Canadian Journal of Microbiology
Effect of toluene on Pseudomonas stutzeri ST -9 morphology: Plasmolysis, cell size and formation of outer membrane vesicles
Journal: Canadian Journal of Microbiology
Manuscript ID cjm-2016-0099.R1
Manuscript Type: Article
Date Submitted by the Author: 18-Mar-2016
Complete List of Authors: Michael, Esti; Ariel University, Chemical Engineering Nitzan, YeshayahuDraft ; Bar-Ilan University Langzam, Yakov; Bar-Ilan University, The Mina & Everard Goodman Faculty of Life Sciences Luboshits, Galia ; Ariel University, Cahan, Rivka; Ariel University, Chemical Engineering
Keyword: Pseudomonas, plasmolysis, toluene, outer membrane vesicles, morphology
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Effect of toluene on Pseudomonas stutzeri ST-9 morphology: Plasmolysis, cell size and 1
formation of outer membrane vesicles 2
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Esti Michael 1,2 , Yeshayahu Nitzan 2, Yakov Langzam 2, Galia Luboshits 1 and Rivka Cahan 1* 4
1Department of Chemical Engineering, Ariel University, Ariel 40700, Israel 5
2The Mina & Everard Goodman Faculty of Life Sciences, Bar Ilan University, 6
Ramat Gan 52900, Israel 7
*Corresponding author 8
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Abstract 1
Isolated toluene degrading Pseudomonas stutzeri ST 9 bacteria were grown in a minimal 2 medium containing toluene (100 mg L 1) (MMT) or glucose (MMG) as the sole carbon source, 3 with specific growth rates of 0.019 h 1 and 0.042 h 1, respectively. Scanning (SEM) as well as 4 transmission (TEM) electron microscope analyses showed that the bacterial cells grown to mid 5 log in the presence of toluene possess a plasmolysis space. TEM analysis revealed that bacterial 6 cells that were grown in MMT were surrounded by an additional "material" with small vesicles in 7 between. Membrane integrity was analyzed by leakage of 260 nm absorbing material and 8 demonstrated only 7% and 8% leakage from cultures grown in MMT compared to MMG. X ray 9 microanalysis showed a 4.3 fold increase in Mg and a 3 fold increase in P compared to cells 10 grown in MMG. FACS analysis indicated that the permeability of the membrane to propidium 11 iodide was 12.6% and 19.6% when Draftthe cultures were grown in MMG and MMT, respectively. 12
The bacterial cell length increased by 8.5%±0.1 and 17% ±2 as measured using SEM images and 13
FACS analysis, respectively. The results obtained in this research show that the presence of 14 toluene led to morphology changes such as plasmolysis, cell size and formation of outer 15 membrane vesicles. However, it does not cause significant damage to membrane integrity. 16
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Key words: Pseudomonas , plasmolysis, toluene, outer membrane vesicles, morphology 21
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Introduction 1
Bacterial cells have evolved various biochemical and physiological adaptation 2
mechanisms, which protect them from the lethal effects of organic solvents such as benzene, 3
ethylbenzene, toluene and xylene. These adaptation mechanisms include: (1) alteration of the 4
properties of the bacterial cell membrane (Tsitko et al. 1999); (2) morphological changes such as 5
bacterial cell size and formation of outer membrane vesicles (OMV) (Kobayashi et al. 2000); (3) 6
changes in the bacterial proteome which include stress proteins and proteins involved in energy 7
metabolism, efflux pumps and porins (Segura et al. 2005). 8
The bacterial cell envelope is the first target for the organic solvents. The cell wall of 9
Gram negative bacteria is composed of inner and outer membranes. The inner lipid bilayer 10
membrane contains mainly phosphatidylethanolamine. The outer membrane consists of an 11
asymmetric bilayer, where the innerDraft leaflet is enriched with phosphatidylethanolamine and the 12
outer leaflet is enriched with lipopolysaccharides (LPS). The inner and outer leaflets are separated 13
by a periplasmic space that contains a hydrophilic peptidoglycan which provides additional 14
stability (Schirmer 1998; Van Gelder et al. 2000). The outer membrane of Gram negative bacteria 15
is a hydrophobic barrier, which facilitates the passage of ions and molecules. Molecules with a 16
molecular mass higher than 600 1,000 Da cannot penetrate through the outer membrane into the 17
periplasm. The low permeability of the outer membrane to hydrophobic molecules is ascribed to 18
the presence of the LPS (Sikkema et al. 1995). 19
Several Gram negative bacteria constantly discharge outer membrane vesicles (OMVs) 20
during their growth. OMVs are bilayered spherical membranous structures, with a diameter 21
ranging between 50 and 250 nm. They contain LPS, outer membrane proteins, phospholipids and 22
periplasmic constituents. OMVs are a secretion and delivery structure through which soluble 23
molecules are released in a complex with insoluble material (Kulp and Kuehn 2010). The OMVs 24
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provide a route for delivery of virulence factors such as phospholipase C, proteases, proelastase, 1 and hemolysins (Kadurugamuwa and Beveridge 1995). 2
The phenomenon of a plasmolysis space pertains to spaces along the length of the bacterial 3 cell where the cytoplasmic membrane shrinks and retracts from the wall (Woldringh 1994). 4
Bacterial plasmolysis is known to occur in response to osmotic stress (20 30% sucrose) and 5 commonly occurs in Gram negative species. However, they have also been documented in Gram 6 positive bacteria (Korber et al. 1996; Schall et al. 1981; Woldringh 1994). A NaCl osmolality of 7
0.03 caused plasmolysis of E. coli which led to a reduction in cytoplasmic and cell water to 20% 8 and 50% of their original values, respectively, and increased periplasmic water by 300% (Cayley 9 et al. 2000). 10
Pseudomonas stutzeri bacterium belongs to the Gamma proteobacteria class, which is 11 widely distributed in a variety of Draft environments, such as soil, plants, water, air and clinical 12 specimens. This is a ubiquitous Gram negative bacterium with a high degree of physiological and 13 genetic adaptability (Rius et al. 2001; Sikorski et al. 2002). Taxonomically, P. stutzeri strains have 14 been grouped into 22 genomovars (Sikorski et al. 2005; Zhang et al. 2014). The isolated toluene 15 degrading bacterium P. stutzeri ST 9 belongs to genomovar 3 and its draft genome was recently 16 published (Gomila et al. 2015). 17
In the present study, P. stutzeri ST 9 was grown in a minimal medium with toluene as the 18 sole carbon source. SEM analysis showed that when P. stutzeri ST 9 was grown in the presence 19 of toluene, the cells possess "hole like" structures. Further investigation using TEM analysis 20 showed these "hole like" structures are plasmolysis spaces where the cytoplasmic membrane 21 shrinks and retracts from the wall. In order to determine whether this phenomenon causes damage 22 to the bacterial cell envelope, the membrane integrity was analyzed by leakage of 260 nm 23 absorbing material, X ray microanalysis and FACS. A change in cell size and OMV formation 24 were also observed. 25
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Materials and methods 1
Minimal Medium (MM): 1 liter MM was composed of 2.44 g Na 2HPO 4, 1.52 g KH 2PO 4, 0.5 g 2
(NH 4)2SO 4, 0.2 g MgSO 4·7H 2O, 0.05 g CaCl 2·2H 2O and 10 mL Trace element solution SL 4. 3
Trace element solution SL-4 was composed of 0.2 g FeSO 4·7H 2O, 0.5 g EDTA and 100 mL 4
Trace element solution SL 6. Trace element solution SL-6 was composed of 0.1 g 5
ZnSO 4·7H 2O, 0.03 g MnCl 2·4H 2O, 0.3 g H 3BO 3, 0.01 g CuCl 2·2H 2O 0.03 g Na 2MoO 4·2H 2O, 6
0.02 g NiCl 2·6H 2O, 0.2 g CoCl 2·6H 2O (457 mineral medium DSMZ). MMG- MM with (1%) 7
glucose as the sole carbon source. MMT- MM with toluene (100 mg L 1) as the sole carbon 8
source. 9
Bacterial strains: P. stutzeri ST 9, was previously isolated from toluene contaminated soil 10
(Gomila et al. 2015). Pseudomonas putida F1 (DSM 6899) was purchased from DSMZ, 11
Germany. Draft 12
Growth conditions . P. stutzeri ST 9 as well as P. putida F1 were inoculated to 0.15 OD 590 nm in 13
MMG or MMT at 30 ºC with shaking at 170 rpm. For growth curve examination, samples were 14
measured using a spectrophotometer (Thermo Electron Genesys 10UV, USA) at 590 nm. 15
Scanning Electron Microscopy (SEM) . P. stutzeri ST 9 was washed and fixed with Karnovsky 16
solution (2.5% glutaraldehyde and 2% paraformaldehyde) for 1 h or was fixed with a diluted 17
fixative (containing a smaller concentration of glutaraldehyde and paraformaldehyde) solution 18
containing 0.5% formaldehyde and 0.1% glutaraldehyde, followed by 1% osmium tetroxide. The 19
cells were then dehydrated by incubation in increasing ethanol concentrations. The specimens 20
were gold coated using a Polaron device. Scanning was performed with a JEOL 840 scanning 21
electron microscope at an accelerating voltage of 20 kV. 22
Transmission electron microscopy (TEM). P. stutzeri ST 9 was washed and fixed with 23
Karnovsky solution (2.5% glutaraldehyde and 2% paraformaldehyde) for 1 h or was fixed with a 24
diluted fixative of 0.5% formaldehyde and 0.1% glutaraldehyde, at room temperature, and washed 25
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three times in 0.1 M sodium cacodylate buffer. The samples were post fixed with 1% OsO 4 for 1 1 h followed by dehydration with solutions of increasing concentrations of ethanol (50%, 70%, 90% 2 and 100%) and propylene oxide. Afterwards, the samples were embedded in Agar 100 resin, Agar 3
Scientific, UK. Thin sections (70 nm) were cut, stained with uranyl acetate and lead citrate, then 4 observed under transmission electron microscope FEI Tecnai 120 kV. 5
Determination of membrane integrity by leakage of 260 nm absorbing material, X-ray 6 microanalysis and FACS: 7
Leakage of 260 nm absorbing material analysis. Nucleic acids absorb UV light at a wavelength 8 of 260 nm. The presence of nucleic acids in a suspension can be used as an indicator of damage to 9 the cell membrane caused by leakage of materials into the surrounding. P. stutzeri ST 9 was 10 grown in MMG or MMT to the earlyDraft and mid log phase and the release of nucleic acid 11 components into the medium during growth was measured. At indicated times, the cultures were 12 adjusted with MM to achieve a bacterial suspensions of 0.15 OD 590 nm. The suspensions were 13 harvested (8,000 g 10 min at 4°C) and the absorbance of the cell free supernatant was recorded at 14
260 nm (Gaur and Khare 2009; Je and Kim 2006; Oliva et al., 2004; Vanhauteghem et al. 2013). 15
Since toluene absorbs at 260 nm, similarly to nucleic acids, pretreatment of the toluene remaining 16 in the supernatant (of the culture growing in MMT) was carried out by extracting the toluene twice 17 using hexane. A culture of bacterial cells that was grown in MMG as in the experiment, but which 18 was treated with ethanol before harvesting, served as a positive control (calculated as 100% of 19 nucleic acids released). Absorbance at 260 nm was measured using a Thermo Electron Genesys 20
10UV Spectrophotometer (USA). 21
X-ray microanalysis (XRMA) . P. stutzeri ST 9 grown in MMG or MMT to mid log phase were 22 harvested, washed twice with 0.1 M ammonium acetate and resuspended in 30 µL ammonium 23 acetate. Each suspension (20 µL) was attached to an aluminum grid, air dried at room temperature 24 for at least 24 h and then coated with a layer of carbon (Cahan et al. 2008; Daniel Hoffmann et al. 25
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2012; Fern´andez et al., 2005; Katowsky et al. 1991; Raetz et al. 2007; Ramos et al., 1995). 1
XRMA was performed using an eXL Link X ray system attached to a JEOL 840 scanning electron 2
microscope. Each spectrum was determined with approximately 10 6 cells. The background level 3
was the same during all measurements. 4
FACS analysis. The propidium iodide (PI) dye is excluded by intact membranes of viable cells. 5
The presence of the dye within the cell therefore indicates disruption of the cell membrane. Flow 6
cytometry (FACS) analysis provides an opportunity to make rapid and quantitative measurements 7
within a few minutes of PI uptake by individual bacterial cells (thousands of cells) (Gottfredsson 8
et al. 1998; Jiang et al. 2014; Lv et al. 2014;Vanhauteghem et al. 2013). To determine membrane 9
integrity, P. stutzeri ST 9 was grown in MMT as well as MMG to the mid log phase. The cells 10 were harvested (8,000 g, 10 min, Draft 4°C) and the pellet was resuspended in 1 mL PBS (cell 11 concentration of 3*10 8 cells mL 1). Followed by staining with propidium iodide (PI) (1 mg/mL 12
stock solution; Sigma Aldrich, USA) for 5 min. PI fluorescence intensity was measured by FACS 13
Calibur (Becton Dickinson, USA) using excitation with an argon laser at 488 nm and emission 14
was collected in the FL2 cytometric channel. 15
Statistics. Data were expressed as means ± SD (standard deviation) of three replicates and were 16
statistically analyzed by ANOVA single factor analysis. The difference between the results was 17
considered significant if the P value was less than 0.05. 18
Results and discussion 19
Toluene degrading bacteria were isolated from toluene contaminated soil and were 20
identified using biochemical and molecular biology methods, as P. stutzeri genomovar 3, and 21
designated as P. stutzeri ST 9 (Gomila et al. 2015). The bacterial cells were grown in a minimal 22
medium containing toluene (100 mg L 1) (MMT) or glucose (MMG) as the sole carbon source 23
(Fig 1). The culture that was grown in MMG reached a maximum OD of about 1.1 at 590 nm after 24
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84 h and a specific growth rate of 0.042 h 1, whereas the culture that was grown in MMT reached 1 a maximum OD of about 0.6 at 590 nm after 96 h and a specific growth rate of 0.019 h 1. 2
The effect of toluene on the bacterial morphology was examined at the log phase and was 3 compared to a control culture, which was grown in MMG. Samples of bacterial cells were washed 4 and fixed in a Karnovsky solution (2.5% glutaraldehyde and 2% paraformaldehyde) and examined 5 using SEM analysis (Fig 2 A, B). As can be seen, P. stutzeri ST 9 bacterial cells that were grown 6 in MMG (Fig 2 A) showed a normal structure, exhibiting a typical rod shape with smooth 7 surfaces. However, when the bacterial cells were grown in MMT, plasmolysis spaces were 8 observed (Fig 2 B). In an attempt to verify that the plasmolysis spaces were not due to an effect of 9 the Karnovsky solution which contains relatively high fixative concentrations (2.5% 10 glutaraldehyde and 2% paraformaldehyde), the same experiment was performed, but with a 11 diluted fixative solution which containedDraft a smaller concentration of glutaraldehyde and 12 formaldehyde (0.5% formaldehyde and 0.1% glutaraldehyde) (Fig 2 C, D). As can be seen, the 13 diluted fixative solution led to the same results, where the bacterial cells which were grown in the 14 presence of glucose (Fig 2 C) exhibited a normal structure, whereas the culture that was grown in 15 the presence of toluene (Fig 2 D), demonstrated plasmolysis spaces (indicated by arrows). The 16 effect of toluene on the morphology of P. putida, which is known as a toluene degrader (Cho et al. 17
2000), was performed in order to clarify whether the plasmolysis induced by toluene is a 18 phenomenon unique to P. stutzeri ST 9. 19
P. putida was grown in MMG as well as MMT and fixed using Karnovsky solution. Plasmolysis 20 spaces also appeared when P. putida cultures were grown in MMT (Fig 2 F). When these bacterial 21 cells were grown in MMG (Fig 2 E), the cells exhibited a normal structure. 22
Further morphology investigation of the effect of toluene on P. stutzeri ST 9 was carried 23 out using TEM analysis. The bacterial cells were harvested at the log phase and fixed with 24
Karnovsky solution (Fig 3 A E). The control bacterial cells, which were grown in MMG (Fig 3 25
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A), exhibited a normal intracellular structure with diffuse DNA and integrity of the outer and 1
inner membranes. In the presence of toluene (MMT), several phenomena were observed: 1 2
plasmolysis spaces (indicated by arrows and designated "P", Fig 3 B); 2- bacterial cells 3
surrounded by an additional "material" (indicated by arrows and designated "M") with small 4
vesicles in between (indicated by arrows and designated "V") (Fig 3 C and D); 3- bacterial cells 5
surrounded by a "wide material" (indicated by arrows and designated "WM" Fig 3 E). Larger 6
pictures were added to Supplementary Information (supplemental 1 and supplemental 2). In order 7
to determine whether the relatively high fixatives concentration in the Karnovsky solution led to 8
plasmolysis, the bacterial cells were also prepared for TEM analysis, with the diluted fixative 9
solution (Fig 3 F). Plasmolysis was also observed when the samples were prepared with the 10
diluted fixative. 11
The phenomenon of bacterialDraft plasmolysis as a consequence of osmotic stress is well 12
known (Alemohammad and Knowles 1974; Korber et al. 1996; Woldringh 1994). However, 13
bacterial plasmolysis due to organic solvents is less known. Using SEM analysis, Park and co 14
authors showed "destructive openings on the cell envelopes" in Pseudomonas sp. DJ 12 bacterial 15
cells that were exposed to aromatic hydrocarbons such as biphenyl, 4 chlorobiphenyl (4CB) 4 16
hydroxybenzoate (4HBA) (Park et al. 2001). Duque and co authors examined a mutant cyoB 17
strain of the solvent tolerant P. putida DOT T1E after exposure to solvents. SEM analysis 18
showed plasmolysis in the mutant cells but not in the wild type (Duque et al. 2004). 19
The vesicles, which are seen in Fig 3 D, were also reported in several studies. OMVs 20
increase bacterial survival by removing a variety of harmful intrinsic or extrinsic agents from the 21
bacterial population (McBroom and Kuehn 2007). The toluene tolerant microorganism P. putida 22
H 2000 was found to release OMVs in the range of about 80 to 100 nm, in response to toluene 23
exposure. The toluene molecules, which adhere to the bacterial outer membrane, were eliminated 24
by OMVs formation. These OMVs contained a higher concentration of toluene molecules than the 25
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concentration found in the cell membrane. The maximum amount of toluene associated with the 1
OMVs was 0.625 mol/mol of lipid at 0.5 h after the addition of toluene, which decreased to 0.172 2 mol/mol at 6.0 h (Kobayashi et al. 2000). The adherence of toluene to OMVs is an ideal model of 3 organic solvent tolerance. This model supports the hypothesis that the outer membrane of Gram 4 negative bacteria is an active eliminator of toxic organic solvents, and not just a simple barrier. 5
We assume that the additional "material" shown in Fig 3 C E is exopolysaccharides 6
(EPS) which is known to be produced by many Pseudomonas species (Kachlany et al. 2001). 7
Polysaccharides are believed to protect bacterial cells from desiccation, heavy metals, organic 8 compounds or other environmental stresses. The strain, the culture conditions, and the type of 9 carbon source influence the amount and the composition of microbial EPS that is produced by a 10 certain species (Onbasli and Aslim 2009). 11
The phenomenon of plasmolysis,Draft which was observed using SEM and TEM analysis, led 12 to a further investigation of membrane integrity. Determination of the membrane integrity was 13 performed using analysis of leakage of 260 nm absorbing material (Fig 4), XRAM (Fig 5) and 14
FACS (Fig 6). 15
The main materials that absorb UV light at 260 nm are nucleic acids. The presence of 16 nucleic acids in a medium can serve as an indicator of damage to the cell membrane (Je and Kim 17
2006). Nucleic acid leakage into the surrounding was determined for cultures that were grown in 18 the presence of toluene and compared with cultures that were grown in the presence of glucose. 19
The cultures were harvested at the early and mid log phase. A positive control of cells with 20 significant membrane damage was performed by treatment with ethanol prior to harvesting (Fig 21
4). The absorbance of the positive control was 0.45 and 0.48 OD 260 nm (calculated as 100% 22 release of nucleic acids) at the early and mid log, respectively. The absorbance of cultures grown 23 in the presence of toluene at the early and mid log phase was 0.107 and 0.119 OD, respectively. 24
This was only 8% and 7% higher than the corresponding cultures grown in the presence of 25
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glucose. The calculated P value of the difference in the percentage of nucleic acids release from 1
cells that were grown in the presence of toluene compared to those grown in glucose, at the early 2
and mid log phase, was 0.07 and 0.08, respectively. These results show that there is no significant 3
leakage of nucleic acids from cells that were grown in the presence of toluene. This finding may 4
be correlated to membrane integrity. It was reported that when bacterial cells were grown in a 5
high solvent concentration (cyclohexane 33% v v), release of nucleic acid components into the 6
medium during the growth of a solvent tolerant strain Pseudomonas aeruginosa PseA was a 7
function of time and showed an absorbance of 0.5 OD at 260 nm (Gaur and Khare 2009). 8
The integrity of the bacterial membrane was also demonstrated by XRMA (Fig. 5). The 9
bacterial cells were grown in the presence of glucose or toluene as the sole carbon source (MMG 10
or MMT, respectively). The cultures were harvested at the mid log phase and were fixed for 11
XRMA. The ion composition shownDraft in Fig 5 revealed a 4.3 fold increase in the peak of Mg 12
(2.45±0.24) as well as a 3 fold increase in P (8.81±0.04) in cells grown in the presence of toluene 13
compared to those grown in glucose. The increase in Mg and P shows that the integrity of the 14
membrane was preserved, since damage to the membrane would possibly lead to ion leakage. The 15
increase in Mg 2+ was reported by several studies that showed that divalent cations such as Mg 2+ 16
and Ca 2+ can help stabilize and maintain the integrity of the outer membrane by binding between 17
adjacent LPS molecules (Katowsky et al. 1991; Raetz et al. 2007). It was found that in P. putida 18
DOT T1 grown in the presence of toluene, the addition of Mg 2+ led to an increase in membrane 19
integrity (Ramos et al., 1995). In the case of Pi accumulation, it was reported that many 20
microorganisms accumulate excess Pi in response to nutrient stress (Alcántara et al. 2014). 21
The integrity of the membrane was also demonstrated using FACS analysis (Fig 6 A). 22
The bacterial cells were grown to the mid log phase, in MMT or MMG. The bacterial cells were 23
stained with PI, which binds to double stranded DNA, but cannot penetrate the intact cell 24
membrane of the bacterial cells. The results show that in the presence of toluene, 19.6% of cells 25
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were stained with PI compared to 12.6% when the cells were grown in the presence of glucose. 1
This result again demonstrates that there was no significant damage to the bacterial cell 2 membrane when the cells were grown in the presence of toluene. 3
Examination of bacterial size in response to exposure to toluene was determined by 4 measuring the bacterial size from at least 10 different SEM fields. The total number of bacteria 5 that were counted from those which were grown in the presence of toluene or glucose were 40 6 and 60, respectively (all cells that were in a clear dividing stage were not taken into account). The 7 results show that the length of bacterial cells grown in the presence of toluene was 8.5%±0.1 8 higher than the length of bacterial cells grown in the presence of glucose. This result was 9 supported by FACS (Fig 6 B) analysis that revealed that the size of the bacterial cells that were 10 exposed to toluene was 17%±2 higher than the size of cells that were grown in the presence of 11 glucose. In the FACS analysis, all Draft cells in a sample of about 6,000 were taken into account, 12 including those in a dividing stage. 13
Changes in bacterial cell size were reported as a result of exposure to toxic hydrocarbons. 14
It was found that the cell size of Enterobacter sp. VKGH12 increased with increasing 15 concentrations of n butanol. This was observed only after the addition of non lethal 16 concentrations. The decrease in the surface area to cell volume ratio in response to the presence of 17 organic solvents leads to a reduction in the cell surface area, and especially the cytoplasmic 18 membrane, which is the major target for the toxic effects. The reduction of the relative surface 19 area represents an adaptive mechanism to the presence of toxic hydrocarbons (Neumann et al. 20
2005). On the other hand, a decrease in cell size was observed with Bacillus subtilis which 21 reduced their cell size when treated with aromatic hydrocarbons (Pinkart et al. 1996). 22
Mycobacterium species grown in a two phase aqueous organic and organic media tended to 23 shrink and decrease their surface roughness, indicating that they increased their surface area 24
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(Carvalho and Fonseca 2004). In this case, the increase in the cell surface area may enhance their 1
ability for uptake of potential growth substrates. 2
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Conclusions 4
Isolated toluene degrading P. stutzeri ST 9 was grown in a minimal medium containing 5
toluene or glucose as the sole carbon source. Toluene induced plasmolysis was shown using SEM 6
and TEM analysis. In order to verify whether the relative high fixatives concentration in the 7
Karnovsky solution (2.5% glutaraldehyde and 2% paraformaldehyde) led to plasmolysis, the 8
bacterial cells were also prepared for SEM and TEM analysis with a diluted fixative solution 9
(0.5% formaldehyde and 0.1% glutaraldehyde). Plasmolysis was observed also when the samples 10
were prepared with the diluted fixative. The membrane integrity was analyzed by leakage of 260 11
nm absorbing material, X ray microanalysisDraft and FACS, and revealed no significant damage to the 12
membrane. The bacterial cell length was increased by 8.5%±0.1 or 17% ±2 as measured using 13
SEM images and FACS analysis, respectively. In addition, OMV formation was observed as a 14
function of exposure to toluene. 15
Acknowledgements: This research was supported in part by the Samaria and Jordan Rift Valley 16
Regional R&D Center, the Research Authority of the Ariel University Center of Samaria and the 17
Rappaport Foundation for Medical Microbiology, Bar Ilan University (to Y.N.). 18
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Fig. 1 P. stutzeri ST 9 growth curve in the presence of toluene (A); glucose (B). 5
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Fig 2. SEM images of P. stutzeri ST 9 bacterial cells grown in MMG (A and C) and MMT (B and 7
D) and fixed using Karnovsky solution (2% glutaraldehyde and 2% paraformaldehyde) (A and B) or 8 with the diluted fixative (C and D). SEM images of P. putida bacterial cells grown in MMG (E) as 9 well as MMT (F) and fixed using Karnovsky solution. Arrows show plasmolysis spaces. 10
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Fig 3 . TEM images of P. stutzeri ST 9 grownDraft in MMT or MMG. A control bacteria grown in MMG; 12
B E bacterial cells that were grown in MMT and fixed using a Karnovsky solution. F bacterial cells 13 that were grown in MMT and fixed using a diluted fixative. The capital letters: P, V, M, WM 14 designated plasmolysis, vesicles, material and wide material, respectively. 15
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Fig. 4. The leakage of 260 nm absorbing material in cultures grown to early and mid log phase, in 17
MM with glucose as the sole carbon source ( ) or toluene ( ). A positive control that was treated 18 with ethanol prior of harvesting ( ). 19
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Fig. 5. X ray elemental spectra of P. stutzeri ST 9 bacterial cells grown to mid log in the 21 presence of toluene (A) and glucose (b). 22
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Fig 6: Flow cytometric analysis. Membrane permeability of P. stutzeri ST 9 cells measured using flow 24 cytometry with propidium iodide. The shaded area: MMT; bright area: MMG (A) . Histograms 25
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showing an indicative of size and peak height distribution (shown as FSC H) of P. stutzeri ST 9 in 1
toluene (MMT) (solid lines) or glucose (MMG) (dotted lines) (B). 2
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0 0 0 12 24 36 48 60 72 84 96 0 12 24 36 48 60 72 84 96 Time (h) Time (h) Fig. 1 P. stutzeri ST-9 growth curve in the presence of toluene (A); glucose (B). Draft
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Fig 2. SEM images of P. stutzeri ST-9 bacterial cells grown in MMG (A and C) and 4
MMT (B and D) and fixed using Karnovsky solution (2% glutaraldehyde and 2% 5
paraformaldehyde) (A and B) or with the diluted fixative (C and D). SEM images of P. putida 6
bacterial cells grown in MMG (E) as well as MMT (F) and fixed using Karnovsky solution. 7
Arrows show plasmolysis spaces. 8
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Fig 3. TEM images of P. stutzeri ST-9 grown in MMT or MMG. A-control bacteria grown in MMG; B-
E- bacterial cells that were grown in MMT and fixed using a Karnovsky solution. F-bacterial cells that were grown in MMT and fixed using a diluted fixative. The capital letters: P, V, M, WM designated plasmolysis, vesicles, material and wide material, respectively.
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Fig. 4. The leakage of 260 nm absorbing material in cultures grown to early and mid log phase, in 3 MM with glucose as the sole carbonDraft source ( ) or toluene ( ). A positive control that was 4 treated with ethanol prior of harvesting ( ). 5
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Fig. 5. X-ray elemental spectra of P. stutzeri ST-9 bacterial cells grown to mid log in the presence of toluene (A) and glucose (b).
Draft
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B A
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Fig 6: Flow cytometric analysis. Membrane permeability of P. stutzeri ST-9 cells measured using 2
flow cytometry with propidium iodide. The shaded area: MMT; bright area: MMG (A) . Histograms 3
showing an indicative of size and peakDraft height distribution (shown as FSC-H) of P. stutzeri ST-9 in 4
toluene (MMT) (solid lines) or glucose (MMG) (dotted lines) (B). 5
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