Appl Microbiol Biotechnol (2014) 98:5599–5606 DOI 10.1007/s00253-014-5549-2
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Rapid adaptation of Rhodococcus erythropolis cells to salt stress by synthesizing polyunsaturated fatty acids Carla C. C. R. de Carvalho & Marco P. C. Marques & Nancy Hachicho & Hermann J. Heipieper
Received: 13 January 2014 / Accepted: 14 January 2014 / Published online: 6 March 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Bacterial cells are known to adapt to challenging environmental conditions such as osmotic stress. However, most of the work done in this field describes the adaptation of growing populations where the new generations acquire traits that improve their ability to survive. In the present study, the responses of Rhodococcus erythropolis cells within the first 30 min after exposure to osmotic stress caused by sodium chloride were studied. The cells changed the total lipid fatty acid composition and also the net surface charge in the 30 min following exposure. Surprisingly, the cells produced a high percentage of polyunsaturated fatty acids. In the presence of 7.5 % NaCl, these polyunsaturated fatty acids, mainly eicosapentaenoic acid (C20:5ω3), arachidonic acid (C20:4ω6) and docosapentaenoic acid (C22:5ω3), comprise more than 36 % of the total fatty acids. The possible function of these very uncommon fatty acids in bacteria could be the decrease in the number of negatively charged groups in ion channels resulting in a repellence of the NaCl. Keywords Stress . Adaptation . Lipids . NaCl . PUFA Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-5549-2) contains supplementary material, which is available to authorized users. C. C. C. R. de Carvalho (*) : M. P. C. Marques Institute of Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal e-mail: [email protected] N. Hachicho : H. J. Heipieper Department of Environmental Biotechnology, Helmholtz Centre for Environmental Research—UFZ, Permoserstraße 15, 04318 Leipzig, Germany Present Address: M. P. C. Marques Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK
Introduction The ability of a bacterial population to survive and thrive under stress conditions depends on the ability of the individual cells to survive the initial shock. Rapid changes in cell composition and physiology have to occur before a given trait can be passed to the next generation, which means that the cells have to use adaptive mechanisms that do not require gene induction and de novo synthesis, e.g. of membrane phospholipid fatty acids (PLFAs). The genus Rhodococcus contains species capable of degrading/converting a plethora of compounds (Bell et al. 1998; de Carvalho and da Fonseca 2005; Finnerty 1992; Larkin et al. 2005; Solyanikova and Golovleva 2011; Warhurst and Fewson 1994), even under conditions which may be considered extreme (de Carvalho 2012; Matys et al. 1998). The remarkable features of these cells may be applied for the bioremediation of xenobiotics in contaminated environments, such as those near oilfields. Petroleum is thought to be a product resulting mainly from the decay of marine organisms, and oil deposits often involve aquifers containing high amounts of NaCl. Oilfield-produced water is usually a major source of salt and, in particular, sodium contamination (Fakhru’l-Razi et al. 2009). In a previous study, strain Rhodococcus erythropolis DCL14 could be successfully adapted to grow and degrade alkanes and alcohols in the presence of up to 7.5 % NaCl, but adaptation involved growth under stepwise increasing concentrations of salt since the non-adapted cells were unable to grow in a medium containing salt concentrations higher than 5.5 % (de Carvalho 2012). The membrane phospholipid fatty acid composition of strain DCL14 changed with increasing sodium chloride concentrations so that a reduction in membrane fluidity and permeability was observed, similarly to the observation in the case of halotolerant strains from the Antarctica (Nicolaus et al. 2001; Ventosa et al. 1998).
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The aim of this study was to evaluate the first response of R. erythropolis cells when exposed to different concentrations of sodium chloride. Contrarily to the large majority of the published studies on bacterial membrane adaptation, the cells in this study were not given time for de novo synthesis of fatty acids and the analysed population was nearly the same during the 30-min assays. Strain R. erythropolis DSM 1069 was used as a model bacterium since it has a broad substrate specificity of aliphatic alcohols (Schenkels and Duine 2000), is able to degrade methoxylated aromatic compounds (Eggeling and Sahm 1980) and light crude oil on a seawater-based medium (Iwabuchi et al. 2000).
Materials and methods Strain R. erythropolis DSM 1069 was obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. The bacterium was originally isolated from a Finnish soil for its ability to decompose lignin and use lignin degradation compounds as a carbon source (Trojanowski et al. 1977). Determination of growth rates in the presence of sodium chloride Cell growth was carried out in cylindrical 250-mL closed flasks containing 20 mL of elemental mineral medium, 0.25 % (v/v) ethanol as a carbon source, and 1, 2.5, 3.5, 5.5 or 7.5 % (w/w) of sodium chloride. The mineral medium contained the following compounds per litre of demineralised water: 0.01 g EDTA, 0.002 g ZnSO4 · 7H2O, 0.001 g CaCl2 · 2H2O, 0.005 g FeSO4 · 7H2O, 0.0002 g Na2MoO4 · 2H2O, 0.0002 g CuSO4 · 5H2O, 0.0004 g CoCl2 · 6H2O, 0.001 g MnCl2 · 4H2O, 0.1 g MgCl2 · 6H2O, 2 g (NH4)2SO4, and 1.55 g K2HPO4 and 0.85 g NaH2PO4 · H2O for buffering (all chemicals were from Sigma-Aldrich). The flasks were inoculated with an exponential growing culture to an initial optical density of ca. 0.1 and incubated at 28 °C and 200 rpm. Growth was monitored by measuring the optical density at 560 nm. All assays were carried out at least in triplicate. Assessment of cell adaptation to sodium chloride R. erythropolis cells were grown in cylindrical 250-mL closed flasks containing 20 mL of elemental mineral medium and 0.25 % (v/v) ethanol as a carbon source at 28 °C and 200 rpm. Optical density measurements at 560 nm were used to monitor cell growth. When the cells reached mid-exponential phase, sodium chloride was added to achieve concentrations of 1, 2.5, 3.5, 5.5 and 7.5 % (w/w). One-millilitre samples of cell
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suspension were taken 6, 12, 20 and 35 min after salt exposure. The samples were immediately centrifuged for 3 min at 14,000 rpm, the supernatant was discharged and the lipids were extracted as described below to prevent further exposure to salt. Assays were carried out at least in duplicate. Lipid extraction and determination of fatty acid composition Bacterial lipids were extracted and fatty acids were methylated in one step using a sulphuric acid–methanol method based on the procedure described by Antolín et al (2008). In summary, 0.75 mL of 2.5 % H2SO4 in methanol was added to the cell pellet and the mixture was heated at 80 °C for 1.5 h. After cooling the mixture at room temperature, 0.35 mL of n-hexane and 0.35 mL of 0.98 % NaCl in water were added. The mixture was stirred for 1 min and centrifuged at 10,000 rpm for 1 min to separate the solvent phases, and the n-hexane phase was recovered. The produced fatty acid methyl esters (FAMEs) were analysed on a 6890N gas chromatograph from Agilent Technologies (Palo Alto, CA, USA), with a flame ionisation detector and a 7683 B series injector, equipped with a 25-m-long Agilent J&W Ultra 2 capillary column from Agilent. Peak identification was achieved by the Sherlock® software version 6.2 (MIDI, Inc.) and by using a qualitative standard of bacterial fatty acid methyl esters and one of polyunsaturated fatty acids, both from Supelco, and a methyl cis-11-octadecenoate standard solution from Sigma-Aldrich. Peak identification was confirmed (Electronic Supplementary Material, Fig. S1) using the NIST/EPA/NIH Mass Spectral Library version 2.0 by injecting both standard and selected samples on a gas chromatograph mass spectrometer (5975B inlet MSD from Agilent) equipped with a DB-1 column from J&W Scientific. The unsaturation index (UI) of the cell membrane fatty acids was defined as the sum of the percentage of each unsaturated fatty acid multiplied by the number of double bonds in the molecule, as previously described for different yeast strains (Kaszycki et al. 2013). Zeta potential Concomitantly to sampling for FAMEs, cells were also collected in mid-exponential phase for zeta potential measurements. They were washed three times and suspended in 10 mM KNO 3 . The electrophoretic mobility of the R. erythropolis cell suspensions was determined in a Doppler electrophoretic light scattering analyser (Zetasizer Nano ZS, Malvern Instruments Ltd.) using a clear disposable zeta cell. The zeta potential (z) was calculated using the electrophoretic mobility as an indirect measure of cell surface charge, according to the method of Helmholtz and Smoluchowski (Hiemenz and Rajagopalan 1997). Calculations were automatically made using the Zetasizer software 6.20, from Malvern Instruments Ltd.
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Error analysis The average error associated with the GC quantification of each FAME was ±2.2 %, quoted for a confidence interval of 99.5 %. Errors were calculated based on seven independently prepared standard solutions.
Results Cell growth in the presence of sodium chloride Maximum growth rate of R. erythropolis DSM 1069 cells in the presence of sodium chloride was observed for a concentration of 2.5 %, reaching 0.20±0.008 h−1. Lower growth rates were observed either when no salt was present or with increasing salt concentrations (Fig. 1). Still the cells could grow in the presence of 7.5 % (w/w) NaCl, at a rate of 0.05± 0.004 h−1. Fast adaptations to the presence of sodium chloride Under the fastest growth condition tested, the doubling time of strain DSM 1069 was 3.5 h (growth rate, μ, 0.20±0.008 h−1 in the presence of 2.5 % NaCl). The assays performed to evaluate the fast response of the cells when exposed to sodium chloride were carried out in the first 35 min after the addition of NaCl so that the response of those individual cells that were challenged could be assessed. Most of the studies published on bacterial adaptation concern the growth of stressed populations with analyses being performed on new cell generations. In the present study, the total lipids of non-stressed cells were mainly composed of the fatty acids C17:0 (8.2 %), C15:0 (13.3 %), C18:1ω9c (14.0 %), C16:1ω7 (20.6 %) and C16:0 (31.7 %) (data not shown). The degree of saturation of these cells, defined as the ratio between saturated and 0.25
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monounsaturated fatty acids, was 1.7 whilst the UI was 0.38 (data not shown). The sum of fatty acids other than saturated straight and monounsaturated reached only 0.9 % of the total lipid composition. When cells were exposed to different concentrations of sodium chloride, they responded by changing the fatty acid composition (Fig. 2). The concentrations allowing the highest growth rates, 2.5 and 3.5 % NaCl, induced a lower extent of changes than lower or higher concentrations. Nevertheless, these cells started producing hydroxy-substituted (HSFAs), saturated methyl-branch ed (SMBFAs), saturated cyclopropyl-branched (SCBFAs) and polyunsaturated (PUFAs) fatty acids in larger quantities than unchallenged cells. After 35 min in the presence of these two concentrations, R. erythropolis cells produced between 6 and 24-fold more of these fatty acids then their non-exposed controls (Fig. 2, 35 min). The sodium chloride concentrations tested induced modifications in the fatty acid profile of the cells already after only 6 min (Fig. 2, 6 min). The amount of monounsaturated fatty acids decreased, in general, to approximately half of the amount observed in cells with no salt present whilst the amount of SMBFAs, SCBFAs, HSFAs and PUFAs became significant, ranging in total between 7.8 and 14.7 % of the total fatty acids depending on the salt concentration. After 12 min, major changes were observed in cells in the presence of 5.5 % NaCl and these cells produced 14.4 % of total fatty acids as PUFAs (Fig. 2, 12 min). In these cells, MUFAs decreased to 20.8 %, when compared to control cells, which is exactly the same increase observed in the sum of all other classes of fatty acids (straight-chain saturated fatty acids (SSFAs), HSFAs, SMBFAs, SCBFAs and PUFAs). The major decreases were observed in the percentage of C18:1ω9c and C16:1ω7 (data not shown). The main polyunsaturated fatty acids produced by these cells in the presence of sodium chloride were eicosapentaenoic acid (EPA, C20:5ω3), arachidonic acid (ARA, C20:4ω6) and docosapentaenoic acid (DPA, C22:5ω3). After 20 min of exposure, R. erythropolis cells in the presence of 1 and 7.5 % NaCl produced also significant amounts of PUFAs (Fig. 2, 20 min). In fact, cells in the presence of 1 % NaCl presented a fatty acid profile similar to those exposed to 5.5 % and 7.5 %, resulting in an UI of ca. 1. In contrast, cells exposed to 0, 2.5 and 3.5 % NaCl presented an UI of around 0.4. The differences observed between the conditions allowing the highest growth rates and those observed in cells in the presence of 1, 2.5 and 3.5 % NaCl were highest after 35 min of exposure (Fig. 2, 35 min). The former cells presented a high percentage (over 70 %) of SSFAs, whilst the latter cells presented over 25 % of PUFAs and less than 53 % of SSFAs. No significant differences were observed during longer exposures under the doubling time (data not shown), indicating that the cells had attained the best
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adjustment in the membrane composition for each sodium chloride concentration in the first 30 min of exposure. A negative correlation between the UI and the maximum growth rate observed for the tested salt concentrations could be observed (Fig. 3). Concentrations of 2.5 and 3.5 % NaCl allowed the highest growth rates and lowest unsaturation indexes. The opposite behaviour in each parameter was observed for both lower and higher salt concentrations. This could indicate an increased metabolic burden when the cells
had to change the fatty acid composition to survive the tested salt concentrations, resulting in lower growth rates when the changes were more extent. The net surface charge of the R. erythropolis DSM 1069 cells, represented by the zeta potential, increased both with time and with sodium chloride concentration (Fig. 3). After only 6 min of exposure to salt, the zeta potential of the cells increased from −34.1 mV observed for control cells to −20.9 mV observed with cells in the presence of 7.5 % NaCl. However, the increase was only significant for concentrations higher than 3 %. Similar results were obtained after 12 min. For concentrations lower than 3.5 % NaCl, the zeta potential after 35 min was lower than that observed after 20 min, but for 35 min, a plateau at around −10.9 mV was measured for the highest concentrations. After 2.25 h, the zeta potential of the cells varied between −20.8 and −17.5 mV for NaCl concentrations of 1 and 7.5 %, respectively (data not shown). A relation between the amount of PUFAs present in the cells and the zeta potential of the cells could be observed for each salt concentration: in general, higher amounts of PUFAs were observed in cells presenting a less negative net surface charge (Fig. 5). Furthermore, the MUFA content was kept nearly constant for each tested NaCl concentration, although the cells changed the net surface charge (data not shown).
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Discussion Zeta potential, mV
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out by Okuyama et al. (2007), from the fact that the best studied bacterial species in terms of physiology, biochemistry and molecular biology were mesophilic species such as Escherichia coli containing no PUFAs. It is now accepted that long-chain PUFAs, such as EPA, DHA and ARA, are preferentially produced by marine bacteria (de Carvalho and Caramujo 2012; DeLong and Yayanos 1986; Hamamoto et al. 1995; Nichols 2003; Russell and Nichols 1999). PUFAs may also easily be decomposed by autoxidation during analytical procedures or by harsh transesterification methods, e.g. with boron trifluoride-methanol or high temperatures, and may not be detected (Eder 1995). The synthesis of PUFAs has also been observed during the adaptation of R. erythropolis DCL14 to extreme conditions (de Carvalho 2012). In the presence of sodium chloride, strain DCL14 produced a nearly constant amount of PUFAs reaching ca. 0.32 % of the total fatty acids, whilst in the presence of 1 % CuSO4, the cells synthesised 1.41 % of PUFAs. However, the amounts of PUFAs synthesised under
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R. erythropolis cells are able to perform a series of biotransformations and degradations that might be relevant for future biotechnological processes (de Carvalho and da Fonseca 2002, 2005; de Carvalho et al. 2007, 2009; Liu et al. 2012; Pirog et al. 2013; Schreiberova et al. 2012). However, if bioremediation and, in particular, bioaugmentation is envisaged, the cells must be able to endure environmental conditions far from optimal. These cells are good degraders of both aliphatic (de Carvalho et al. 2005, 2009; Liu et al. 2012) and aromatic compounds (de Carvalho et al. 2007; Eggeling and Sahm 1980; Schreiberova et al. 2012), are able to desulfurise dibenzothiophenes (Izumi et al. 1994; Ohshiro et al. 1996; Wang and Krawiec 1996; Yu et al. 2006) and may be used in the bioremediation of fuels (de Carvalho and da Fonseca 2005; Lee et al. 1999; Michel et al. 2004). Since most oil deposits and oilfield-produced water contain high concentrations of salt, the cells should be able to withstand salt shocks. R. erythropolis DSM 1069 showed a maximum growth rate in the presence of 2.5 % NaCl (Fig. 1), which is typical for many marine bacteria (Mutnuri et al. 2005). It was thus expected, as the conditions were further away from the optimum salt concentration, that the cells would produce larger changes in fatty acid composition. However, the responses of these cells during the first moments of exposure were, at the beginning of this study, unknown. In fact, the initial response of R. erythropolis cells to both low and high concentrations of sodium chloride showed significant increases of the unsaturation index of the lipids when compared to cells exposed to 2.5 % NaCl (Figs. 2 and 3). A very surprising finding of this study was the observed synthesis of PUFAs. Especially the fact that PUFAs were detected already after 6 min of exposure to NaCl and that their abundance reached up to 36.3 % of the total lipids in bacteria exposed for 35 min to 5.5 % NaCl has not been described in bacteria before. This appearance of PUFAs was accompanied by a concomitantly decrease in the percentage of MUFAs. Therefore, constitutively expressed fatty acid desaturases could be responsible in this fast post de novo synthetic modification of saturated and monounsaturated fatty acids to synthesise PUFAs. In fact, genes for such desaturases have been identified in other R. erythropolis: strain SK121 produces stearoyl-CoA 9-desaturases (Sebastian et al. 2009) whilst strain PR4 produces acyl-CoA desaturases (Takarada et al. 2005). Desaturases are specific for the position of the double bond they insert relative to the carboxyl end of the substrate molecule, and during PUFA production, a series of desaturases should be involved (Metz et al. 2001; Russell and Nichols 1999). Until the 1990s, it was considered that, with the exception of selected cyanobacteria, bacteria were unable to produce PUFAs. This assumption was derived probably, as pointed
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these conditions were about 1 order of magnitude lower than those observed in the present study. Fulco demonstrated that bacilli and mycobacteria could produce dienoic fatty acids by aerobic desaturation of fatty acids (Fulco 1974), but recent studies link the fatty acid composition to the bacterial ecological niche (de Carvalho and Caramujo 2012; de Carvalho and Fernandes 2010; Nichols 2003). As previously mentioned, it is generally accepted that only few bacteria are capable of synthesising PUFAs, whereby most of them are marine bacteria, in particular from cold, deep-sea sediments, fish and water (Hamamoto et al. 1995; Yano et al. 1998; Yano et al. 1997). Under such conditions, PUFAs are presumed to allow the maintenance of appropriate cytoplasmic membrane fluidity (Valentine and Valentine 2004), although in Shewanella livingstonensis, no significant difference was detected between the parental strain and an EPA-less mutant (Kawamoto et al. 2009). The best studied species regarding PUFA production in marine bacteria are members of the genus Shewanella. In these cells, eight open-reading frames (ORFs) were identified in a 38-kb genomic fragment: three were similar to fatty acid synthases and the remaining five were presumed to be effectors of C20 PUFA biosynthesis (Takeyama et al. 1997). However, Metz and co-workers have also described an alternative pathway for the synthesis of long PUFAs that does not require multiple desaturases and elongases but a polyketide synthaselike gene cluster (Metz et al. 2001). In Rhodococcus sp., polyketide synthase genes have also been found, e.g. strain RHA1 contains seven polyketide synthase genes (McLeod et al. 2006) and R. erythropolis PR4 contains a sequence encoding for a putative polyketide synthase with 2059 AA (Takarada et al. 2005). As pointed out by McLeod and colleagues, the high number of polyketide synthase genes in strain RHA1 was not expected but it evidences extensive secondary metabolism in these cells (McLeod et al. 2006). One of the main functions of these enzymes is the synthesis of mycolic acids in Rhodococcus strains and in other mycolic acid-containing bacteria (Portevin et al. 2004). Mycolic acids in Rhodococcus are known to also contain polyunsaturated aliphatic chains (Tomiyasu and Yano 1984). However, a relation between these genes, their necessity to be constitutively expressed and their function in the fast synthesis of PUFAs remains to be studied in rhodococci. The R. erythropolis DSM 1069 studied presented higher zeta potential values with increasing sodium chloride concentrations, and for each concentration, the value also increased with time (Fig. 4). Remarkably, the percentage of polyunsaturated fatty acids was highest for cells presenting the highest values of zeta potential (Fig. 5). It has been shown that PUFAs directly affect the activity of membrane proteins such as voltage-gated ion channels and free PUFAs may affect different membrane proteins including ion channels (Börjesson and Elinder 2011). Additionally, the multiple double bonds in
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these fatty acids may influence the bilayer organisation of membranes including, e.g. lateral pressure, microviscosity, curvature, permeability and elasticity which affect membrane proteins (Shaikh and Edidin 2008). PUFAs can also promptly inhibit sodium ion channels, which may be used to prevent cardiac arrhythmias in mammals (Xiao et al. 1995; Xiao et al. 2005). In R. erythropolis ATCC 15592, a channel-forming protein was described and the cell wall channel was found to be wide, water-filled and mainly permeable for cations due to the presence of negatively charged groups at the opening of the channel (Lichtinger et al. 2000). The relation between PUFAs and net surface charge observed in strain DSM 1069 could thus contribute to a lower number of negatively charged groups in ion channels and to an inhibition of sodium ions’ entrance to the cell. This is clearly an indication of a cleaver adaptation mechanism by this R. erythropolis strain. In conclusion, R. erythropolis cells were shown to be capable of very rapidly adapting high concentrations of sodium chloride. Hereby, modifications in the total lipid fatty acid content were observed already after only 6 min of exposure. The most remarkable adaptive response was the synthesis of polyunsaturated fatty acids, which are very unusual in mesophilic bacteria. Thus, bacteria of the genus Rhodococcus were shown to be able to promptly adapt to stress conditions. This is another proof for the remarkable feature of these bacteria, making them perfect biocatalysts both in terms of bioremediation and whole-cell biotransformation. Acknowledgments CCCR de Carvalho and MPC Marques would like to thank the Fundação para a Ciência e a Tecnologia (Portugal) for financial support (programs "Ciência 2007" and "FCT Investigators 2013", and SFRH/BPD/64160/2009, respectively) and Prof. Susete M. Dias and Joana Duarte for the help in the GC-MS analysis. This work was partially supported by a Portuguese-German integrated action (A-15/11) awarded by the Conselho de Reitores das Universidades Portuguesas (CRUP) and the Deutscher Akademischer Austauschdienst (DAAD).
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APPLIED MICROBIAL AND CELL PHYSIOLOGY
Rapid adaptation of Rhodococcus erythropolis cells to salt stress by synthesizing polyunsaturated fatty acids Carla C. C. R. de Carvalho & Marco P. C. Marques & Nancy Hachicho & Hermann J. Heipieper
Received: 13 January 2014 / Accepted: 14 January 2014 / Published online: 6 March 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Bacterial cells are known to adapt to challenging environmental conditions such as osmotic stress. However, most of the work done in this field describes the adaptation of growing populations where the new generations acquire traits that improve their ability to survive. In the present study, the responses of Rhodococcus erythropolis cells within the first 30 min after exposure to osmotic stress caused by sodium chloride were studied. The cells changed the total lipid fatty acid composition and also the net surface charge in the 30 min following exposure. Surprisingly, the cells produced a high percentage of polyunsaturated fatty acids. In the presence of 7.5 % NaCl, these polyunsaturated fatty acids, mainly eicosapentaenoic acid (C20:5ω3), arachidonic acid (C20:4ω6) and docosapentaenoic acid (C22:5ω3), comprise more than 36 % of the total fatty acids. The possible function of these very uncommon fatty acids in bacteria could be the decrease in the number of negatively charged groups in ion channels resulting in a repellence of the NaCl. Keywords Stress . Adaptation . Lipids . NaCl . PUFA Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-5549-2) contains supplementary material, which is available to authorized users. C. C. C. R. de Carvalho (*) : M. P. C. Marques Institute of Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal e-mail: [email protected] N. Hachicho : H. J. Heipieper Department of Environmental Biotechnology, Helmholtz Centre for Environmental Research—UFZ, Permoserstraße 15, 04318 Leipzig, Germany Present Address: M. P. C. Marques Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK
Introduction The ability of a bacterial population to survive and thrive under stress conditions depends on the ability of the individual cells to survive the initial shock. Rapid changes in cell composition and physiology have to occur before a given trait can be passed to the next generation, which means that the cells have to use adaptive mechanisms that do not require gene induction and de novo synthesis, e.g. of membrane phospholipid fatty acids (PLFAs). The genus Rhodococcus contains species capable of degrading/converting a plethora of compounds (Bell et al. 1998; de Carvalho and da Fonseca 2005; Finnerty 1992; Larkin et al. 2005; Solyanikova and Golovleva 2011; Warhurst and Fewson 1994), even under conditions which may be considered extreme (de Carvalho 2012; Matys et al. 1998). The remarkable features of these cells may be applied for the bioremediation of xenobiotics in contaminated environments, such as those near oilfields. Petroleum is thought to be a product resulting mainly from the decay of marine organisms, and oil deposits often involve aquifers containing high amounts of NaCl. Oilfield-produced water is usually a major source of salt and, in particular, sodium contamination (Fakhru’l-Razi et al. 2009). In a previous study, strain Rhodococcus erythropolis DCL14 could be successfully adapted to grow and degrade alkanes and alcohols in the presence of up to 7.5 % NaCl, but adaptation involved growth under stepwise increasing concentrations of salt since the non-adapted cells were unable to grow in a medium containing salt concentrations higher than 5.5 % (de Carvalho 2012). The membrane phospholipid fatty acid composition of strain DCL14 changed with increasing sodium chloride concentrations so that a reduction in membrane fluidity and permeability was observed, similarly to the observation in the case of halotolerant strains from the Antarctica (Nicolaus et al. 2001; Ventosa et al. 1998).
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The aim of this study was to evaluate the first response of R. erythropolis cells when exposed to different concentrations of sodium chloride. Contrarily to the large majority of the published studies on bacterial membrane adaptation, the cells in this study were not given time for de novo synthesis of fatty acids and the analysed population was nearly the same during the 30-min assays. Strain R. erythropolis DSM 1069 was used as a model bacterium since it has a broad substrate specificity of aliphatic alcohols (Schenkels and Duine 2000), is able to degrade methoxylated aromatic compounds (Eggeling and Sahm 1980) and light crude oil on a seawater-based medium (Iwabuchi et al. 2000).
Materials and methods Strain R. erythropolis DSM 1069 was obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. The bacterium was originally isolated from a Finnish soil for its ability to decompose lignin and use lignin degradation compounds as a carbon source (Trojanowski et al. 1977). Determination of growth rates in the presence of sodium chloride Cell growth was carried out in cylindrical 250-mL closed flasks containing 20 mL of elemental mineral medium, 0.25 % (v/v) ethanol as a carbon source, and 1, 2.5, 3.5, 5.5 or 7.5 % (w/w) of sodium chloride. The mineral medium contained the following compounds per litre of demineralised water: 0.01 g EDTA, 0.002 g ZnSO4 · 7H2O, 0.001 g CaCl2 · 2H2O, 0.005 g FeSO4 · 7H2O, 0.0002 g Na2MoO4 · 2H2O, 0.0002 g CuSO4 · 5H2O, 0.0004 g CoCl2 · 6H2O, 0.001 g MnCl2 · 4H2O, 0.1 g MgCl2 · 6H2O, 2 g (NH4)2SO4, and 1.55 g K2HPO4 and 0.85 g NaH2PO4 · H2O for buffering (all chemicals were from Sigma-Aldrich). The flasks were inoculated with an exponential growing culture to an initial optical density of ca. 0.1 and incubated at 28 °C and 200 rpm. Growth was monitored by measuring the optical density at 560 nm. All assays were carried out at least in triplicate. Assessment of cell adaptation to sodium chloride R. erythropolis cells were grown in cylindrical 250-mL closed flasks containing 20 mL of elemental mineral medium and 0.25 % (v/v) ethanol as a carbon source at 28 °C and 200 rpm. Optical density measurements at 560 nm were used to monitor cell growth. When the cells reached mid-exponential phase, sodium chloride was added to achieve concentrations of 1, 2.5, 3.5, 5.5 and 7.5 % (w/w). One-millilitre samples of cell
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suspension were taken 6, 12, 20 and 35 min after salt exposure. The samples were immediately centrifuged for 3 min at 14,000 rpm, the supernatant was discharged and the lipids were extracted as described below to prevent further exposure to salt. Assays were carried out at least in duplicate. Lipid extraction and determination of fatty acid composition Bacterial lipids were extracted and fatty acids were methylated in one step using a sulphuric acid–methanol method based on the procedure described by Antolín et al (2008). In summary, 0.75 mL of 2.5 % H2SO4 in methanol was added to the cell pellet and the mixture was heated at 80 °C for 1.5 h. After cooling the mixture at room temperature, 0.35 mL of n-hexane and 0.35 mL of 0.98 % NaCl in water were added. The mixture was stirred for 1 min and centrifuged at 10,000 rpm for 1 min to separate the solvent phases, and the n-hexane phase was recovered. The produced fatty acid methyl esters (FAMEs) were analysed on a 6890N gas chromatograph from Agilent Technologies (Palo Alto, CA, USA), with a flame ionisation detector and a 7683 B series injector, equipped with a 25-m-long Agilent J&W Ultra 2 capillary column from Agilent. Peak identification was achieved by the Sherlock® software version 6.2 (MIDI, Inc.) and by using a qualitative standard of bacterial fatty acid methyl esters and one of polyunsaturated fatty acids, both from Supelco, and a methyl cis-11-octadecenoate standard solution from Sigma-Aldrich. Peak identification was confirmed (Electronic Supplementary Material, Fig. S1) using the NIST/EPA/NIH Mass Spectral Library version 2.0 by injecting both standard and selected samples on a gas chromatograph mass spectrometer (5975B inlet MSD from Agilent) equipped with a DB-1 column from J&W Scientific. The unsaturation index (UI) of the cell membrane fatty acids was defined as the sum of the percentage of each unsaturated fatty acid multiplied by the number of double bonds in the molecule, as previously described for different yeast strains (Kaszycki et al. 2013). Zeta potential Concomitantly to sampling for FAMEs, cells were also collected in mid-exponential phase for zeta potential measurements. They were washed three times and suspended in 10 mM KNO 3 . The electrophoretic mobility of the R. erythropolis cell suspensions was determined in a Doppler electrophoretic light scattering analyser (Zetasizer Nano ZS, Malvern Instruments Ltd.) using a clear disposable zeta cell. The zeta potential (z) was calculated using the electrophoretic mobility as an indirect measure of cell surface charge, according to the method of Helmholtz and Smoluchowski (Hiemenz and Rajagopalan 1997). Calculations were automatically made using the Zetasizer software 6.20, from Malvern Instruments Ltd.
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Error analysis The average error associated with the GC quantification of each FAME was ±2.2 %, quoted for a confidence interval of 99.5 %. Errors were calculated based on seven independently prepared standard solutions.
Results Cell growth in the presence of sodium chloride Maximum growth rate of R. erythropolis DSM 1069 cells in the presence of sodium chloride was observed for a concentration of 2.5 %, reaching 0.20±0.008 h−1. Lower growth rates were observed either when no salt was present or with increasing salt concentrations (Fig. 1). Still the cells could grow in the presence of 7.5 % (w/w) NaCl, at a rate of 0.05± 0.004 h−1. Fast adaptations to the presence of sodium chloride Under the fastest growth condition tested, the doubling time of strain DSM 1069 was 3.5 h (growth rate, μ, 0.20±0.008 h−1 in the presence of 2.5 % NaCl). The assays performed to evaluate the fast response of the cells when exposed to sodium chloride were carried out in the first 35 min after the addition of NaCl so that the response of those individual cells that were challenged could be assessed. Most of the studies published on bacterial adaptation concern the growth of stressed populations with analyses being performed on new cell generations. In the present study, the total lipids of non-stressed cells were mainly composed of the fatty acids C17:0 (8.2 %), C15:0 (13.3 %), C18:1ω9c (14.0 %), C16:1ω7 (20.6 %) and C16:0 (31.7 %) (data not shown). The degree of saturation of these cells, defined as the ratio between saturated and 0.25
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monounsaturated fatty acids, was 1.7 whilst the UI was 0.38 (data not shown). The sum of fatty acids other than saturated straight and monounsaturated reached only 0.9 % of the total lipid composition. When cells were exposed to different concentrations of sodium chloride, they responded by changing the fatty acid composition (Fig. 2). The concentrations allowing the highest growth rates, 2.5 and 3.5 % NaCl, induced a lower extent of changes than lower or higher concentrations. Nevertheless, these cells started producing hydroxy-substituted (HSFAs), saturated methyl-branch ed (SMBFAs), saturated cyclopropyl-branched (SCBFAs) and polyunsaturated (PUFAs) fatty acids in larger quantities than unchallenged cells. After 35 min in the presence of these two concentrations, R. erythropolis cells produced between 6 and 24-fold more of these fatty acids then their non-exposed controls (Fig. 2, 35 min). The sodium chloride concentrations tested induced modifications in the fatty acid profile of the cells already after only 6 min (Fig. 2, 6 min). The amount of monounsaturated fatty acids decreased, in general, to approximately half of the amount observed in cells with no salt present whilst the amount of SMBFAs, SCBFAs, HSFAs and PUFAs became significant, ranging in total between 7.8 and 14.7 % of the total fatty acids depending on the salt concentration. After 12 min, major changes were observed in cells in the presence of 5.5 % NaCl and these cells produced 14.4 % of total fatty acids as PUFAs (Fig. 2, 12 min). In these cells, MUFAs decreased to 20.8 %, when compared to control cells, which is exactly the same increase observed in the sum of all other classes of fatty acids (straight-chain saturated fatty acids (SSFAs), HSFAs, SMBFAs, SCBFAs and PUFAs). The major decreases were observed in the percentage of C18:1ω9c and C16:1ω7 (data not shown). The main polyunsaturated fatty acids produced by these cells in the presence of sodium chloride were eicosapentaenoic acid (EPA, C20:5ω3), arachidonic acid (ARA, C20:4ω6) and docosapentaenoic acid (DPA, C22:5ω3). After 20 min of exposure, R. erythropolis cells in the presence of 1 and 7.5 % NaCl produced also significant amounts of PUFAs (Fig. 2, 20 min). In fact, cells in the presence of 1 % NaCl presented a fatty acid profile similar to those exposed to 5.5 % and 7.5 %, resulting in an UI of ca. 1. In contrast, cells exposed to 0, 2.5 and 3.5 % NaCl presented an UI of around 0.4. The differences observed between the conditions allowing the highest growth rates and those observed in cells in the presence of 1, 2.5 and 3.5 % NaCl were highest after 35 min of exposure (Fig. 2, 35 min). The former cells presented a high percentage (over 70 %) of SSFAs, whilst the latter cells presented over 25 % of PUFAs and less than 53 % of SSFAs. No significant differences were observed during longer exposures under the doubling time (data not shown), indicating that the cells had attained the best
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branched fatty acids, SCBFAs saturated cyclopropyl-branched fatty acids, MUFAs monounsaturated fatty acids, HSFAs hydroxy-substituted fatty acids, PUFAs polyunsaturated fatty acids
adjustment in the membrane composition for each sodium chloride concentration in the first 30 min of exposure. A negative correlation between the UI and the maximum growth rate observed for the tested salt concentrations could be observed (Fig. 3). Concentrations of 2.5 and 3.5 % NaCl allowed the highest growth rates and lowest unsaturation indexes. The opposite behaviour in each parameter was observed for both lower and higher salt concentrations. This could indicate an increased metabolic burden when the cells
had to change the fatty acid composition to survive the tested salt concentrations, resulting in lower growth rates when the changes were more extent. The net surface charge of the R. erythropolis DSM 1069 cells, represented by the zeta potential, increased both with time and with sodium chloride concentration (Fig. 3). After only 6 min of exposure to salt, the zeta potential of the cells increased from −34.1 mV observed for control cells to −20.9 mV observed with cells in the presence of 7.5 % NaCl. However, the increase was only significant for concentrations higher than 3 %. Similar results were obtained after 12 min. For concentrations lower than 3.5 % NaCl, the zeta potential after 35 min was lower than that observed after 20 min, but for 35 min, a plateau at around −10.9 mV was measured for the highest concentrations. After 2.25 h, the zeta potential of the cells varied between −20.8 and −17.5 mV for NaCl concentrations of 1 and 7.5 %, respectively (data not shown). A relation between the amount of PUFAs present in the cells and the zeta potential of the cells could be observed for each salt concentration: in general, higher amounts of PUFAs were observed in cells presenting a less negative net surface charge (Fig. 5). Furthermore, the MUFA content was kept nearly constant for each tested NaCl concentration, although the cells changed the net surface charge (data not shown).
0.25
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Unsaturation index
Fig. 2 Fatty acid composition of R. erythropolis cells exposed to sodium chloride (bars) and corresponding calculated unsaturation index (line). SSFAs straight-chain saturated fatty acids, SMBFAs saturated methyl-
0.00 0
1
2
3
4
5
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7
8
NaCl, %(w/w)
Fig. 3 Unsaturation index (after 35 min of exposure) (open diamonds) and growth rate (filled squares) dependence on sodium chloride concentration
Appl Microbiol Biotechnol (2014) 98:5599–5606
5603 0
Discussion Zeta potential, mV
12 min 20 min
-10
35 min -20
-30
-40 0
2
4
6
8
NaCl, %
Fig. 4 Zeta potential of R. erythropolis cells after 6, 12, 20 and 35 min of exposure to different concentrations of sodium chloride
out by Okuyama et al. (2007), from the fact that the best studied bacterial species in terms of physiology, biochemistry and molecular biology were mesophilic species such as Escherichia coli containing no PUFAs. It is now accepted that long-chain PUFAs, such as EPA, DHA and ARA, are preferentially produced by marine bacteria (de Carvalho and Caramujo 2012; DeLong and Yayanos 1986; Hamamoto et al. 1995; Nichols 2003; Russell and Nichols 1999). PUFAs may also easily be decomposed by autoxidation during analytical procedures or by harsh transesterification methods, e.g. with boron trifluoride-methanol or high temperatures, and may not be detected (Eder 1995). The synthesis of PUFAs has also been observed during the adaptation of R. erythropolis DCL14 to extreme conditions (de Carvalho 2012). In the presence of sodium chloride, strain DCL14 produced a nearly constant amount of PUFAs reaching ca. 0.32 % of the total fatty acids, whilst in the presence of 1 % CuSO4, the cells synthesised 1.41 % of PUFAs. However, the amounts of PUFAs synthesised under
50
PUFA (% of total fatty acids)
R. erythropolis cells are able to perform a series of biotransformations and degradations that might be relevant for future biotechnological processes (de Carvalho and da Fonseca 2002, 2005; de Carvalho et al. 2007, 2009; Liu et al. 2012; Pirog et al. 2013; Schreiberova et al. 2012). However, if bioremediation and, in particular, bioaugmentation is envisaged, the cells must be able to endure environmental conditions far from optimal. These cells are good degraders of both aliphatic (de Carvalho et al. 2005, 2009; Liu et al. 2012) and aromatic compounds (de Carvalho et al. 2007; Eggeling and Sahm 1980; Schreiberova et al. 2012), are able to desulfurise dibenzothiophenes (Izumi et al. 1994; Ohshiro et al. 1996; Wang and Krawiec 1996; Yu et al. 2006) and may be used in the bioremediation of fuels (de Carvalho and da Fonseca 2005; Lee et al. 1999; Michel et al. 2004). Since most oil deposits and oilfield-produced water contain high concentrations of salt, the cells should be able to withstand salt shocks. R. erythropolis DSM 1069 showed a maximum growth rate in the presence of 2.5 % NaCl (Fig. 1), which is typical for many marine bacteria (Mutnuri et al. 2005). It was thus expected, as the conditions were further away from the optimum salt concentration, that the cells would produce larger changes in fatty acid composition. However, the responses of these cells during the first moments of exposure were, at the beginning of this study, unknown. In fact, the initial response of R. erythropolis cells to both low and high concentrations of sodium chloride showed significant increases of the unsaturation index of the lipids when compared to cells exposed to 2.5 % NaCl (Figs. 2 and 3). A very surprising finding of this study was the observed synthesis of PUFAs. Especially the fact that PUFAs were detected already after 6 min of exposure to NaCl and that their abundance reached up to 36.3 % of the total lipids in bacteria exposed for 35 min to 5.5 % NaCl has not been described in bacteria before. This appearance of PUFAs was accompanied by a concomitantly decrease in the percentage of MUFAs. Therefore, constitutively expressed fatty acid desaturases could be responsible in this fast post de novo synthetic modification of saturated and monounsaturated fatty acids to synthesise PUFAs. In fact, genes for such desaturases have been identified in other R. erythropolis: strain SK121 produces stearoyl-CoA 9-desaturases (Sebastian et al. 2009) whilst strain PR4 produces acyl-CoA desaturases (Takarada et al. 2005). Desaturases are specific for the position of the double bond they insert relative to the carboxyl end of the substrate molecule, and during PUFA production, a series of desaturases should be involved (Metz et al. 2001; Russell and Nichols 1999). Until the 1990s, it was considered that, with the exception of selected cyanobacteria, bacteria were unable to produce PUFAs. This assumption was derived probably, as pointed
6 min
1.0 % 40
30
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20
10 0 -40
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Fig. 5 Variation of the content of PUFA in relation to the zeta potential of the R. erythropolis cells exposed to 1–7.5 % (w/w) NaCl
5604
these conditions were about 1 order of magnitude lower than those observed in the present study. Fulco demonstrated that bacilli and mycobacteria could produce dienoic fatty acids by aerobic desaturation of fatty acids (Fulco 1974), but recent studies link the fatty acid composition to the bacterial ecological niche (de Carvalho and Caramujo 2012; de Carvalho and Fernandes 2010; Nichols 2003). As previously mentioned, it is generally accepted that only few bacteria are capable of synthesising PUFAs, whereby most of them are marine bacteria, in particular from cold, deep-sea sediments, fish and water (Hamamoto et al. 1995; Yano et al. 1998; Yano et al. 1997). Under such conditions, PUFAs are presumed to allow the maintenance of appropriate cytoplasmic membrane fluidity (Valentine and Valentine 2004), although in Shewanella livingstonensis, no significant difference was detected between the parental strain and an EPA-less mutant (Kawamoto et al. 2009). The best studied species regarding PUFA production in marine bacteria are members of the genus Shewanella. In these cells, eight open-reading frames (ORFs) were identified in a 38-kb genomic fragment: three were similar to fatty acid synthases and the remaining five were presumed to be effectors of C20 PUFA biosynthesis (Takeyama et al. 1997). However, Metz and co-workers have also described an alternative pathway for the synthesis of long PUFAs that does not require multiple desaturases and elongases but a polyketide synthaselike gene cluster (Metz et al. 2001). In Rhodococcus sp., polyketide synthase genes have also been found, e.g. strain RHA1 contains seven polyketide synthase genes (McLeod et al. 2006) and R. erythropolis PR4 contains a sequence encoding for a putative polyketide synthase with 2059 AA (Takarada et al. 2005). As pointed out by McLeod and colleagues, the high number of polyketide synthase genes in strain RHA1 was not expected but it evidences extensive secondary metabolism in these cells (McLeod et al. 2006). One of the main functions of these enzymes is the synthesis of mycolic acids in Rhodococcus strains and in other mycolic acid-containing bacteria (Portevin et al. 2004). Mycolic acids in Rhodococcus are known to also contain polyunsaturated aliphatic chains (Tomiyasu and Yano 1984). However, a relation between these genes, their necessity to be constitutively expressed and their function in the fast synthesis of PUFAs remains to be studied in rhodococci. The R. erythropolis DSM 1069 studied presented higher zeta potential values with increasing sodium chloride concentrations, and for each concentration, the value also increased with time (Fig. 4). Remarkably, the percentage of polyunsaturated fatty acids was highest for cells presenting the highest values of zeta potential (Fig. 5). It has been shown that PUFAs directly affect the activity of membrane proteins such as voltage-gated ion channels and free PUFAs may affect different membrane proteins including ion channels (Börjesson and Elinder 2011). Additionally, the multiple double bonds in
Appl Microbiol Biotechnol (2014) 98:5599–5606
these fatty acids may influence the bilayer organisation of membranes including, e.g. lateral pressure, microviscosity, curvature, permeability and elasticity which affect membrane proteins (Shaikh and Edidin 2008). PUFAs can also promptly inhibit sodium ion channels, which may be used to prevent cardiac arrhythmias in mammals (Xiao et al. 1995; Xiao et al. 2005). In R. erythropolis ATCC 15592, a channel-forming protein was described and the cell wall channel was found to be wide, water-filled and mainly permeable for cations due to the presence of negatively charged groups at the opening of the channel (Lichtinger et al. 2000). The relation between PUFAs and net surface charge observed in strain DSM 1069 could thus contribute to a lower number of negatively charged groups in ion channels and to an inhibition of sodium ions’ entrance to the cell. This is clearly an indication of a cleaver adaptation mechanism by this R. erythropolis strain. In conclusion, R. erythropolis cells were shown to be capable of very rapidly adapting high concentrations of sodium chloride. Hereby, modifications in the total lipid fatty acid content were observed already after only 6 min of exposure. The most remarkable adaptive response was the synthesis of polyunsaturated fatty acids, which are very unusual in mesophilic bacteria. Thus, bacteria of the genus Rhodococcus were shown to be able to promptly adapt to stress conditions. This is another proof for the remarkable feature of these bacteria, making them perfect biocatalysts both in terms of bioremediation and whole-cell biotransformation. Acknowledgments CCCR de Carvalho and MPC Marques would like to thank the Fundação para a Ciência e a Tecnologia (Portugal) for financial support (programs "Ciência 2007" and "FCT Investigators 2013", and SFRH/BPD/64160/2009, respectively) and Prof. Susete M. Dias and Joana Duarte for the help in the GC-MS analysis. This work was partially supported by a Portuguese-German integrated action (A-15/11) awarded by the Conselho de Reitores das Universidades Portuguesas (CRUP) and the Deutscher Akademischer Austauschdienst (DAAD).
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