ORIGINAL PAPER

Zileena Zahir Kimberley D. Seed Jonathan J. Dennis

Isolation and characterization of novel organic solvent-tolerant bacteria

Received: 15 July 2005 / Accepted: 14 September 2005 / Published online: 20 October 2005 Springer-Verlag 2005

Abstract Some organic solvents are extremely toxic toliving organisms by virtue of their ability to partitioninto and disrupt the normal functioning of biologicalmembranes. In recent years, several bacteria have beendiscovered that are more tolerant to these toxic solventsthan most microorganisms. Using enrichment proce-dures, we have isolated new organic solvent-tolerantbacteria from both hyrdocarbon-contaminated andpristine soil samples. These organisms were character-ized by several dierent experimental proceduresincluding description of their cellular physiology, 16SrDNA homology, organic solvent tolerance range, andsurvival after solvent exposure. The results indicate thatgram-positive bacteria can be isolated from the envi-ronment that are as tolerant to toxic organic solvents, ifnot more so, than the most organic solvent-tolerantgram-negative bacteria.

Keywords Solvent tolerance Organic solvents Bacterial cell envelope Enrichment

Introduction

It is generally thought that organic solvents exhibit ex-treme toxicity toward living microorganisms because oftheir accumulation in hydrophobic biological mem-branes. Because of this toxicity, organic solvents have inthe past been used as permeabilization agents, disinfec-tants, food preservatives, and industrial solvents(Davidson and Barnden 1981; de Bont 1998). Thehydrophobicity of organic solvents can be expressed in

terms of Pow, which represents the ability of a com-pound to partition over an octanol/water two-phasesystem (Sikkema et al. 1994). It has been established thatlog Pow is correlated with the toxicity of a specic or-ganic solvent and that log Pow values, especially betweentwo and four, are highly toxic for microorganisms (Os-borne et al. 1990). Whether a solvent is toxic to a bac-terial cell depends upon its concentration in themembrane, which relates to its water solubility and itsability to partition from the water phase to the mem-brane (de Bont 1998). Solvents with log Pows below twoare generally too hydrophilic to partition into mem-branes well, and solvents with log Pows above four aretoo hydophobic to have high water solubility (Kieboomand de Bont 2000). The mechanisms of membrane tox-icity by organic solvents have been well studied andcomprehensively reviewed (Sikkema et al. 1995). Ingram-negative cells, extensive permeabilization is causedin the cytoplasmic membrane, while the outer membraneremains relatively intact. This preferential partitioningof organic solvents into the cytoplasmic membraneprevents normal functioning of the membrane as aselective barrier, allowing the leakage of such macro-molecules as RNA, phospholipids, and proteins(Woldringh 1973). Perhaps more importantly, organicsolvents produce a loss of ion gradients across thecytoplasmic membrane that destroys energy productionand transduction via proton motive force (Sikkema et al.1994). The accumulation of toxic organic solvents in themembrane increases membrane uidity, increases mem-brane swelling, and reduces the normal functioning ofmembrane-associated proteins (Sikkema et al. 1995;Weber and de Bont 1996). The accumulation of organicsolvents results in the disruption of bilayer stability andmembrane structure, causing a loss of membrane func-tion and ultimately cell death.

Despite this extreme toxicity, organic solvent-tolerantbacteria that are capable of growth in a two-phase wa-tertoluene system have been isolated. Many of thesetolerant bacterial species, including the rst strain iso-lated, were gram-negative bacteria such as Pseudomonas

Communicated by K. Horikoshi

Z. Zahir K. D. Seed J. J. Dennis (&)Department of Biological Sciences, University of Alberta,Edmonton, Alberta, Canada, T6G 2E9E-mail: jon.dennis@ualberta.caTel.: +1-780-4922529Fax: +1-780-4929234

Extremophiles (2006) 10:129138DOI 10.1007/s00792-005-0483-y

putida (Inoue and Horikoshi 1989; Shima et al. 1991;Cruden et al. 1992; Weber et al. 1993; Ramos et al. 1995;Ohta et al. 1996; Kim et al. 1998) or closely relatedPseudomonas sp. (Nakajima et al. 1992; Ogino et al.1994). Gram-positive bacteria such as Bacillus (Moriyaand Horikoshi 1993; Isken and de Bont 1998; Sardessaiand Bhosle 2002b) and Rhodococcus (Paje et al. 1997)have also been found to be organic solvent tolerant,although limited investigation has occurred towardsunderstanding the mechanisms of their organic solventtolerance. Because the rst interaction of a solvent witha cell occurs at the cell membrane, membrane modi-cation was rst studied as a mechanism of solvent tol-erance in gram-negative bacteria (Sikkema et al. 1995).Several investigators have also been able to isolate sol-vent tolerant strains from solvent sensitive bacteria,suggesting that bacteria can adapt at least somewhat tothe presence of organic solvents (Weber et al. 1993).More recently, it has been discovered that toxic organicsolvents are a substrate for RND type multidrug euxpumps found in both solvent-tolerant Pseudomonas sp.(Kieboom et al. 1998a; Ramos et al. 1998) and Escher-ichia coli (White et al. 1997). It is generally accepted thatthe relatively impermeable cell envelope works in con-junction with an energy-driven eux pump to limit theentry of organic solvents into the cell and extrude anyorganic solvents that partition into the inner membranethrough the outer membrane (Kieboom and de Bont2000). However, for gram-positive bacteria, a generallymore permeable cell envelope precludes the eectivenessof a specialized solvent eux pump, suggesting that themechanism of organic solvent tolerance is primarilymembrane modication. In this paper, we present theisolation and characterization of several new gram-negative and gram-positive bacteria obtained throughenrichment procedures that are extremely tolerant totoxic organic solvents.

Materials and methods

Isolation of bacteria

Cells were isolated from a mixed microbial populationby culturing soil samples collected from various loca-tions in rich LuriaBertani (Lennox) media (10 g tryp-tone, 5 g yeast extract and 5 g NaCl per liter) or 1/2 LBmedia (5 g tryptone, 2.5 g yeast extract, and 2.5 g NaClper liter) for several days at 25C with shaking in sealedasks with a known low level of organic solvent, typi-cally toluene. Soil samples were obtained from both oilcontaminated and pristine (rhizosphere) locations. Soilplanted to marigolds was selected because of the po-tential presence of thiophenes (Christensen and Lam1990). After allowing growth in the ask to proceed toopacity, an aliquot was removed and used as an inocu-lum in a new ask with fresh medium and an additional5 mM of organic solvent. Although aqueous solutionsshould be saturated by toluene above 7 mM, at which

point a two-phase system exists, we continued to addadditional solvent in order to limit the eects of anypotential solvent volatilization to the headspace. Afterrepeated subcultures with up to 30 mM toluene, the cellswere plated out under two conditions without organicsolvent: a selective condition on Pseudomonas isolationagar (PIA) that selected for the preferential growth ofpseudomonads, and a non-selective condition wherecells were plated onto 1/2 LB. Four isolates from bothselection conditions were retained for further testing.

Physiological characterization

The morphology of the gram-stained solvent-tolerantbacteria was examined by light microscopy using a ZeissKF-2 light microscope. In order to examine changes tocellular morphology in the presence of organic solvents,cells were grown to log phase in LB medium containingeither no toluene or 50 mM toluene. Samples were takendirectly from the cultures and applied to grids that al-lowed the visualization of cells using a Philips/FEI(Morgagni) Transmission electron microscope withCCD camera in the departments microscopy unit. Ini-tially, samples were negatively stained with 0.2% phos-photungstic acid before visualization. In order tospecically visualize the capsular structure, cells ob-tained from growth with or without solvent were labeledwith polycationic ferritin to produce capsular stabiliza-tion followed by staining with ruthenium red (Sigma,MO, USA) and thin-sectioning prior to observation.

Biolog Microplates (Biolog, Hayward, CA, USA)were used to determine the ability of the strains to utilizeany of a number of dierent carbon sources. Testing wasperformed according to the manufacturers recommen-dations. Carbon utilization was scored positive if thecorresponding well had reduction of tetrazolium dyeproducing a purple color. Gram-negative non-entericbacteria were grown on Biolog universal growth (BUG)Agar with 5% sheeps blood at 30C, and inoculatedinto the GN2 MicroPlate test wells with GN/GP-IF(0.4% NaCl, 0.03% Pluronic F-68 and 0.02% Gellumgum) according to the manufacturers recommendations(Biolog). Gram-positive non-spore forming bacteriawere grown similarly but inoculated using GN/GP-IF +20% thioglycolate to reduce capsule formation, andtested in GP2 MicroPlates (Biolog). Gram-positive sporeforming bacteria were grown in BUG media + 0.25%maltose + thioglycolate and inoculated into GP2 Mi-croplates with GN/GP-IF without added thioglycolate.Microplates were read using Biologs MicroLog3 4.20software and GP 6.0 and GN 6.0 databases after 6 and16 h of reaction time.

Taxonomic classication of bacterial strains

Genomic DNA was extracted from the newly isolatedbacteria using a published protocol employing CTAB

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(Ausubel et al. 1991). PCR reactions were performedusing Invitrogen Pfx polymerase according to the man-ufacturers recommendations (Invitrogen Corp., Carls-bad, CA, USA), and a standardized set of primersdesigned to amplify the bacterial 16S gene (Johnson1994). To be consistent with other database entries, weattempted to obtain 16S DNA sequence in both direc-tions of between 1,000 and 1,500 bp for each isolate. 16SrDNA sequence sizes obtained ranged from 1,160 bp forisolate ZZ5, to 1,498 bp for isolate ZZ7. In general,DNA was manipulated and stored as previously de-scribed (Sambrook et al. 1989). DNA sequencing of thePCR products was performed by our departmentsMolecular Biology Service Unit on an ABI PRISM 3100Genetic Analyzer (Applied Biosystems, Foster City, CA,USA), following purication with a QIAGEN PCRminiprep column (QIAGEN Inc., Mississauga, ON,USA). Sequences were edited and assembled using ABIsEditView and AutoAssembler programs (Applied Bio-systems), and the sequences were compared to GenBankdatabase entries using BLASTN (Altschul et al. 1990).Closely related sequences were selected for use in theconstruction of phylogenetic trees. The raw sequenceswere converted to LaserGenes EditSeq les and alignedusing the CLUSTAL algorithm of LaserGenes Meg-Align program (DNASTAR Inc., Madison, WI, USA).

Growth after solvent shock

In order to observe a cells inherent ability to withstandtoxic organic solvents without adaptation, cells weresubjected to rapid solvent addition producing large-scale

cell death. In this solvent shock growth condition, cellswere initially cultured overnight in the absence of sol-vent to provide a fresh inoculum for a subculture. Cellswere grown to early log phase in a sealed Nephelo ask,shocked with a set amount of solvent, and OD600 wasmeasured as an indicator of cell growth.

Growth in two-phase solvent systems

Growth curves were initially performed in Nepheloasks sealed with either a neoprene stopper or an alu-minum foil-covered neoprene stopper to prevent solventvolatilization. Growth measurements were takenobtaining OD600 readings on a spectrophotometerwithout opening and resealing the asks to prevent theloss of volatilized organic solvents. Cells were subcul-tured every 24 h to new media containing increasingamounts of toluene. An additional 5 mM toluene wasadded each day to the new ask and the experiment wascarried out until the subcultured cells failed to grow inthe new media overnight. Cell death at particular con-centrations of organic solvent in solution indicates thatappreciable amounts of solvent were not being lost tovolatilization. Because solvent shock and solvent growthexperiments suggested that cells were adapting to solventconcentrations well above the limit of solvent solubility,we attempted to compare the maximal solvent concen-trations in which these cells could survive. In theseexperiments, cells were allowed to adapt to the presenceof toxic organic solvents through any possible meansincluding de novo gene expression or mutation. Over-night growth experiments with high concentrations of

Table 1 Characteristics of newly isolated solvent-tolerant bacteria

Strain name 16S ComparisonClosestNeighbor (GenBankaccession number)

Original Sample Toluene tolerancea

Staphylococcussp. strain ZZ1

Staphylococcus sp. LMG-19417(AJ276810)

Solvent contaminated media 100 mM [1% (v/v)]

(1,187/1,187 bp identity)B. cereus strain ZZ2 B. cereus strain ATCC 10987

(AJ577290)Marigold soil (Edmonton, Alberta) 90 mM [0.96% (v/v)]

(1,166/1,166 bp identity)B. cereus strain ZZ3 B. cereus strain E33L (CP000001) Marigold soil (Edmonton, Alberta) 100 mM [1% (v/v)]

(1,414/1,414 bp identity)B. cereus strain ZZ4 B. cereus strain LRN (AY138275) Oil contaminated soil (Northern Alberta) 100 mM [1% (v/v)]

(1,366/1,366 bp identity)Pseudomonas sp. strain ZZ5b Pseudomonas sp.

(biodegradation) (Y13246)Marigold soil (Southern Alberta) 20 mM [0.21% (v/v)]

(1,482/1,482 bp identity)Pseudomonas sp. strain ZZ6b Pseudomonas citronellolis (Z76659) Marigold soil (Southern Alberta) 20 mM [0.21% (v/v)]

(1,156/1,160 bp identity; 99%)S. maltophilia strain ZZ7b S. maltophilia (AB180661) Marigold soil (Edmonton, Alberta) 20 mM [0.21% (v/v)]

(1,487/1,498 bp identity; 99%)B. cepacia strain ZZ8b B. cepacia complex strain

ATCC 17759 (AY741334)Marigold soil (Southern Alberta) 23 mM [0.24% (v/v)]

(1,435/1,468 bp identity; 97%)

aGrowth in LB media plus solventbGram negative bacteria

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organic solvents were performed with 10 ml of LB mediain 125 ml Erlenmeyer asks sealed with foil-coveredneoprene stoppers, shaken at 220 rpm in a 30C incu-bation chamber.

Results

Characterization of solvent-tolerant bacteria

The four isolates from the non-selective were gram-po-sitive bacteria as determined by gram stain, whereas thefour isolates bacteria from PIA were stained as gram-negative bacteria. The gram-positive bacteria were giventhe strain names ZZ1ZZ4, and the gram-negativebacteria were named ZZ5ZZ8. Initial analysis under alight microscope showed ZZ1 to be a gram-positive coccifound in irregular clusters, ZZ2 and ZZ3 to be a gram-positive rods existing as single cells or pairs, and ZZ4 tobe a gram-positive rod existing in regular clusters. Lightmicroscopy showed ZZ5 to be a gram-negative rodexisting in clusters, and ZZ6, ZZ7, and ZZ8 to be gram-negative rods found in pairs or chains.

Taxonomic classication of these isolates was deter-mined by both 16S rDNA comparison and carbonsource utilization comparisons with other bacteria. Asshown in Table 1 and Fig. 1a, the 16S rDNA sequencealignment of the newly isolated gram-negative bacteriawith previously isolated organisms identied a novelPseudomonas sp. (ZZ5) as well as a Pseudomonas sp.

(ZZ6) that is similar to P. citronellolis, an organismfrequently isolated from oil-contaminated soil (Bhat-tacharya et al. 2003). In addition, a Stenotrophomonasmaltophilia isolate named ZZ7 was identied; anorganism that has recently become a clinical problemdue to its high levels of eux mediated antibiotic resis-tance. Finally, we isolated a Burkholderia cepacia com-plex member named ZZ8 from soil planted to marigoldsthat could withstand the presence of slightly more tol-uene than the other bacteria isolated under selectiveconditions. Further taxonomic classication by 16SrDNA sequence comparison indicated that strain ZZ8 isa B. cenocepacia strain (genomovar IIIb).

In the non-selective culture condition, several gram-positive bacteria that exhibited higher solvent tolerancesthan the isolated gram-negative bacteria were isolated(Table 1). This was somewhat surprising given thatgram-negative bacteria are generally more resistant tomany toxic compounds because of their more imperme-able double membrane cell envelope. By 16S rDNAanalysis it was determined that we had isolated an envi-ronmental Staphylococcus sp. (ZZ1), two Bacillus cereusisolates (ZZ2 and ZZ4), and a Bacillus sp. (ZZ3). Thepartial 16S rDNA sequence of Staphylococcus sp. ZZ1was shown to have 100% identity with Staphylococcussp. strain LMG 19417 (AJ276810.1) and other relatedStaphylococcus sp. As shown in Fig. 1b, Staphylococcussp. strain ZZ1 as well as the related Staphylococcus sp.(all reported to be involved in some form of organichydrocarbon degradation), are more closely related to

Fig. 1 a Phylogenetic tree of isolated gram-negative bacteria andclosely related species. Branch distances represent relatednessbetween partial 16S sequences determined for each bacterialspecies. b Phylogenetic tree of isolated gram-positive bacteria

and closely related species. The two major branches in each tree areused for outlier comparison purposes only and do not denote aclose relationship

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Staphylococcus epidermidisRP62A (NC_002976) and theBacillus clade of soil organisms than Staphylococcusaureus. The 16S rDNA sequences reported in this study

have been submitted to the GenBank database with therespective accession numbers for strains ZZ1ZZ8deposited as DQ113448DQ113455.

Fig. 2 Growth curves ofisolated bacteria followingsolvent shock. Set quantities oforganic solvents were added tocells growing in early lag phaseat 4 h after subculture. Cellgrowth was assessed bymeasuring OD600 in sealedNephelo asks to reduce theloss of organic solvent byvolatilization. Growth afterdierent solvent shockquantities are displayed on onegraph, showing an inverserelationship between theamount of solvent added to themedia and the amount of cellgrowth. Shown are organicsolvent shock growth curves forthe, a newly isolated B. cepaciacomplex strain ZZ8, b thepreviously characterized P.putida S12 strain (19), and c thenewly isolated Staphylococcussp. strain ZZ1. The y-axisshows time elapsed in hours

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General physiology of these newly isolated solvent-tolerant bacteria as determined by Biolog MicroLog3metabolic analysis generally conrmed 16S comparisons.Physiological data using Biolog MicroLog3 MicroPlatespredicted isolate ZZ1 to be a Staphylococcus warnerispecies with 100% probability. Isolate ZZ2 was predictedto be a novel Bacillus sp., though with strong similarity toB. cereus or Bacillus thuringenesis (which could not bedistinguished using these tests). Isolates ZZ3 and ZZ4were both predicted to be B. cereus/thuringensis strainswith 100% probability. Even though 16S rDNA analysispredicted ZZ5 and ZZ6 to be closely related, isolate ZZ5was predicted to be a new pseudomonad with the mostsimilarity to P. citronellolis, but exhibited only 43%similarity in substrate metabolism, while isolate ZZ6 waspredicted to be a P. citronellolis strain with 100% prob-ability (84% similarity in substrate utilization). IsolateZZ7 was predicted to be an S. maltophilia strain with100% probability, while isolate ZZ8 was predicted to bea B. cepacia complex member (Burkholderia multivorans;genomovar II) with 100% probability (70% similarity insubstrate utilization). The discrepancy between the ZZ816S rDNA comparison and the physiological data ob-tained through Biolog experiments suggests that ZZ8 is aB. cenocepacia strain with expanded metabolic capabili-ties similar to B. multivorans.

Bacterial growth characteristics in organic solvents

In terms of growth in organic solvents (shown in Ta-ble 1), it is apparent that the gram-positive mechanismof solvent tolerance is more eective than that seen withnewly isolated gram-negative cells. Although solventinsolubility above a certain threshold concentration wasexpected to produce a cell death plateau, this was notobserved. Instead, cell death increased with increasingsolvent concentrations, in both solvent shock andadaptation conditions, even at solvent concentrationswell above the known solvent solubility level. This eectwas observed not only for the newly isolated gram-po-sitive bacteria (Fig. 2c), but also for the gram-negativebacteria (Fig. 2a) including the previously well-charac-terized organic solvent-tolerant P. putida S12 (Fig. 2b,Weber et al. 1993). The nding that gram-positiveorganisms such as Staphylococcus sp. strain ZZ1 weretolerant to higher concentrations of toluene than thegram-negative organisms tested using the exact sameconditions strongly suggests that these gram-positivebacteria can withstand the direct eects of toxic organicsolvents better than most gram-negative bacteria. Thedetection of this killing eect at dierent concentrationsabove maximum solubility suggests that solvent toxicityis more complex than previously described.

Table 2 Range of toxic organic solvent tolerance of gram-positive bacteria

Solvent (concentration) log Pow Staphylococcussp. strain ZZ1

B. cereusstrain ZZ2

B. cereusstrain ZZ3

B. cereusstrain ZZ4

Hexane 100 mM [1.3% (v/v)] 3.5 +++ +++ +++ +++Cyclohexane 100 mM [1% (v/v)] 3.2 +++ +++ +++ +++p-Xylene 100 mM [1.2% (v/v)] 3.0 +++ - -Toluene 50 mM [0.53% (v/v)] 2.5 +++ +++ +++ +++Toluene 100 mM [1% (v/v)] 2.5 +++ +++ +++1-Heptanol 100 mM [1.4% (v/v)] 2.4 - - - -Dimethylphthalate 100 mM [2% (v/v)] 2.3 +++ - +++ +++Fluorobenzene 100 mM [1% (v/v)] 2.2 +++ +++ +++ +++Benzene 100 mM [1% (v/v)] 2.0 +++ +++ +++ +++Phenol 20 mM [0.18% (v/v)] 1.5 +++ - +++ +++

+++ growth overnight (16 h); minimal growth overnight; - no growth

Table 3 Range of toxic organic solvent tolerance of gram-negative bacteria

Solvent (concentration) log Pow Pseudomonassp. strain ZZ5

Pseudomonassp. strain ZZ6

S. maltophiliastrain ZZ7

B. cepaciastrain ZZ8

P. putidastrain S12

Hexane 100 mM [1.3% (v/v)] 3.5 +++ +++ +++ +++ +++Cyclohexane 100 mM [1% (v/v)] 3.2 +++ +++ +++ + +++p-Xylene 100 mM [1.2% (v/v)] 3.0 - - +++Toluene 30 mM [0.32% (v/v)] 2.5 - - - - +++Toluene 100 mM [1% (v/v)] 2.5 - - - - +++1-Heptanol 100 mM [1.4% (v/v)] 2.4 - - - - Dimethylphthalate 20 mM [0.33% (v/v)] 2.3 +++ +++ +++Dimethylphthalate 100 mM [2% (v/v)] 2.3 +++ +++ - - +++Fluorobenzene 100 mM [1% (v/v)] 2.2 - - - - -Benzene 100 mM [1% (v/v)] 2.0 - - - - -Phenol 20 mM [0.18% (v/v)] 1.5 - - - - -

+++ growth overnight (16 h); minimal growth overnight; - no growth

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In the adaptive growth condition, cells were repeat-edly sub-cultured every 24 h into fresh media containingslightly increasing levels of one solvent such as toluene.This experimental procedure was less harsh than thesolvent shock procedure illustrated in Fig. 2, and al-lowed the organisms an opportunity to adapt to thepresence of solvents through mutation or gene expres-sion. Using this procedure it was found that some cells,particularly the gram-positive cells, could withstandmuch higher levels of solvents than in the solvent shockexperiments; for Staphylococcus sp. ZZ1 compare100 mM toluene (Table 1) versus 50 mM toluene(Fig. 2c). In general, we found solvent shock was lesswell-tolerated than growth in solvents suggesting thatcells can at least partially adapt to harsh growth con-ditions given time to either undergo mutation and/orexpress new cellular proteins or structures. In spite of theup-regulation of eux pumps by some gram-negativebacteria to resist the presence of solvents in the solvent

growth conditions (Kieboom et al. 1998b), the isolatedgram-negative bacteria were less well adapted to thepresence of high concentrations of solvents than weregram-positive bacteria (compare the horizontal linesrepresenting no new growth in Fig. 2ac).

In order to determine whether this high level of solventtolerance seen in gram-positive bacteria was specic totoluene, we tested the new bacterial isolates in dierenttoxic organic solvents. As shown in Table 2, all of thenewly isolated gram-positive bacteria exhibited hightolerance in two-phase systems of organic solvents withlog Pow values approaching 2.0, including 20 mMphenol(except ZZ2), 100 mM benzene, and 100 mM uoro-benzene, while the gram-negative bacteria were killed atsimilar solvent concentrations (Table 3). The newly iso-lated gram-negative bacteria were also sensitive to 30 and100 mM toluene as expected whereas the gram-positive

Fig. 3 Electron micrographs of the newly isolated organic solvent-tolerant Staphylococcus sp. ZZ1 grown in LB media underconditions, a without the organic solvent toluene, or b with50 mM toluene in the growth medium. Panel B shows the modiedmembrane of Staphylococcus sp. ZZ1 at 44,000 times magnicationas compared to a 200 nm scale bar. The black region observed inpanel b is an electron micrograph grid section

Fig. 4 Transmission electron micrographs of thin-sectioned Staph-ylococcus sp. ZZ1 for capsule detection. Isolate ZZ1 was grown inthe absence (a) and presence (b) of 50 mm toluene in the growthmedium before xation, cross-sectioning, and staining withruthenium red dye. The cells are shown in comparison to a200 nm scale bar at 44,000 times magnication

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bacteria were tolerant to this solvent concentration.There was some solvent specicity exhibited by bothgram-positive and gram-negative bacteria; the gram-negative cells were sensitive to toluene and solvents withlog Pow values near 2.0, and all bacteria were sensitive to1-heptanol. The data obtained for P. putida S12 is similarto that previously obtained (Kieboom et al. 1998a).

Cellular adaptation to organic solvents

To examine the gram-positive mechanism of solventtolerance more closely, we grew the newly isolated envi-ronmental Staphylococcus sp. ZZ1 in media containingsolvent or no solvent and compared cellular morpho-logies under an electron microscope after staining withphosphotungstic acid. As compared to growth withoutsolvent (Fig. 3a), cells grown in the presence of 50 mMtoluene produce an extracellular capsule (Fig. 3b). Thiscapsule gives the cells a stued or fuzzy appearance, andcovers the coccoid Staphylococcus cells nearly com-pletely, except at the attachment site to neighboring cells.It is apparent that this capsule extends some distancefrom the cell membrane as observed by the occlusion ofbackground detail as shown in Fig. 3b. As shown inFig. 4a and b, a more detailed visualization of this cap-sule was produced upon staining the organic solventgrown cells with ruthenium red and cross-sectioning thecells prior to EM visualization (Vanrobaeys et al. 1999).

Discussion

Bacterial tolerance to toxic compounds is thought to bethe result of an interplay between a relatively imper-meable membrane that limits the diusion of thesecompounds into the cell, and energy-dependent euxmechanisms that extrude any noxious agents out of thecell that have managed to bypass the membrane barrier.Since it has been demonstrated that RND eux pumpsfrom gram-negative cells can accept as substrates bothorganic solvents and multiple classes of antibiotics, itwas anticipated that cells with the highest levels ofantibiotic resistance would also have the highest levels oforganic solvent tolerance. Therefore, it was somewhatsurprising that gram-positive bacteria (with membranesmore permeable to antibiotics) were found to be gener-ally more tolerant to organic solvents than gram-nega-tive bacteria. While bacteria with both types of cellenvelope architecture seem to react similarly with respectto solvent growth or solvent shock (solvent shock con-ditions being more lethal), there is a clear dierence inthe ability of gram-positive cells to withstand higherconcentrations of toxic organic solvents with log Powvalues of approximately 2.0.

By enriching for only those cells able to withstandextreme levels of toxic organic solvents, we anticipatedthat this selection would result in the isolation of bac-teria best able to survive in organic solvents. Although

the gram-negative bacteria we isolated were not as sol-vent tolerant as P. putida S12 (Weber et al. 1993), theywere at least as tolerant as E. coli DH5a, which with-stood 2025 mM toluene using our experimental con-ditions (data not shown). E. coli is a moderately solventtolerant bacterium due to its expression of the AcrAB-TolC eux pump that is capable of both antibiotic andorganic solvent eux (White et al. 1997). In comparison,the data obtained for P. putida S12 in Table 3 is similarto that previously found (Kieboom et al. 1998a), andsuggests that these newly isolated gram-negative bacteriado not contain a specialized solvent eux pump likeSrpABC (Kieboom et al. 1998a).

Other gram-positive bacteria tolerant to organic sol-vents have been previously identied. A Rhodococcus sp.isolated from a chemical-contaminated site in Australiahas been shown to have exceptional tolerance to highlevels of benzene (Paje et al. 1997). A Bacillus sp. strainhas been isolated from soil that tolerates the organicsolvent n-butanol (Sardessai and Bhosle 2002b), and aBacillus sp. strain has been isolated from deep sea with ahigh level of tolerance to benzene (Moriya and Hori-koshi 1993). Isken and de Bont (1998) describe the iso-lation of several Bacillus sp. from normal soilenvironments tolerant to toluene. One particular strainof B. cereus named R1 was subjected to further analysis(Matsumoto et al. 2002). In comparison with the resultsobtained for the B. cereus isolates described in thisstudy, B. cereus R1 was less tolerant of solvents with lowlog Po/w values such as benzene at similar solvent con-centrations (1% v/v). However, B. cereus R1 was toler-ant to similar levels of hexane and toluene, and similarlysensitive to 1-heptanol. Some evidence was shown forthe existence of an active transport system for theexclusion of solvents in B. cereus R1 (Matsumoto et al.2002), however, it is unknown if such a system is in-volved or important to the tolerance observed in all B.cereus strains. While Rhodococcus sp. strain 33 is e-cient at tolerating higher levels of benzene (2% v/v) inpart through benzene metabolism (Paje et al. 1997), thegram-positive cells characterized in this study have beendemonstrated to be almost as tolerant, withstanding atleast 1% (v/v) benzene.

Although several mechanisms such as deactivatingenzymes, endospore protection, or eux pumps havebeen proposed for this tolerance in gram-positive bacte-ria, little information about these mechanisms has beendescribed. We hypothesize that the observed solventtolerance in some gram-positive bacteria is derived fromthe unusual capsular ultrastructure. While gram-positivebacteria have in the past been shown to use extracellularcapsules to resist the eects of the immune system as wellas penetration by antibiotics, to our knowledge this is therst observation of a capsule apparently being used towithstand the eects of noxious environmental chemi-cals. Since organic solvents preferentially partition intothe hydrophobic portion of the phospholipid bilayer, it isanticipated that solvent tolerance can be achievedthrough the production of a hydrophilic carbohydrate

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capsule that repels organic solvents and prevents themfrom reaching the cellular membrane. This capsule isevident in Fig. 3b, where the background detail is ob-scured by a capsule that is dicult to visualize experi-mentally. Ruthenium red staining of the cell surface priorto EM visualization produced somewhat better images ofthis structure (Fig. 4a, b). We are currently working tofurther characterize this gram-positive capsule anddetermine whether it is the sole mechanism responsiblefor the observed solvent tolerance in these bacteria. Be-cause Staphylococcus strain ZZ1 exhibits tolerance totoxic organic solvents under solvent shock conditionswhere no prior adaptation such as capsule productionhas occurred, it suggests that there are also other physi-ological mechanisms that make these gram-positivebacteria solvent tolerant.

Organic solvents in two-phase systems are thought toonly cause cellular toxicity when solubilized in theaqueous phase where cells are expected to exist. Atconcentrations above the threshold level of solvent sol-ubility, it has been proposed that additional solvent willnot cause increased toxicity or cell death. Our resultsappear to contradict these predictions. Our results (fromboth solvent shock and solvent growth conditions) withsolvent concentrations in excess of solvent solubilitymay be the result of our growth conditions, whereshaking asks agitate the two-phase solutions such thatcells are not always maintained in the aqueous phase. Inthis case, the addition of higher concentrations of sol-vents would cause the solvent phase to more frequentlycontact the cells directly with the expected outcome ofmembrane destruction and cell death. Provided that theexperimental conditions are similar for each bacteriumtested, accurate estimates can be made about the relativeorganic solvent tolerance of each bacterium at solventconcentrations above the threshold of solvent solubility(Bar 1988). Moreover, the ability of a bacterium to growin solvents with dierent log Pow values as an indicatorof organic solvent tolerance (as shown in Tables 2, 3)mirror the results obtained in the solvent shock andsolvent growth experiments for the new bacterial isolatesversus previously characterized solvent-tolerant bacteriasuch as P. putida S12. This strongly supports the viewthat these new gram-positive bacteria are at least astolerant, if not more tolerant than previously charac-terized solvent-tolerant gram-negative bacteria.

It has previously been shown that eux pump genescan be transferred between gram-negative strains toconfer increased solvent tolerance on the host (Kieboomet al. 1998a). Because the proper assembly of the tri-partite RND eux pump in the gram-negative cellenvelope is essential to this increased solvent tolerance,it is unlikely that these genes could be transferred togram-positive organisms to produce properly assembledeux pumps in the single bacterial membrane. However,it may be possible to transfer the genes for an extracel-lular polysaccharide to a solvent-tolerant gram-negativebacterium and expect proper assembly on the surface ofthe cell. The combination of an extracellular capsule

with an energy-dependent eux system that can extrudesolvents may confer additional organic solvent toleranceto modied bacteria that could be used industrially(Sardessai and Bhosle 2002a). Modied bacterial cellswith enhanced solvent tolerance would be extremelyuseful in the elds of applied microbiology and bio-technology. Bacteria used in the environmental bio-remediation of toxic wastes could be enhanced towithstand higher concentrations of the pollutants. In theindustrial production of ne chemicals, organic solvent-tolerant bacteria would be better able to withstandextraction of the chemical end-product with a secondphase of organic solvent. The further characterization ofthese specialized organic solvent-tolerant bacteria willprovide us with the knowledge required for their use inbiotechnological applications.

Acknowledgements This work was supported by a grant from theNatural Sciences and Engineering Research Council of Canada.We thank Dr. Julia Foght and Dr. Susan Jensen for helpful dis-cussions, and Rakesh Bhatnagar and the other members of theBiological Sciences Microscopy Unit for assistance.

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