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Pseudomonas aeruginosa IBBML1 Adaptation to High Concentrations of Hydrocarbons

Mihaela Marilena Lazaroaie*

Center of Microbiology,Institute of Biology,Romanian Academy,296 Spl. Independentei St,060031,PO 56-53,Bucharest,Romania

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Abstract

The tolerance of Pseudomonas aeruginosa IBBML1 strain to 5-15% (v/v) pure or mixed saturated and monoaromatic hydrocarbons and the capacity of bacterial strain to use as single source of carbon 5%, 10% (v/v) pure or mixed saturated and monoaromatic hydrocarbons was different, according to the nature of the hydrophobic substrate, and also according to the hydrocarbons concentration. Benzene, toluene, ethylbenzene and cyclohexane, with the logarithm of the partition coefficient of hydrocarbons in octanol-water mixture (log POW) between 2.14 and 3.35, were more toxic for Pseudomonas aeruginosa IBBML1 cells, compared with n-hexane and n-hexadecane, with log POW 3.86 and 9.15, respectively. Pseudomonas aeruginosa IBBML1 presented high tolerance to the presence of mixture of saturated hydrocarbons in the liquid medium, but presented high sensitivity to the presence of mixture of monoaromatic hydrocarbons in the liquid medium. The adhesion of Pseudomonas aeruginosa IBBML1 cells to pure or mixed hydrocarbons depends on the substrate nature and the concentration of hydrophobic substrate, and also according to culture conditions, with values between 0.23% and 65.23%. Transmission electron microscopy, thin-layer chromatography and electrophoresis studies revealed the existence of significant cellular and molecular differences between the Pseudomonas aeruginosa IBBML1 cells grown in the absence of hydrocarbons (control) and the cells grown in the presence of saturated (except n-hexane) and monoaromatic (except ethylbenyene) hydrocarbons. Transmission electron microscopy revealed: wider periplasmic space, outer membrane lesions, cells shape modifications and wavy outer membrane. Thin-layer chromatography studies revealed changes in lipid phospholipid headgroups (elevated levels of phosphatidylglycerol or the absence of phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, and cardiolipin) and glycolipids (elevated level of rhamnolipid methyl ester or the absence of rhamnolipid methyl ester, rhamnolipid acid). Electrophoresis studies revealed strong induction of the synthesis of some proteins, while other proteins were synthesized in barely detectable quantities.

Keywords

Adaptation; Bacteria; Pure or mixed hydrocarbons.

1. Introduction

Petroleum hydrocarbons are important energy resources used in industry and in our daily life. Petroleum hydrocarbons spills occur frequently and they are a major cause of environment pollution. In Romania,in almost 150 years of crude oil extraction and processing,took place massive spills of petroleum and petroleum products,both on extraction and processing areas,and also on the indigenous and imported petroleum transportation routes and stockpiling areas. As a result of the development of the petrochemical industry,Romania,like other countries from Europe,is dealing with serious problems concerning hydrocarbons pollution of soil and water. Great efforts are being carried out to develop strategies and technologies for preventing and controlling global pollution with hydrocarbons. Still,the increasing numbers of accidents that occur in the environment make the pollution with hydrocarbons a major problem for the next decades.

The petroleum hydrocarbons are compounds with high toxicity for the bacterial cells,so that,as a result of their partition in lipid bilayer,they produce significant modifications in the membrane structure and functions [1-13]. The induced modifications differ according to the type of hydrocarbon and to the localization in the lipid bilayer. Nevertheless,bacteria that can grow under these conditions have been found [4,14-16],suggesting that they may have the ability to resist to high concentrations of hydrocarbons. The molecular mechanisms for the resistance of bacteria to hydrocarbons have been studied for some Gram-negative bacteria [4-7,17-23]. Regarding tolerance to hydrocarbons,a number of elements have been suggested as being involved in the response to these toxic compounds: metabolism of hydrocarbons to non-toxic metabolites [7],rigidification of the cell membrane via alteration in the composition of phospholipids [2,8,22,24-26],alteration in the cell surface to make it less permeable by lack of some porins [27-29],formation of vesicles for removing hydrocarbons from the cell [12],modification of lipopolysaccharides [30,31],efflux of hydrocarbons in an energy-dependent process [6,7,9,21,23,32-38],and a flagellar transport system involved in the transfer of some proteins implicated in hydrocarbons exclusion to the periplasmic space or to the outer membrane [38-40].

Although there are numerous studies on cellular and molecular modifications induced by hydrocarbons to different strains of Pseudomonas aeruginosa,still there are few studies on the modifications induced by hydrocarbons mixtures and there is no study to compare the effects of pure hydrocarbons and the effects of mixture of hydrocarbons. The biotechnological importance of the Pseudomonas genus bacteria makes them ideal candidates for cellular and molecular studies. The aim of this study was to test the tolerance and degradative capacity of Pseudomonas aeruginosa IBBML1 to 5-15% (v/v) pure or mixed saturated and monoaromatic hydrocarbons. The modifications induced on cellular and molecular level on Pseudomonas aeruginosa IBBML1,grown in the presence of 5% and 10% (v/v) pure or mixture of saturated and monoaromatic hydrocarbons are also presented in this study.

2. Methods

Bacterial strain. Isolation of IBBML1 bacterial strain from Poeni petroleum sludge (Teleorman County,Romania) was carried out on mineral medium and crude oil 5% (v/v) as single carbon source,using the enriched cultures method. The taxonomic affiliation of IBBML1 bacterial strain was determined based on its phenotypic characteristics and also based on the G+C content of the bacterial chromosome [41]. The identification result for isolated bacterial strain with API profile 1350575 and with G+C content of the DNA 66.2 mol% corresponded to Pseudomonas aeruginosa.

2.1 Tolerance and degradative capacity of Pseudomonas aeruginosa IBBML1 to saturated (n-hexane,n-hexadecane,cyclohexane,mixture of them) and monoaromatic (benzene,toluene,ethylbenzene,mixture of them) hydrocarbons. Bacterial cells were cultivated on liquid LB-Mg medium [24] (control) or liquid M9-Mg medium [4] supplemented with 0.6% (w/v) ferric citrate (control) and on the same media in the presence of 5-15% (v/v) hydrocarbons. Flasks were sealed,incubated 24 hours at 28°C on a rotary shaker (150-200 rpm). The growth of the bacterial strain was determined by spetrophotometric measurement of the optical density (OD660) and by determining the viability of bacterial cells (CFU/ml). Bacterial cells in the exponential phase of growth were also plated on solid LB-Mg medium (control) or solid M9-Mg medium supplemented with 0.6% (w/v) ferric citrate (control) and on the same media that was overlaid with hydrocarbons [42] or supplied with hydrocarbons in the vapor phase [4]. Petri plates were sealed and the formation of hydrocarbonresistant bacterial colonies on the agar was determined after 24 hours incubation at 28°C.

2.2 Cellular and molecular modifications induced by saturated (n-hexane,n-hexadecane,cyclohexane,mixture of them) and monoaromatic (benzene,toluene,ethylbenzene,mixture of them) hydrocarbons on the IBBML1 bacterial strain. Cells were cultivated on liquid LBMg medium (control) and on LB-Mg medium in the presence of 5%,10% (v/v) hydrocarbons. Flasks were sealed,incubated 24 hours at 28°C on a rotary shaker (150-200 rpm).

2.2.1 Modifications induced by hydrocarbons to cell wall hydrophobicity. Bacterial adhesion to hydrocarbons was determined using the method of Rosenberg et al. [43]. The bacterial adhesion to hydrocarbons was also studied on wet mount with the optical microscope.

2.2.2 Modifications induced by hydrocarbons to cell ultrastructure. The samples were prepared using the method indicated by Ramos et al. [4]. Thin sections were fixed on special grids and poststained with uranyl acetate and lead citrate,and examined in a Selmi 125 transmission electron microscope (TEM),at an accelerating voltage of 75 kV.

2.2.3 Modifications induced by hydrocarbons to lipid profile. Lipids were extracted using a modified Bligh-Dyer technique [44]. The samples were spotted onto 20×20cm Silica gel 60 TLC aluminium sheets (Merck),and the separation was performed using a mixture of chloroform-methanol-acetic acidwater (85:22.5:10:4 v/v/v/v) as mobile phase. For phospholipids visualization,the plates were treated with 10% (w/v) molybdatophosphoric acid hydrate in ethanol. For glycolipids visualization,the plates were treated with 0.5% (w/v) -naphthol in methanol-water mixture (1:1 v/v) and sulfuric acidethanol mixture (1:1 v/v). The identification of the investigated components was done based on their motilities (Rf) and their comparison with those of standards.

2.2.4 Modifications induced by hydrocarbons to protein profile. Membrane and periplasmic protein fractions were extracted by resuspending the bacterial cell pellet in 10 mM HEPES-NaOH,pH 7.6,10 mM EDTA,and 10 mM MgCl2. Before the polyacrylamide gel electrophoresis,the samples were solved in Laemmli buffer and denaturated at 95°C,for 5 min. 30 μg of protein per lane were loaded onto a 10% polyacrylamide gel. Broad range protein molecular weight marker (Promega) was used for molecular mass estimation. Gels were stained with Coomassie brilliant blue and destained in ethanol-glacial acetic acid-water (4.5:1:4.5 v/v/v) mixture. Protein content was measured by the method of Bradford [45].

Reagents. n-hexane (96% pure),n-hexadecane (99% pure),cyclohexane (99.7% pure),benzene (99% pure),toluene (99% pure) were obtained from Merck,ethylbenzene (98% pure) was obtained from Sigma-Aldrich. Other reagents used were procured from Merck (E. Merck,Darmstadt,Germany),Sigma-Aldrich (Saint-Quentin-Fallavier,France),Difco Laboratories,(Detroit,Michigan,USA),Promega (Promega GmbH,Mannheim,Germany) or bioMérieux (Marcy-l’Etoile,France).

3. Results and Discussion

Most of our knowledge about pollutants and the way they are biodegraded in the environment has previously been shaped by laboratory studies using isolated hydrocarbon-degrading bacterial strains.

3.1 Tolerance and degradative capacity of Pseudomonas aeruginosa IBBML1 to saturated and monoaromatic hydrocarbons. The key factor for hydrocarbons degradation is represented by the tolerance that some microorganisms exhibit toward these compounds. Pseudomonas aeruginosa IBBML1 bacterial strain was able to grow on liquid and solid LB-Mg medium,in the presence of saturated and monoaromatic hydrocarbons. The intensity of bacterial growth on liquid medium,estimated through the degree of turbidity of the culture liquids (OD660),was different according to the nature of the hydrophobic substrate,and also according to the hydrocarbons concentration (5- 15% v/v),with values between 0.010 and 1.526 (Table 1). The maintenance or loss of viability of the cells grown on reach liquid medium in the presence of hydrocarbons,estimated by determining the most probable number of bacterial cells on solid medium,were different according to the nature of the hydrophobic substrate,and also according to the hydrocarbons concentration (5-15% v/v),with values between 0 and 2.5×1010 CFU/ml (data not shown). The microorganisms’ sensitivity to hydrocarbons depends on the logarithm of the partition coefficient of hydrocarbons in octanol-water mixture (logPOW). The toxicity of hydrocarbons is generally in inverse correlation with logPOW,thus hydrocarbons with logPOW between 1.5 and 3.5 are extremely toxic for microorganisms [2]. However,hydrocarbon toxicity depends not only on the inherent toxicity of the compound,but also on the intrinsic tolerance of the bacterial strain [7]. Benzene,toluene,ethylbenzene and cyclohexane,with log POW 2.14,2.64,3.17 and 3.35,respectively,were more toxic (OD660 = 0.016-0.608) for Pseudomonas aeruginosa IBBML1 cells,compared with n-hexane and n-hexadecane (OD660 = 0.495-1.456),with log POW 3.86 and 9.15,respectively. Pseudomonas aeruginosa IBBML1 presented high sensitivity (OD660 = 0.016-0.218) to the presence in the liquid medium of benzene,in concentrations of 5%,10% (v/v) and especially 15% (v/v). Pseudomonas aeruginosa IBBML1 presented higher tolerance to the presence in the liquid medium of mixture of saturated hydrocarbons (OD660 = 1.260-1.526),compared with the tolerance to the presence of individual saturated hydrocarbons (OD660 = 0.354-1.456). Bacterial cells presented higher sensitivity to the presence in the liquid medium of mixture of monoaromatice hydrocarbons (OD660 = 0.010-0.166),compared with the sensitivity to the presence of individual monoaromatic hydrocarbons (OD660 = 0.016-0.506). The growth of Pseudomonas aeruginosa IBBML1 strain on agar LB-Mg medium overlaid with hydrocarbons was 50-100% in the presence of pure or mixed saturated hydrocarbons,and 50% in the presence of ethylbenzene. The growth of Pseudomonas aeruginosa IBBML1 strain on agar LBMg medium supplied with hydrocarbons in the vapor phase was 50-100% in the presence of pure or mixed saturated hydrocarbons,and 100% in the presence of ethylbenzene. It can be observed also,like in the previous assay,the high sensitivity of Pseudomonas aeruginosa IBBML1 strain to the presence of benzene,mixture of monoaromatic hydrocarbons,and also to the presence of toluene (Table 1).

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The tolerance to different hydrocarbons does not seem to be related with degradation. A lot of microorganisms are able to tolerate a wide range of hydrocarbons but they are not capable to degrade or to transform them [8;17;33;46]. Thus I decided to test the capacity of Pseudomonas aeruginosa IBBML1 bacterial strain to use saturated and monoaromatic hydrocarbons as single source of carbon. Pseudomonas aeruginosa IBBML1 was able to use 5% and 10% (v/v) n-hexane,n-hexadecane,cyclohexane,and mixture of saturated hydrocarbons as single source of carbon (OD660 = 0.214-0.562) (Table 1). Bacterial strain was able to use 5% (v/v) benzene,toluene,ethylbenzene,mixture of monoaromatic hydrocarbons (OD660 = 0.096-0.303),and 10% (v/v) toluene,ethylbenzene (OD660 = 0.136,0.213) as single source of carbon. The maintenance or loss of viability of the cells grown on minimal liquid medium in the presence of hydrocarbons,estimated by determining the most probable number of bacterial cells on solid medium (with values between 0 and 1.2×107 CFU/ml,data not shown) were correlated with growth (with values between 0.020 and 0.562).

The growth of Pseudomonas aeruginosa IBBML1 strain on agar M9-Mg medium overlaid with hydrocarbons was 10-25% in the presence of pure or mixed saturated hydrocarbons,and was below 0.01% in the presence of pure or mixed monoaromatic hydrocarbons (Table 1). Although it was able to use 5% (v/v) benzene,toluene,ethylbenzene,mixture of monoaromatic hydrocarbons and 10% (v/v) toluene and ethylbenzene in liquid medium,the Pseudomonas aeruginosa IBBML1 bacterial strain did not use monoaromatic hydrocarbons as single carbon source in solid medium. This can be explained by the fragmentation of liquid medium surface film in small droplets,more available to bacterial degradation,due to the stirring (150-200 rpm). The growth of Pseudomonas aeruginosa IBBML1 strain on agar M9-Mg medium,supplied with hydrocarbons in the vapor phase,was 10-25% in the presence of pure or mixed saturated hydrocarbons,and 25% in the presence of ethylbenzene (Table 1).

3.2 Cellular and molecular modifications induced by saturated and monoaromatic hydrocarbons. According to the literature [2,4-9,11,12,18,22,23,47-49] hydrocarbons are generally toxic to microbial cells,as a result of their partition in the lipid bilayer,where they can cause significant modifications in the membrane structure (appearance of wider periplasmic space,appearance of outer membrane vesicles which contain hydrocarbons,appearance of inclusions which contain hydrocarbons,disorganization of the cytoplasmic membrane,deformation of the cell wall) and functions (loss of ions,metabolites,lipids and proteins,dissipation of the pH gradient and electrical potential,inhibition of membrane proteins functions). This is often followed by cell lysis and death.

3.2.1 Modifications induced by hydrocarbons to cell wall hydrophobicity. The adhesion of Pseudomonas aeruginosa IBBML1 cells to pure or mixed hydrocarbons depended on the substrate nature and concentration of hydrophobic substrate,and also on the culture conditions,with values between 0.23% and 65.23% (Table 2). Although the hydrocarbons are compounds with relatively low water solubility,the solubility rate may increase by increasing their specific surface,as a result of the mechanical dispersion realized by stirring the tubes containing the aqueous phase (cell suspension) and the organic phase (hydrocarbon) [50,51]. The strong stirring of the tubes allows the contact between the hydrocarbons microdroplets (obtained through mechanical dispersion,as a result of stirring) and the bacterial cells. The hydrocarbon microdroplets represent a hydrophobic substrate for the bacterial cell adhesion. The hydrocarbons microdroplets to which the bacterial cells adhered are stable and can be found at the interface between the aqueous and organic layer,causing a decrease of the turbidity (OD660) in the aqueous phase. Pseudomonas aeruginosa IBBML1 cells presented lower hydrophobicity (0.23%-29.41%) when the growth was done on LB-Mg medium with 5%,10% (v/v) hydrocarbons,compared with cell hydrophobicity (2.51%-65.23%) when the growth was done on LBMg medium without hydrocarbons (in the case of control) (Table 2). The low hydrophobicity of the cell envelope represents a defensive mechanism,which prevents the hydrocarbons accumulation in high concentration in the bacterial cell membranes [7,11,31,41,51].

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3.2.2 Modifications induced by hydrocarbons to cell ultrastructure. The transmission electron microscopy (TEM) studies did not make evident the existence of some significant structural differences between the Pseudomonas aeruginosa IBBML1 cells grown in the absence of hydrocarbons (Figure 1a) and the cells grown in the presence of 5%,10% (v/v) n-hexane (Figure 1b),ethylbenzene (Figure 1h). There were significant structural differences between the Pseudomonas aeruginosa IBBML1 cells grown in the absence of hydrocarbons (Figure 1a) and the cells grown in the presence of other saturated and monoaromatic hydrocarbons,used in concentration of 5%,10% (v/v). The structural modifications differed according to the nature of the hydrophobic substrate,but for the same hydrocarbon there were not differences for concentrations of 5% and 10% (v/v). To the Pseudomonas aeruginosa IBBML1 cells,grown on LB-Mg medium in the presence of 5%,10% (v/v) nhexadecane (Figure 1c),mixture of saturated hydrocarbons (Figure 1e),it was observed the appearance of a wider periplasmic space than in the cells grown on LB-Mg medium in the absence of hydrocarbon. To the Pseudomonas aeruginosa IBBML1 cells,grown on LB-Mg medium in the presence of 10% (v/v) mixture of saturated hydrocarbons (Figure 1e),5%,10% (v/v) benzene (Figure 1f) and toluene (Figure 1g),it was observed the appearance of some lesions at the outer membrane level. To the Pseudomonas aeruginosa IBBML1 cells,grown on LB-Mg medium in the presence of 5%,10% (v/v) cyclohexane (Figure 1d),benzene (Figure 1f) and toluene (Figure 1g),it was observed that the cells are surrounded with a material,of exopolysaccharides nature probably. To the Pseudomonas aeruginosa IBBML1 cells grown on LB-Mg medium in the presence of 5%,10% (v/v) benzene (Figure 1f),and toluene (Figure 1g),there were observed modifications of the cells shape. The cell wall of Pseudomonas aeruginosa IBBML1 cells grown on LB-Mg medium in the absence of hydrocarbon is smooth,while the cell wall of the cells grown on LB-Mg medium in the presence of 5%,10% (v/v) mixture of monoaromatic hydrocarbons (Figure 1i) is deformed and the outer membrane is wavy.

electronic-biology-Pseudomonas-aeruginosa

Figure 1. The cell structure modifications of Pseudomonas aeruginosa IBBML1 in the presence of saturated and monoaromatic hydrocarbons 10% (v/v). Control (a); n-hexane (b); n-hexadecane (c); cyclohexane (d); mixture of saturated hydrocarbons (e); benzene (f); toluene (g); ethylbenzene (h); mixture of monoaromatic hydrocarbons (i); wider periplasmic space (wPPS); exopolysaccharides material (EPSm); outer membrane lesions (OMl); cells shape modifications (CSm); wavy outer membrane (wOM).

3.2.3 Modifications induced by hydrocarbons to lipid profile. The phospholipids found,based on their Rf values (Figure 2 a),in Pseudomonas aeruginosa IBBML1 cells grown in the absence of hydrocarbons and in the presence of hydrocarbons were phosphatidylcholine (PC with Rf 0.37-0.41),phosphatidylethanolamine (PE with Rf 0.65-0.71),phosphatidylglycerol (PG with Rf 0.71-0.76),and cardiolipin/diphosphatidylglycerol (CL with Rf 0.83- 0.89). It was not observed the presence of phosphatidylserine (PS). The thin-layer chromatography (TLC) studies did not make evident the existence of some significant differences between lipid profile of the Pseudomonas aeruginosa IBBML1 cells grown in the absence of hydrocarbons and the cells grown in the presence of 5%,10% (v/v) n-hexane,5% (v/v) cyclohexane,5%,10% (v/v) ethylbenzene,mixture of monoaromatic hydrocarbons. Pseudomonas aeruginosa IBBML1 cells grown in the presence of 5%,10% (v/v) n-hexadecane,5% (v/v) mixture of saturated hydrocarbons had elevated levels of PG. Pseudomonas aeruginosa IBBML1 cells grown in the presence of 10% (v/v) cyclohexane exhibit the absence of PC. Pseudomonas aeruginosa IBBML1 cells grown in the presence of 10% (v/v) mixture of saturated hydrocarbons,5%,10% (v/v) benzene,toluene exhibits the absence of PE,PC,PG and CL. This was expected,because the electron microscopy studies made evident the appearance of some lesions at the outer membrane level (Figures 1e to 1g).

electronic-biology-glycolipids-modifications

Figure 2. The lipid profile (phospholipids, glycolipids) modifications of Pseudomonas aeruginosa IBBML1 in the presence of saturated and monoaromatic hydrocarbons. Control (C); 5% (1), 10% (2) n-hexane; 5% (3), 10% (4) n-hexadecane; 5% (5), 10% (6) cyclohexane; 5% (7), 10% (8) mixture of saturated hydrocarbons; 5% (9), 10% (10) benzene; 5% (11), 10% (12) toluene; 5% (13), 10% (14) ethylbenzene; 5% (15), 10% (16) mixture of monoaromatic hydrocarbons; origin (O); solvent front (F); phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL); rhamnolipid acid (R-A), rhamnolipid methyl ester (R-Me).

According with the literature the initial damage caused by some hydrocarbons on bacterial cells occurs by disruption of phospholipids membranes [2,11]. Hydrocarbons can modify saturated-tounsaturated fatty acids ratio or induce cis-to-trans isomerization of unsaturated fatty acids and also can produce changes in phospholipid headgroups [5,7,8,19,52-55]. These changes are interpreted as a way for bacteria to maintain membrane fluidity and impermeability or to restore membrane integrity and to reduce hydrocarbons partitioning in the membrane [2,7,49].

The glycolipids found,based on their Rf values (Figure 2b),in Pseudomonas aeruginosa IBBML1 cells grown in the absence of hydrocarbons and in the presence of saturated or aromatic hydrocarbons were rhamnolipid acid (R-A lower spots with Rf 0.36-0.38) and rhamnolipid methyl ester (R-Me higher spots with Rf 0.70-0.76). For the Pseudomonas aeruginosa IBBML1 cells,the production of rhamnolipid methyl ester increased in the presence of 5%,10% (v/v) n-hexadecane,and 5% (v/v) mixture of saturated hydrocarbons. Pseudomonas aeruginosa IBBML1 cells grown in the presence of 10% (v/v) mixture of saturated hydrocarbons,benzene,toluene exhibited the absence of rhamnolipid methyl ester,and the cells grown in the presence of 10% (v/v) mixture of saturated hydrocarbons,and 5%,10% (v/v) benzene,toluene,etylbenzene,mixture of monoaromatic hydrocarbons exhibited the absence of rhamnolipid acid. It was observed that rhamnolipids (monorhamnolipid acid,monorhamnolipid methyl ester,dirhamnolipid acid,dirhamnolipid methyl ester) are not absolutely required for the utilization of hydrocarbons (hexadecane,octadecane),but they facilitate the use of these substrates,leading to the mediated uptake of solubilized hydrocarbons [56-58].

3.2.4 Modifications induced by hydrocarbons to membrane and periplasmic protein profile. To investigate the modifications induced by hydrocarbons to membrane and periplasmic protein profile,one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D SDS-PAGE) was used. Many more types of variation in protein sequences can be distinguished on one-dimensional gels in the absence of denaturants such as urea used in two-dimensional electrophoresis [59]. Onedimensional gel electrophoresis showed the existence of the proteins with estimated molecular weights of 110-,105-,87-,77-,68-,60-,54-,50-,40-,33-,30-,28-,13- and 10-kDa in the membrane and periplasmic protein profile of the Pseudomonas aeruginosa IBBML1 cells grown in the absence of hydrocarbons (Figure 3 ). The electrophoresis studies showed the existence of some differences in the membrane and periplasmic protein profile of the Pseudomonas aeruginosa IBBML1 cells grown in the absence of hydrocarbons (control),compared with the membrane and periplasmic protein profile of the cells grown in the presence of saturated and monoaromatic hydrocarbons. It was observed strong induction of the synthesis of some proteins in the membrane and periplasmic protein profile of the Pseudomonas aeruginosa IBBML1 cells grown in the presence of 5% (v/v) n-hexadecane,5%,10% (v/v) mixture of saturated hydrocarbons,5%,10% (v/v) benzene,while it was observed repression of the synthesis of some proteins in the presence of 10% (v/v) n-hexadecane,5%,10% (v/v) cyclohexane,toluene. No modifications were observed in the membrane and periplasmic protein profile of the Pseudomonas aeruginosa IBBML1 cells grown in the presence of 5%,10% (v/v) n-hexane,ethylbenzene,mixture of monoaromatic hydrocarbons.

electronic-biology-benzene-toluene

Figure 3. The protein profile modifications of Pseudomonas aeruginosa IBBML1 in the presence of saturated and monoaromatic hydrocarbons. Broad range protein molecular weight marker, Promega (M); control (C); 5% (1), 10% (2) n-hexane; 5% (3), 10% (4) n-hexadecane; 5% (5), 10% (6) cyclohexane; 5% (7), 10% (8) mixture of saturated hydrocarbons; 5% (9), 10% (10) benzene; 5% (11), 10% (12) toluene; 5% (13), 10% (14) ethylbenzene; 5 % (15), 10% (16) mixture of monoaromatic hydrocarbons.

Similar modifications were revealed by Segura et al. [23]. The presence of toluene in the culture medium in which Pseudomonas putida DOT-T1E has been cultured provoked drastic changes in the protein pattern,indicating a complex response of bacterial cells to hydrocarbons. As part of this response,new proteins involved in hydrocarbon tolerance were synthesized (i.e. proteins involved in the catabolism of toluene,proteins involved in the channeling of metabolic intermediates to the Krebs cycle and activation of purine biosynthesis,proteins involved in sugar transport,stress-related proteins) and an increased expression of some preexisting proteins (i.e. efflux pumps belonging to the resistancenodulation- cell division family) also took place,counteracting the decrease in activity due to membrane structural damage caused by the presence of toluene inside the cell.

4. Conclusions

Pseudomonas aeruginosa IBBML1 was able to tolerate and also to use saturated (n-hexane,nhexadecane,cyclohexane,mixture of them) and monoaromatic (benzene,toluene,ethylbenzene,mixture of them) hydrocarbons as single source of carbon. Benzene,toluene,ethylbenzene and cyclohexane,with log POW between 2.14 and 3.35,were more toxic for Pseudomonas aeruginosa IBBML1 cells,compared with n-hexane and nhexadecane,with log POW 3.86 and 9.15,respectively. Pseudomonas aeruginosa IBBML1 presented high tolerance to the presence in the liquid medium of mixture of saturated hydrocarbons,but presented high sensitivity to the presence of mixture of monoaromatic hydrocarbons. It was observed a cumulative effect of the cell toxicity only when it was used the mixture of aromatic hydrocarbons. The adhesion of Pseudomonas aeruginosa IBBML1 cells to pure or mixed hydrocarbons depended on the substrate nature and concentration of hydrophobic substrate,and on the culture conditions,with values between 0.23% and 65.23%. TEM,TLC and 1D SDS-PAGE studies revealed the existence of significant cellular and molecular differences between the Pseudomonas aeruginosa IBBML1 cells grown in the absence of hydrocarbons (control) and the cells grown in the presence of some saturated (n-hexadecane,cyclohexane,mixture of saturated) and monoaromatic (benzene,toluene,mixture of monoaromatic) hydrocarbons. This is only a starting point. Further studies will be carried out,as well as the genomic DNA will be screened by PCR for the presence of catabolic genes involved in known hydrocarbons biodegradative pathways.

Acknowledgements

This study was supported by the grant of the Romanian Academy of Science.

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