseudomonas strains tolerant to supersaturating concentrations of toluene did not use this compound as a carbon source (Inoue and Horikoshi, 1989). (ii) Pseudomonas putida DOT-T1E is a toluene-tolerant strain that degrades and uses this chemical via the toluene–dioxygenase pathway. Mutants unable to metabolize this compound were generated and shown to be as tolerant of toluene as the wild type (Mosqueda et al., 1999). This therefore establishes a neat difference between the metabolism of a chemical and tolerance to it. Instead, lipid membrane ridigification in response to toxic compounds and efflux of the chemicals are key elements and, although their role in solvent tolerance has not the same extent, below we will review the most critical features that suggest that they play a role in solvent tolerance. The initial stage of damage caused by many solvents when bacterial cells are exposed to organic solvents is the binding and penetration of the lipid bilayer (Sikkema et al., 1995). As a consequence, membrane fluidity is affected, and bacteria undertake appropriate responses to diminish its disruptive effect. This is achieved by readjusting fluidity, primarily by altering the composition of the lipid bilayer. This response is apparent in almost all membrane constituents. The compensation mechanisms resemble some of those observed in response to physical and chemical changes imposed by the environment (Russell and Fukanaga, 1990). The major responses to solvent exposure with regard to membrane composition are discussed below. There are two major mechanisms for changing the ester-linked fatty acid composition, and thus membrane fluidity, in bacterial lipid bilayers: the cis/trans isomerization of fatty acids as a short-term response; and the change in the saturated–unsaturated fatty acid ratio as a long-term response to solvent exposure. Additionally, the ratio of long-chain to short-chain fatty acids can also be altered to regulate membrane fluidity. Figure 1 shows that the steric behaviour of trans fatty acids and saturated fatty acids is very similar, as both possess a long extended conformation allowing a denser packing of the membrane. In contrast, the cis configuration of the acyl-chain has a non-movable bend of 30°, which causes steric hindrance and disturbs the highly ordered fatty acid package. The cis fatty acids have a lower phase transition temperature than the corresponding trans isomers (Keweloh and Heipieper, 1996). . Effect of cis/trans isomerization on membrane structure. The exposure to solvents triggers the conversion of cis-unsaturated fatty acids (blue) to trans-unsaturated fatty acids (red) and thus allows a denser molecular packing of the phospholipids. Saturated fatty acids are shown in yellow. Until recently, the cis acyl-chain configuration of membrane-located esterified fatty acid has been regarded as the sole isomer. However, the occurrence of trans isomers in cellular lipids has now been reported for a number of microorganisms, e.g. Pseudomonas, Vibrio sp. and methylothrophic bacteria (reviewed by Keweloh and Heipieper, 1996). Various Pseudomonas putida strains have been described as responding by shifting their cis/trans ratio when exposed to organic solvents (phenol, 4-chlorophenol, toluene, xylenes or alcohols of different chain length; Heipieper et al., 1992; Weber et al., 1994; Ramos et al., 1995; 1997; Pinkart and White, 1997). It has been shown that the cis/trans isomerization of ester-linked fatty acid is a post-synthetic enzyme modification in several P. putida strains (Diefenbach and Keweloh, 1994). All strains contain mainly oleic acid (C16:1,9) and vaccenic acid (C18:1,11) as trans isomers, and these are synthesized directly from the cis isomer without shifting the position of the double bond (Loffeld and Keweloh, 1996). Organic solvents increase membrane fluidity, and an increment in trans fatty acid content could counteract the alteration in membrane fluidity. The cis/trans isomerases of P. putida P8, called CtiP8 (Holtwick et al., 1997), and P. putida DOT-T1, called CtiT1 (Junker and Ramos, submitted), have been cloned and sequenced recently; they are 95% identical at the amino acid sequence level. The CtiT1 enzyme was shown to be synthesized constitutively. The molecular basis by which the Cti isomerase becomes active in response to toluene and other organic solvents is still unknown. To understand the impact of the cis/trans conversion on solvent resistance in P. putida DOT-T1, an isogenic CtiT1 knockout mutant was generated and characterized (Junker and Ramos, submitted). The mutant CtiT1 strain displayed a longer lag phase in comparison with the parental strain when transferred to minimal medium with toluene supplied in the gas phase as the sole source of carbon and energy. A lower survival rate of the mutant strain was also observed when shocked with 0.08% (v/v) toluene in LB liquid medium (Junker and Ramos, submitted). Thus, the cis/trans isomerization of fatty acids in phospholipids improves the survival of this strain in response to toluene. The cis/trans isomerase in the non-solvent-resistant P. putida 2440 was sequenced as well, and the DNA sequence of the gene and promoter region showed only minor differences from the ctiT1 gene. Consequently, it was concluded that the solvent-resistant character of strain DOT-T1 is not only caused by the cis/trans isomerase but also by other mechanisms (see section on pumps). The major importance of the cis/trans conversion lies in the fact that the constitutively expressed enzyme Cti can thus trigger an immediate emergency response to overcome initial membrane damage. By doing this, the cells gain time for a more precise adjustment to the new environmental conditions. In the meantime, a long-term response can be triggered. The increase in unsaturated fatty acids of phospholipids, a well-known response to temperature changes and solvent exposure (Cronan, 1968; Suutari and Laakso, 1994; Loffeld and Keweloh, 1996), has been observed in, for example, E. coli in the presence of long-chain alcohols and aromatic compounds (Ingram, 1977). A temperature increase, which has a fluidizing effect on the lipid bilayer like some organic solvents, has been shown to lead to an upregulation of saturated fatty acids in the psychrophilic Vibrio sp., E. coli and P. putida P8, whereas a decrease in temperature promoted the formation of unsaturated fatty acids (Cronan, 1968; Hamamoto et al., 1994; Loffeld and Keweloh, 1996). This widespread strategy to regulate membrane fluidity depends on the de novo synthesis of fatty acids, a process that is time-consuming and might not be quick enough to enable bacteria to survive a solvent or heat shock, especially if they are slow growing. An augmentation in the saturated fatty acid content in P. putida strain Idaho was observed as late as 15 min after solvent exposure (Pinkart and White, 1997); this adaptation mechanism is considered to be a long-term response. Other Pseudomonas strains do not alter their unsaturated/saturated fatty acid ratio in response to organic solvents (Weber et al., 1994; Ramos et al., 1997). The change in phospholipid headgroups is a less well- studied phenomenon, and few data on it are available. The composition of the phospholipid headgroup influences membrane fluidity (Weber and de Bont, 1996). In the presence of toluene, changes in the phospholipid headgroup composition were observed for P. putida S-12 in a chemostat. In the phospholipid headgroup, phosphatidylethanolamine (PE) decreased and diphosphatidylglycerol (DPG) as well as phosphatidylglycerol (PG) increased. DPG has a higher transition temperature than PE (10°C higher), which lowers membrane fluidity, producing a stabilizing effect (Weber and de Bont, 1996). Similar results were reported for P. putida DOT-T1 (Ramos et al., 1997). In P. putida strain Idaho (Pinkart and White, 1997), the total amount of membrane phospholipid fatty acids increased when exposed to o-xylene. A detailed analysis of the phospholipid headgroup biosynthesis in the presence of xylene in P. putida strain Idaho revealed an increase in the level of PE and a decrease in the level of PG. PE has a higher melting point than PG and, thus, its increase tends to stabilize the cell membrane. Therefore, different Pseudomonas strains seem to have developed different strategies for changing phospholipid headgroup composition to increase membrane rigidity and, in this way, to overcome the damaging effects of solvents. Changes in outer membrane proteins and lipopolysaccharides (LPS) after exposure to solvents have been monitored in various bacteria (Pinkart et al., 1996; Weber and de Bont, 1996). LPS molecules are made up of a polysaccharide chain and several saturated fatty acids and have low permeability for hydrophobic compounds. The LPS structure of P. putida strain Idaho changed in response to xylene exposure, which possibly reduces the permeability for xylene (Pinkart et al., 1996). However, there is no conclusive evidence yet that can explain satisfactorily the role of the outer membrane LPS in solvent resistance. The presence of divalent cations (e.g. Mg2+ and Ca2+ ions) was found to improve survival when added to the growth medium supplemented with organic solvents in several P. putida strains (Inoue et al., 1991; Ramos et al., 1995; Weber and de Bont, 1996). It is likely that the divalent cations electrostatically linked adjacent polyanionic LPS molecules and reduced charge repulsion. This allowed a denser packing of the anionic membrane molecules, and the membrane became more hydrophobic, which affected membrane stability and the access of solvents to the membrane. An increase in the protein–lipid content has been observed in E. coli when exposed to ethanol or phenol (Ingram, 1977; Keweloh et al., 1990). A higher protein content has a rigidifying effect on membranes, as proteins hinder lipid motion (Weber and de Bont, 1996). During evolutionary history, bacteria have been exposed to different toxic compounds, such as natural toxins, endogenous metabolic end-products, antibiotics, etc. To protect themselves, microorganisms have evolved devices that detoxify and extrude these substances. This phenomenon leads to the occurrence of multidrug resistance (MDR), which is a unidirectional efflux system that catalyses the active extrusion of a large number of structurally and functionally unrelated compounds from the bacterial cytoplasm (or internal membrane) to the external medium. Some of the substrates of these MDR pumps are xenobiotics, which do not resemble specific natural substrates that these cells may have encountered during evolution. The data available so far indicate that the physical characteristics of the compounds (charge, hydrophobicity or amphipathicity), rather than their structures, could be the determining factor in the specificities of these multidrug efflux systems (Paulsen et al., 1996; Bolhuis et al., 1997). MDR pumps are thought to be a major cause of antimicrobial multiresistance in bacteria (Levy, 1992), although the usual function and the evolution of these MDR pumps is still an enigma. There are four families of MDR transporters identified in bacteria: (i) the major facilitator superfamily (MFS); (ii) the small multidrug resistance; (iii) the ATP-binding cassette; and (iv) the RND family of pumps. The efflux pumps for organic solvents identified so far in Gram-negative bacteria belong to the last family. Multidrug efflux systems have been the subject of recent reviews (Paulsen et al., 1996; Bolhuis et al., 1997; Lewis et al., 1997). The RND pumps of Gram-negative bacteria are complex transporters that export toxic substances across the two membranes of the cell envelope in a single energy-coupled step. The process requires a cytoplasmic membrane export system that acts as an energy-dependent extrusion pump, a membrane fusion protein (MFP) and an outer membrane factor (OMF). The energy-dependent pump contains a number of transmembrane α-helices and two large domains projecting into the periplasm. MFPs form the ‘link’ between the cytoplasmic membrane-located transporter and the OMF. MFPs are anchored to the cytoplasmic membrane, and they possibly interact with the corresponding cytoplasmic membrane transporter through a less hydrophobic part. The C-terminal region of MFPs is well conserved and is predicted to consist of β-structures, suggesting that it may insert into the outer membrane and interact with the OMF. OMFs are fairly uniform in size (398–495 and they may form of up to (Paulsen et al., 1997). and of E. coli strain showed tolerance to organic solvents such as or The results that some system determining resistance been made active in the organic This was the a between organic solvent tolerance and resistance (Aono et al., 1995). The most system in E. coli efflux of organic solvents is the system, in which the protein is the (Aono et al., 1996), with being the and a of the RND family of transporters et al., It was observed that the level of was high in several of the organic tolerant Some of the were in the gene et al., 1997). The gene of the which is by the protein that to the promoter region in and 1997). The protein is a that a of and and that E. coli cells with resistance to a large number of and It has been shown that the of for at position in the region of tolerant was for the et al., et al., 1997). It is likely that the mutant protein was unable to the In this way, the protein was and it the response to and organic solvents. The and gene products can in the of and in increased organic solvent tolerance in E. coli et al., of the gene decreased the organic solvent tolerance of E. coli The growth characteristics indicate functions of the and and in their to organic solvents. of increased to and organic solvents in or the was linked to of the and the system was identified as the major pump for the resistant to organic solvents and et al., 1996; et al., 1997). with the toluene-tolerant P. putida strains and DOT-T1 indicate that the amount of and in cells in the presence of toluene was to lower than in the the chain or the was the results showed in the presence of in higher of of the aromatic in P. putida The results the in these cells growing in the presence of a organic solvent could be an energy-dependent system that may decrease the level of solvent in the membranes and de Bont, 1996; Ramos et al., 1997; The evidence for the of an efflux system for organic solvents in both P. putida strains was the and molecular of two but efflux pumps involved in solvent tolerance et al., Ramos et al., In both the of as the for of the solvent efflux systems The amino acid of the proteins by the of P. putida and the tolerance of P. putida DOT-T1 have a high with the multidrug efflux system of P. from to to the proteins and the RND family of multidrug resistance. The and the proteins that as a between the membrane transporter in these systems by and and the outer membrane protein by and In P. putida a pump to the has been reported et al., The of P. putida can be transferred to a P. putida producing an increase in solvent resistance in the The solvent resistance is on the level of of the et al., evidence in organic solvents was by the fact that the P. putida strain was unable to extrude from the cell membrane when in the of organic solvents. However, the system in the mutant P. putida when cells were on LB with toluene in the gas suggesting the of a pump, which seems to be an pump to the . of the pump of P. putida The multidrug efflux systems described in P. and are able to some level of tolerance to organic solvents, such as and the being the system et al., P. strains of as well as the system, showed tolerance to and whereas strains in which these were or showed no resistance. The data suggested that the multidrug efflux systems of P. can organic solvents, as well as It is that the efflux of organic solvents by a system to the RND family is of the main mechanisms resistance to organic solvents in several E. coli and Pseudomonas sp. In Gram-negative bacteria, a number of responses have been found to counteract the effect of organic solvents that are the rigidification of the cell membrane and the extrusion of the toxic In a number of Pseudomonas a response to is the isomerization of the synthesized of unsaturated lipids to the a by a Cti exposure of Pseudomonas E. coli and other microorganisms to concentrations of solvents often leads to an increase in the total amount of phospholipids and to a higher of saturated long-chain changes are with in the level of different phospholipid All the changes are the rigidification of the cell membrane. pumps of the RND family are critical for solvent tolerance. The molecular for the are but a by all of them is that they a A number of efflux pumps for organic solvents have been identified in Pseudomonas and and in E. coli consist of an energy-dependent transporter to the membrane in all the an outer membrane and and a membrane fusion protein that through the A in all the and seems to the pump and the Mutants in the efflux pumps are more to solvents than their parental although different of to different solvents can be found in different which may pumps or the of as yet elements involved in solvent tolerance. Regarding is to the role of phospholipids, LPS and membrane proteins in the rigidification of the cell membranes and their specific if in tolerance to solvents. the efflux pumps function their and they solvents that are in the membrane or in the cytoplasm need detailed in The the presence of the solvent and the of and long-term responses the of such as the cis to trans or the of that lead to altered phospholipid composition, as well as the of different pumps are of high that In solvent tolerance in bacteria is an that to the molecular basis of this and to this in such as the of toxic and compounds in chemicals of added and the removal of pollutants from with large of toxic solvents. in the was by from the and the