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Appl. Environ. Microbiol.-1996-Beller-1188-96Isolation and Characterization of a Novel Toluene-Degrading, Sulfate-reducing Bacterium

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  1996, 62(4):1188. Appl. Environ. Microbiol.  H R Beller, A M Spormann, P K Sharma, J R Cole and M Reinhard   toluene-degrading, sulfate-reducing bacterium. Isolation and characterization of a novel http://aem.asm.org/content/62/4/1188 Updated information and services can be found at: These include: CONTENT ALERTS more» cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new articles http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint o
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    1996, 62(4):1188. Appl. Environ. Microbiol. H R Beller, A M Spormann, P K Sharma, J R Cole and M Reinhard  toluene-degrading, sulfate-reducing bacterium.Isolation and characterization of a novel http://aem.asm.org/content/62/4/1188Updated information and services can be found at: These include:  CONTENT ALERTS  more»cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new articles http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders:  http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to:  onA  pr i  l  1  3  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om  onA  pr i  l  1  3  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om    A  PPLIED AND  E NVIRONMENTAL   M ICROBIOLOGY , Apr. 1996, p. 1188–1196 Vol. 62, No. 40099-2240/96/$04.00  0Copyright    1996, American Society for Microbiology Isolation and Characterization of a Novel Toluene-Degrading,Sulfate-Reducing Bacterium HARRY R. BELLER, 1 * ALFRED M. SPORMANN, 1 PRAMOD K. SHARMA, 1 JAMES R. COLE, 2  AND  MARTIN REINHARD 1  Environmental Engineering and Science, Department of Civil Engineering, Stanford University, Stanford,California 94305-4020, 1  and Center for Microbial Ecology, Michigan State University, East Lansing, Michigan 48824 2 Received 27 November 1995/Accepted 31 January 1996  A novel sulfate-reducing bacterium isolated from fuel-contaminated subsurface soil, strain PRTOL1, min-eralizes toluene as the sole electron donor and carbon source under strictly anaerobic conditions. Themineralization of 80% of toluene carbon to CO 2  was demonstrated in experiments with [  ring  -U- 14 C]toluene;15% of toluene carbon was converted to biomass and nonvolatile metabolic by-products, primarily the former.The observed stoichiometric ratio of moles of sulfate consumed per mole of toluene consumed was consistent with the theoretical ratio for mineralization of toluene coupled with the reduction of sulfate to hydrogen sulfide.Strain PRTOL1 also transforms  o - and  p -xylene to metabolic products when grown with toluene. However,xylene transformation by PRTOL1 is slow relative to toluene degradation and cannot be sustained over time.Stable isotope-labeled substrates were used in conjunction with gas chromatography-mass spectrometry toinvestigate the by-products of toluene and xylene metabolism. The predominant by-products from toluene,  o -xylene, and  p -xylene were benzylsuccinic acid, (2-methylbenzyl)succinic acid, and 4-methylbenzoic acid (or  p -toluic acid), respectively. Metabolic by-products accounted for nearly all of the  o -xylene consumed. Enzymeassays indicated that acetyl coenzyme A oxidation proceeded via the carbon monoxide dehydrogenase pathway.Compared with the only other reported toluene-degrading, sulfate-reducing bacterium, strain PRTOL1 isdistinct in that it has a novel 16S rRNA gene sequence and was derived from a freshwater rather than marineenvironment. Leakage of gasoline from underground fuel storage tanks isa pervasive source of groundwater contamination in the UnitedStates (41). Bioremediation is one of a limited number of op-tions for restoring aquifers contaminated with the hazardous,relatively water-soluble aromatic hydrocarbons that occur inunleaded gasoline, such as benzene, toluene, and xylenes. Be-cause many contaminated aquifers are anaerobic as a result of oxygen depletion by indigenous aerobic microorganisms, hy-drocarbon degradation by indigenous anaerobic bacteria mer-its serious consideration at some sites as a method of ground- water restoration. Partially in response to such environmentalconcerns, knowledge of the metabolic capabilities of anaerobicbacteria has expanded dramatically in the past decade, partic-ularly with respect to single-ring aromatic hydrocarbons andclosely related oxygenated compounds. For example, 18 purecultures capable of anaerobic toluene degradation have beenreported since 1989, including 16 denitrifying cultures (10, 14,19, 34, 36, 48), one ferric iron-reducing culture (25, 26), andone marine sulfate-reducing culture (33); fermentative-metha-nogenic mixed enrichment cultures that degrade toluene havealso been reported (12, 21, 43). More than 10 sulfate-reducingcultures that can grow on aromatic substrates have been re-ported since 1980 (3, 4, 8, 9, 11, 22, 23, 32, 33, 35, 39, 40, 46);however, only one of these can metabolize an aromatic hydro-carbon (  Desulfobacula toluolica ) (33). In this article, we reportthe isolation of strain PRTOL1, the second sulfate-reducingbacterium known to degrade an aromatic hydrocarbon and thefirst such organism from a freshwater environment. MATERIALS AND METHODS Chemicals.  The chemicals used in this study were of the highest purity avail-able (generally  99%) and were used as received. Most organic chemicals werepurchased from Aldrich Chemical Co., Inc. (Milwaukee, Wis.), and most inor-ganic chemicals were purchased from J. T. Baker, Inc. (Phillipsburg, N.J.). Stableisotope-labeled chemicals included toluene-  d 8  (  99 atom%; Aldrich ChemicalCo.);  o -xylene-  d 10  and  p -xylene-  d 10  (99   atom%; Aldrich Chemical Co.); andbenzaldehyde-  - 13 C,  d 1  (98% purity [D] and 99% purity [ 13 C]; Isotec, Inc., Mi-amisburg, Ohio). Gas chromatography-mass spectrometry (GC-MS) analyses of deuterium-labeled alkylbenzenes demonstrated that they were of high purity andcontained none of the metabolites reported in this study. For radiolabeled assays,[  ring  -U- 14 C]toluene (  98% purity, 10.2 mCi/mmol; Sigma Chemical Co., St.Louis, Mo.) was diluted into unlabeled toluene (  99.9% purity; Aldrich Chem-ical Co.) to a final specific activity of 75.1  Ci/mmol.Only a few of the compounds reported as metabolic by-products in this study were commercially available: benzylsuccinic acid (Sigma Chemical Co.), 3-ben-zoylpropionic acid (Aldrich Chemical Co.), and  p -toluic acid (Aldrich ChemicalCo.). The basis for identification of other by-products without authentic stan-dards is presented in later sections. However, the identifications of benzylfumaricacid and (2-methylbenzyl)fumaric acid require additional discussion. The firstreport of benzylfumaric acid from anaerobic toluene metabolism was based onthe use of a geometric isomer, benzylmaleic acid, as a standard (13). A recentstudy in which benzylfumaric acid and three structurally similar isomers [benzyl-maleic acid and (  E )- and (  Z )-phenylitaconic acid] were synthesized indicated thatthe four isomers could be distinguished by their GC retention times but not bytheir mass spectra (20). Thus, all four isomers would have to be available to makedefinitive identifications of such metabolic by-products. An analogous situationprobably applies to the compound previously identified as (2-methylbenzyl)fu-maric acid (13). For the sake of consistency with previous studies and in theabsence of the applicable standards, we will retain the names of the compoundspreviously reported as benzylfumaric acid and (2-methylbenzyl)fumaric acid withthe understanding that the closely related phenylitaconic or benzylmaleic acidisomers may actually apply. Source of bacteria.  The organism described in this article was isolated fromsoil collected at the Naval Air Station, Patuxent River, Md. The site was exten-sively contaminated with aviation fuel. The sample was collected from a Qua-ternary stratum of an outcrop through which hydrocarbon-contaminated ground- water was seeping. Microcosms inoculated with Patuxent River soil and * Corresponding author. Phone: (415) 723-0315. Fax: (415) 725-3162.1188   onA  pr i  l  1  3  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om   enrichment cultures developed from these microcosms were described previously(5, 7). Growth medium and conditions of isolation and cultivation.  The basal mineralcomponent of the medium used for isolation and maintenance of strain PRTOL1 was similar to the medium used for Patuxent River microcosms and enrichmentcultures (5); however, vitamins and trace elements were added for isolation andmaintenance of the culture. The basal mineral component included the followingcompounds added at the concentrations (mM) specified in parentheses:NaHCO 3 (30), NH 4 Cl (28), NaH 2 PO 4    H 2 O (4.4), FeSO 4    7H 2 O (3), NaCl (1.7), KCl(1.3), CaCl 2    2H 2 O (0.68), MgCl 2    6H 2 O (0.49), MnCl 2    4H 2 O (0.025), andNa 2 MoO 4    2H 2 O (0.004). The medium (excluding FeSO 4  and NaHCO 3 ) wasautoclaved at 121  C for 20 min and then aseptically purged with an oxygen-freemixture of 79% N 2 –21% CO 2  for 45 min. After purging, bicarbonate was addedto the medium as a sterile, anaerobic 1.0 M solution (45) in addition to anaer-obic, filtered ferrous sulfate. Also added were anaerobic and sterile vitamin,trace element, and selenite-tungstate solutions that were prepared as describedby Widdel and Bak (45) (stock solutions 1, 4, 6, 7, and 8). Prior to inoculation,the medium was prereduced with 150 to 200   M sodium sulfide added from afiltered 0.1 M stock solution. Highly purified water (Milli-Q; Millipore Corp.,Marlborough, Mass.) was used to prepare all the aqueous solutions used in thisstudy; all stock solutions were prepared with Milli-Q water that had been auto-claved at 121  C for 20 min and then aseptically purged with oxygen-free N 2 . Thefinal pH of the medium was approximately 7. All preparation and incubation of enrichment cultures, serial dilutions, andpure culture suspensions were performed at 35  C under strictly anaerobic con-ditions in an anaerobic glove box (Coy Laboratory Products, Inc., Ann Arbor,Mich.) with a gas composition of approximately 90% N 2 –7.5% CO 2 –2.5% H 2 .Glass, plastic, and stainless steel materials used to contain or manipulate thecultures were sterile (either autoclaved or purchased sterile) and were allowed todegas in the anaerobic glove box before use. Preparation of enrichment cultureshas been described previously (5). At the time that serial dilutions were initiated,the enrichment cultures had been maintained in the laboratory with toluene andsulfate as the sole electron donor and acceptor, respectively, for over 3 years.Microscopically pure cultures were obtained by repeated serial dilution of enrichment cultures with liquid medium in crimp-top serum culture tubes (18 by150 mm; Bellco Glass, Inc., Vineland, N.J.) that were sealed with polytetrafluo-roethylene (PTFE)-coated butyl rubber liners (Alltech Associates, Inc., Deer-field, Ill.). Toluene (  99.9% purity, glass-distilled, filtered [0.5-  m-pore-sizefilter]; Aldrich Chemical Co.) was added into the medium as a pure liquid witha 10-  l syringe. The syringe was sterilized with ethanol and allowed to dry beforebeing used for toluene. Toluene concentrations were kept at or below 50   Mduring serial dilution to preclude toxicity. Because Patuxent River enrichmentcultures were found to be highly sensitive to sulfide (with inhibition in the rangeof 1 to 3 mM) (6), FeSO 4  rather than MgSO 4  was used as a sulfate source tominimize the dissolved sulfide concentration. The 10  6 -diluted culture from thefirst dilution series was grown with toluene for several months before being usedas the inoculum for a second dilution series. The 10  8 -diluted culture from thissecond series, which appeared homogenous by microscopic examination, wasgrown with toluene for several months before serving as the inoculum for furtherdilution series. Agar dilution series (45) were performed by using the serially diluted liquidculture as an inoculum. Benzoate (1 mM) was used rather than toluene in theagar dilution series to expedite growth. Colonies of PRTOL1 were brown andlens shaped. Isolated colonies were transferred to liquid medium with toluene asthe sole carbon source.Cultures were maintained in amber glass, screw-cap bottles that were sealed with PTFE Mininert valves (Alltech Associates, Inc.). Toluene was monitoredand added when depleted; filter-sterilized, anaerobic FeSO 4  was added via sy-ringe when sulfate was depleted (as indicated by cessation of toluene consump-tion). Cultures growing on toluene were examined microscopically for purity ona regular basis. To independently test the purity of the cultures used in this study,complex medium that included 0.5% nutrient broth was inoculated withPRTOL1 (5 to 10% inoculum) and was examined microscopically after 2 to 3 weeks of incubation. No microbial contaminants were observed in these tests. Catabolic studies.  For catabolic studies, PRTOL1 cells were grown with tol-uene and sulfate in a 2-liter glass reactor sealed with Mininert valves. Cells wereharvested anaerobically by centrifugation (5,000    g   for 40 min at 20  C) and were resuspended in the anaerobic growth medium described previously. Allexperiments with volatile aromatic compounds, such as toluene, were conductedin screw-cap glass containers sealed with Mininert valves; for all other com-pounds, PTFE-faced silicone septa inside open-hole screw caps were used to sealbottles. Aromatic compounds that are liquids at room temperature (e.g., ben-zene, toluene, ethylbenzene, xylene isomers, benzyl alcohol, benzaldehyde, andcresol isomers) were added as pure liquids with a 10-  l syringe. All othercompounds were added as anaerobic, filtered aqueous stock solutions; no or-ganic carrier phases were used for amending the cultures. Catabolic studies wereperformed in duplicate with appropriate controls, as described in later sections. Enzyme assays.  All harvesting and manipulation of cells for enzyme assays were conducted under anaerobic conditions. Benzoate-grown cells (400 ml) wereharvested by centrifugation, washed in an anaerobic MOPS (morpholinepro-panesulfonic acid) buffer (50 mM, pH 7.0) containing 5 mM dithiothreitol and 5mM MgCl 2 , and then resuspended in 1.5 ml of the MOPS buffer. Assays wereperformed with permeabilized cells (2% Triton X-100 [vol/vol]) under anaerobicconditions in stoppered glass cuvettes at 35  C. Carbon monoxide dehydrogenaseand formate dehydrogenase activities were assayed by following the reduction of methyl viologen at 578 nm, as described elsewhere (38). 2-Oxoglutarate dehy-drogenase activity was also assayed with methyl viologen as the electron accep-tor; a tricine buffer (100 mM, pH 8.5) containing 5 mM dithiothreitol and 1 mMmethyl viologen was used. The protein concentration in the permeabilized cellpreparation was estimated by using the measured cell density and estimated values for cell mass and composition taken from Neidhardt (29); the proteincontent of the cell preparation could not be measured directly because of inter-ferences caused by the presence of FeS.  Analytical methods. (i) Aromatic hydrocarbon analysis.  Toluene and othersingle-ring aromatic hydrocarbons were measured by a static headspace tech-nique using a model 5890A gas chromatograph (Hewlett-Packard Company,Palo Alto, Calif.) with a model PI 52-02A photoionization detector (10.2 eVlamp; HNU Systems, Inc., Newton, Mass.) and a DB-624 fused silica capillarycolumn (30-m length, 0.53-mm inner diameter, 3.0-  m film thickness; J & WScientific, Folsom, Calif.). Analyses were isothermal (80  C) with splitless injec-tion (the split was turned on after 0.5 min). The method is described in detailelsewhere (5). The detachable stainless steel needles for the gas-tight syringesused for headspace sampling were autoclaved before each use. Peak integrationfor aromatic hydrocarbons (as well as for sulfate and oxygenated aromatic com-pounds [described below]) was performed with a Nelson analytical chromatog-raphy software system (model 2600; Perkin-Elmer, Cupertino, Calif.). (ii) Oxygenated aromatic compound analysis.  Concentrations of oxygenatedaromatic compounds tested individually as electron donors in catabolic studies with PRTOL1 (phenylpropionate, phenylacetate, benzylsuccinate, benzyl alco-hol, benzaldehyde, benzoate,  o - and  p -cresol,  p -hydroxybenzoate, and  p -toluate) were determined in the supernatant of centrifuged samples by reverse-phasehigh-performance liquid chromatography (HPLC) with a Perkin-Elmer series400 liquid chromatograph connected to a Hewlett-Packard model 1050 variable wavelength detector. The mobile phase was a 65:35 (vol/vol) mixture of methanoland 50 mM acetate buffer (pH 3.5) flowing isocratically at 1 ml/min through an Adsorbosphere HS C 18  column (5-  m particle size, 250 mm by 4.6 mm innerdiameter; Alltech). Compounds were quantified with a wavelength of either 265nm (for the first four compounds listed above) or 280 nm. For the aqueousHPLC standards, benzyl alcohol, benzaldehyde, and cresols were added as pureliquids; benzoate and  p -hydroxybenzoate were added as sodium salts; and theremaining four compounds were added from methanolic stock solutions. Themaximum methanol concentration in the HPLC standards was 0.06% (vol/vol). (iii) Sulfate analysis.  Sulfate in filtered samples (0.2-  m-pore-size syringefilters) was determined with an ion chromatograph (series 4000i, Dionex Cor-poration, Sunnyvale, Calif.) equipped with an HPIC-AS4A column, an anionmicromembrane suppressor, and a conductivity detector. Analyses were iso-cratic, with a 0.75 mM sodium bicarbonate–2.2 mM sodium carbonate eluantflowing at a rate of 2 ml/min. (iv)  14 C analysis.  14 C-labeled toluene was used in PRTOL1 cultures to inves-tigate the extent of toluene mineralization and incorporation into biomass. Anal- ysis of   14 C activity in culture liquid was performed with a Tri-Carb model 2500TR/AB liquid scintillation analyzer (Packard Instruments Co., Inc., DownersGrove, Ill.). All samples were automatically blank corrected and were correctedfor sample-specific quenching by using an external standard method (with a 133 Ba gamma source) and a quenching curve developed from a series of quenched standards.Sample-processing methods were very similar to those described previously(5), except that headspace was not sampled in the present study because exper-iments were designed to have negligible headspace (  1.5% of the total volume).By this method, three fractions of   14 C activity were determined in culture sam-ples:  14 CO 2 , nonvolatile carbon (including biomass and nonvolatile metabolites),and volatile carbon (including toluene and volatile metabolites). (v) Nonvolatile metabolite analysis.  Samples of culture (20 to 50 ml) wereacidified to a pH of   2 with solvent-cleaned HCl and extracted three times in aseparatory funnel with diethyl ether (Ultra Resi-Analyzed, distilled-in-glass; J. T.Baker). The extracts were dried with anhydrous sodium sulfate, derivatized withethereal diazomethane (15), concentrated under a gentle stream of high-puritynitrogen at room temperature, exchanged into dichloromethane (Ultra Resi- Analyzed, distilled-in-glass; J. T. Baker, Inc.), and analyzed by GC-MS. GC-MSanalyses were performed with a model 5890A GC (Hewlett-Packard Company) with a DB-5 fused silica capillary column (30-m length, 0.32-mm inner diameter,0.25-  m film thickness; J & W Scientific) coupled to an HP 5970 series massselective detector; data analysis was performed with Hewlett-Packard G1034Csoftware designed for the MS ChemStation. The GC oven was programmed from45  C (held for 2 min) to 110  C at 8  C/min and then from 110  C to 250  C at4  C/min (held for 5 min); the injection port temperature was 275  C, and thetransfer line temperature was 280  C. Injections were splitless, with the splitturned on after 0.5 min. For data acquisition, the MS scanned from 50 to 350atomic mass units at a rate of two scans per s. Determination of growth.  Growth was determined by two methods: micro-scopic cell counts and optical density. Growth experiments were conducted in thedark with clear glass tubes (13 by 100 mm) that were sealed with PTFE-facedsilicone septa inside open-hole screw caps. The medium used in these experi-ments contained MgSO 4  rather than FeSO 4  to preclude the formation of FeCO 3 V OL  . 62, 1996 SULFIDOGENIC TOLUENE-DEGRADING BACTERIUM 1189   onA  pr i  l  1  3  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om   and FeS precipitates. Cell counting was performed by phase-contrast microscopyat 400-fold total magnification with a Petroff-Hausser counting chamber(Hausser Scientific, Blue Bell, Pa.). Because the cell densities being counted were generally between 10 6 and 5  10 7 cells per ml, samples were concentratedfivefold by centrifugation before being counted. A more rapid method used to assess growth was determining the opticaldensity (absorbance) at 600 nm with a Spectronic model 20D single-beam spec-trophotometer (Milton Roy, Rochester, N.Y.). When measurements were made,the sealed tubes were removed from the glove box, analyzed within 10 min, andreturned to the glove box. A determination that growth had occurred in a given sample was based onpositive results relative to controls for both optical density and cell counts. Morespecifically, the two criteria for growth were (i) an optical density value greaterthan or equal to that of the positive control and (ii) a cell count greater than orequal to four times that of the negative control. In experiments testing potentialelectron donors, the positive controls included benzoate and the negative con-trols had no added electron donor. In experiments testing potential electronacceptors, the positive controls included sulfate and the negative controls had noadded electron acceptor. Growth was not determined for most of the experi-ments that assessed potential electron donors for PRTOL1. In these experi-ments, only metabolism (as indicated by the consumption of the electron donorand/or acceptor) was assessed because the relatively long generation time andlow growth yield of PRTOL1 made growth difficult to quantify reliably. 16S rRNA gene isolation, sequencing, and analysis.  Total DNA was isolatedfrom PRTOL1 by a method shown to work for diverse bacteria (42). A portionof this DNA (ca. 0.1  g) was used in the PCR to amplify most of the 16S rRNA gene. The primers used to amplify near full-length 16S rRNA gene sequences(5  -AGAGTTTGATCCTGGCTCAG-3   and 5  -AAGGAGGTGATCCAGCC-3  ) were modified versions of the primers fD1 and rD1 used by Weisburg et al.(44). The PCR mixture consisted of 1.5 mM MgCl 2 , 0.2 mM each deoxynucleo-side triphosphate (dNTP), 0.25  M each primer, 1  Taq  polymerase buffer, and0.75 U of   Taq  polymerase (Promega Corp., Madison, Wis.) in a volume of 30  l. Amplification was carried out by using a GeneAmp PCR System 9600 ThermalCycler (Perkin-Elmer Corp., Norwalk, Conn.) with a program consisting of aninitial denaturation at 92  C for 130 s; 30 cycles of 94  C for 15 s, 55  C for 30 s, and72  C for 130 s; and a final elongation cycle at 72  C for 370 s.The resulting PCR product was purified by gel electrophoresis with a 1%agarose gel and was recovered using Gene Clean purification resin according tothe manufacturer’s suggestions (Bio 101, Inc., La Jolla, Calif.). The purified PCRproduct was cloned in the vector pCRII by using a TA Cloning Kit (Invitrogen,San Diego, Calif.) according to the manufacturer’s directions. Plasmid DNA containing the 16S rRNA gene insert was isolated from one clone by using theQiagen plasmid mini kit according to the manufacturer’s directions (Qiagen,Chatsworth, Calif.).The DNA sequence of the insert was determined by automated fluorescentdye terminator sequencing with an ABI Catalyst 800 laboratory robot and ABI373A sequencer (Applied Biosystems, Foster City, Calif.). The primers that wereused corresponded to conserved regions of the 16S rRNA sequence (47). Ap-proximately 95% of the insert sequence was determined in both directions.Related sequences were obtained from the Ribosomal Database Project (24). A maximum-likelihood phylogenetic tree was created with the program fast-DNAml (30), by using a weighting mask to include only unambiguously alignedpositions with all other program options at their default values. This analysis wasrepeated on 100 bootstrap samples to obtain confidence estimates of the branch-ing order (16). The program CONSENSE from the PHYLIP program package(17) was used to determine the number of times that each group shown in thefinal tree was monophyletic in the bootstrap analysis. Nucleotide sequence accession number.  The 16S rRNA gene sequence of strain PRTOL1 has been assigned GenBank accession number U49429. RESULTSMorphology.  Cells of PRTOL1 are oval, 2 to 3  m long, and1.2 to 1.7   m in diameter and stain gram negative. A phase-contrast photomicrograph of PRTOL1 cells growing on tolu-ene is presented in Fig. 1. Microscopic examination did notreveal any evidence of spores or of swimming motility. Phylogenetic classification based on 16S rRNA.  Phyloge-netic relationships based on 16S rRNA gene sequences aredepicted in Fig. 2. Strain PRTOL1 is classified in the delta FIG. 1. Phase contrast micrograph of PRTOL1 cells growing on toluene. Bar, 5  m.FIG. 2. Maximum-likelihood phylogenetic tree of PRTOL1 and representa-tive delta  Proteobacteria . The bar represents nucleotide changes per position.Numbers at internal nodes are the percentage of 100 bootstrap samples in whichthe organisms to the right of the node were monophyletic. 1190 BELLER ET AL. A  PPL  . E NVIRON . M ICROBIOL  .   onA  pr i  l  1  3  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   a em. a s m. or  g /  D  ownl   o a d  e d f  r  om 
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