Numerical and Genetic Analysis of Polycyclic Aromatic Hydrocarbon-Degrading Mycobacteria

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Numerical and Genetic Analysis of Polycyclic AromaticHydrocarbon-Degrading Mycobacteria

Yong-Hak Kim1, Karl-H. Engesser2 and Carl E. Cerniglia1

(1) Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079(2) Abteilung Biologische Abluftreinigung, Institut fur Siedlungswasserbau, Wasserguteund Abfallwirtschaft (ISWA), Universitat Stuttgart, Bandtale 2, 70569Stuttgart, Germany

Received: 21 June 2004 / Accepted: 4 October 2004 / Online publication: 19 August 2005

Abstract

Ability to degrade high molecular weight polycyclicaromatic hydrocarbons (PAHs) has been found in diversespecies of fast-growing mycobacteria. This study includedseveral PAH-degrading mycobacteria from heavily con-taminated sites and an uncontaminated humus soil in theNatural Park, Schwabische Alb, Germany. The numericalanalysis with a total of 131 tests showed that isolates fromhumus soil and contaminated sites had similar substrateutilization patterns for primary alcohols from ethanol topentanol, 1,4-butanediol, benzyl alcohol, hexadecane,ethyl acetate, fluoranthene, phenanthrene, and pyrene asthe sole carbon and energy (C/E) sources. Significantdifferences between the two subgroups isolated fromhumus soil and contaminated sites were observed in theutilization of polyalcoholic sugars, including adonitol, D-arabitol, L-arabitol, erythritol, inositol, rhamnose, sorbi-tol, and xylitol. Among isolates from humus soil, strainPYR100 showed high similarity in 16S rDNA sequencewith M. vanbaalenii strain PYR-1 (=DSM 7251, 100%)and M. austroafricanum ATCC 33464 (99.9%). In addi-tion to the numerical analysis, the 16S–23S intergenicspacer sequence was useful for discriminating betweenthe closely related strains PYR100 and PYR-1 (98%similarity). The patterns of the variable V2 and V3 re-gions in the ribosomal RNA gene corresponding to Esc-herichia coli positions 179 to 197 and 1006 to 1023,respectively, were useful for dividing fast-growing andthermosensitive PAH-degrading mycobacteria into tensubgroups consistent with the phylogenetic positions.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) exist ubiqui-tously as natural constituents and combustion products offossil fuels. Biodegradation of high molecular weight PAHswith four or more condensed aromatic rings has beenintensively studied. PAH-degrading mycobacteria play aunique role in the degradation of a variety of PAHs,including naphthalene, acenaphthene, anthracene, fluo-rene, phenanthrene, pyrene, fluoranthene, benz[a]antha-cene, and benzo [a] pyrene [2, 4, 7, 13, 18, 33, 35, 36, 39].

Generally, PAH-degrading mycobacteria are fast-growing strains that mineralize fluoranthene, phenan-threne, and pyrene. In case of a Mycobacterium sp. iso-lated from PAH-containing enrichment culture ofcontaminated river sediment, concurrent degradation ofphenanthrene and pyrene occurred after the preliminaryexposure to either compound [32]. The rates of PAHdegradation are influenced by chemical and physicalproperties of PAHs, e. g., water solubility and dissolutionrate [2, 42], as well as by environmental factors, such asorganic content, pH, oxygen concentration, and tem-perature for growth [10, 13, Kim et al., unpublisheddata]. We recently reported that PAH-degradingMycobacterium spp. strains PYR-1 (=DSM 7251) andPYR100 have similar enzyme activities, not only for PAHdegradation but also for detoxification of PAH catecholsand quinones to prevent the redox cycle generatingreactive oxygen species which affect the cell survival andviability [25].

Among PAH-degrading mycobacterial species, mosthave been isolated from heavily contaminated sites, be-cause there is public interest in potential use of thesespecific microorganisms for restoration of oil- and PAH-contaminated sites [2, 13, 17, 31]. In contrast, Kim [23]isolated several PAH-degrading Mycobacterium strainsfrom uncontaminated humus soil. The bacterial com-positions may differ according to the physico-chemical

Correspondence to: Yong-Hak Kim at present address: School of Bio-logical Sciences, Seoul National University, San 56-1 Shinrim-dong,Kwanak-ku, Seoul 151-747, Republic of Korea; E-mail: yhkim660628@hotmail.com

110 DOI: 10.1007/s00248-004-0126-3 d Volume 50, 110–119 (2005) d � Springer Science+Business Media, Inc. 2005

properties of the environment, as well as dynamicchanges in the physiological and metabolic activities ofthe biological components in the presence of environ-mental pollutants. To classify microorganisms that areabundant and culturable under certain conditions, thenumerical taxonomy is based on classical morphological,physiological, and biochemical phenotypes to distinguishspecies and subspecies in comparison with known taxo-nomic references [37, 38, 43]. Moreover, the comparativeanalysis of 16S ribosomal RNA gene (rDNA) sequences isgenerally accepted for bacterial systematics [47].

We present here the numerical and genetic analysisof PAH-degrading mycobacteria isolated from heavilycontaminated sites and uncontaminated humus soil. Thenumerical analysis and sequencing the 16S–23S IGS wereuseful for distinguishing between closely related strains.The comparative analysis of the 16S rDNA sequenceamong members of fast-growing Mycobacterium speciesrevealed the two variable regions at positions of 179 to197 (V2 region in Escherichia coli numbering system) and1006 to 1023 (V3 region), which were useful for theclassification of PAH-degrading mycobacteria consistentwith the phylogenetic positions.

Methods

Chemicals. Chemicals used for carbon and energy (C/E) and carbon and nitrogen (C/N) tests (Table 1) wereobtained from Aldrich (Steinheim), Serva (Heidelberg),and Sigma (Deisenhof) in Germany. A redox indicator,tetrazolium violet, was purchased from Sigma (Deisen-hof, Germany). Other medium components and reagentswere from Oxoid (Wesel) and Merck (Darmstadt), Ger-many.

Microorganisms and Culture Conditions. Thisstudy included 14 PAH-degrading Mycobacterium strainsisolated from pyrene-containing enrichment cultures ofvarious soil and sediment samples: strain PYR-1 (=DSM7251) originated from oil-contaminated sediments,Redfish Bay, Texas, USA [17]; 11 isolates from anuncontaminated humus soil sample obtained from theNational Natural Park, Schwabische Alb, Germany; oneisolate (strain S10) from a pyrene-degrading mixed cul-ture provided by the Fraunhofer Gesellschaft, Stuttgart,Germany; and one isolate (strain PYR400) from an oldcoal gasification plant site, Germany. Strain PYR GCK(=ATCC 700033), which had been classified asM. flavescens based on the biochemical and fatty acidprofiles [7], was included for the 16S rDNA sequenceanalysis. The PAH-degrading mycobacterial cultures weremaintained on phosphate-based minimal (PBM) med-ium supplemented with 1–2 mM of phenanthrene orpyrene as the sole carbon source [24]. All strains used inthis study were deposited in the Strain Collection of

Abteilung Biologische Abluftreinigung, ISWA, Universi-tat Stuttgart, Germany, and Division of Microbiology,National Center for Toxicological Research, US FDA,Jefferson, Arkansas.

Characterization of Strains. Morphological prop-erties were determined by phase-contrast microscopy.Gram staining, acid-alcohol fastness, colony morphology,optimal conditions for growth at various temperatures(20�, 25�, 30�, 35�, 40�, and 45�C), NaCl concentrations(0, 0.5%, 1%, 2%, 4%, 7.5%, and 15%), pH (4.5, 5.5, 6.5,7, 7.5, 8.5, and 9.5) adjusted with 5 M HC1 or 5 MNaOH, and pigment production were examined in amodified R2A-medium (0.5 g yeast extract, 0.5 g pan-creatic peptone, 0.5 g casein hydrolysate, 1 g D-glucose,0.3 g sodium pyruvate, 0.3 g sodium acetate, 0.3 gpotassium phosphate dibasic, and 0.05 g MgSO4 in 1000ml distilled water) and on tryptic soy agar plates (30 gtryptic soy broth + 15 g Bacto-agar in 1000 ml distilledwater). All tests were performed at 30�C, except fordetermination of optimal temperature.

Numerical Analysis. The catalase test was carriedout with a loopful of colonies grown on tryptic soy agar(TSA) plates mixed with a drop of 3% hydrogen peroxideon a glass slide. The oxidase test was performed usingoxidase-test sticks (Baktident Oxidase, Merck) accordingto the user�s manual. Hydrolase assays for casein, cellu-lose, chitin, gelatin, poly-b-hydroxybutyrate, starch, andTween 20/80 were performed according to the Manual ofMethods for General Bacteriology [11], and the Microbio-logical Methods [5]. A modified R2A-medium was usedby mixing separately autoclaved polymer substrate (finalconcentration, 2%) at 50�C. In case of Tween 20/80 (1:1,v/v), CaCl2 solution was separately autoclaved and addedto the test medium at the final concentration of 0.1%. Aclear zone of hydrolyzed pectin was visualized after intactpectin had precipitated for several minutes with additionof 1 ml of 3.2% HCl to 20 ml agar medium.

For biochemical fingerprinting, test chemicals listedin Table 1 were used as the sole sources of carbon andenergy (C/E) and carbon and nitrogen (C/N). Each testchemical dissolved in distilled water or dimethylsulfoxide(DMSO) was sterilized through a 0.2-lm membrane fil-ter. For C/N sources, no inorganic nitrogen source (i.e.,ammonium sulfate and sodium nitrate) was added tophosphate-based minimal (PBM) media. Cells wereharvested by centrifugation at 4000 rpm for 15 min, andwere suspended in sterile 50 mM phosphate buffer (pH7.0).The cell suspension was incubated at 30�C and 108rpm for 2–4 h to avoid a false positive reaction of tet-razolium violet (final concentration 10 lg ml)1). Theresting cells were harvested by centrifugation at 4�C and4000 rpm for 15 min and re-suspended in PBM medium.The initial cell density (OD546) was adjusted to 0.05–0.1

Y-H. KIM ET AL.: NUMERIC AND GENETIC ANALYSIS 111

cm)1. Aliquots of the cell suspension were dispensed tosterile 20-ml bottles (WGA, Dtisseldorf, Germany) tomake the final volume of 2 ml with addition of a sole C/Eor C/N at the final concentration of 0.1% by volume orweight. The bottles were tightly closed with Teflon-linedrubber septa and aluminum caps. The optical density at546 nm (OD546) was measured with each 1 ml sampletaken at 24 h and 48 h during incubation at 30�C and 108rpm. Cell growth and color change of redox dye weredetermined in comparison to test controls withoutaddition of C/E or C/N source. Every test was at leastduplicated, and the results were scored using a numericalsystem, {)1, 0, 1}. The ‘‘1’’ value was given for more than50% optical density difference (DOD546) compared to thetest control. The ‘‘0’’ value resulted from a significant dyecolor change with less than 50% DOD546. The ‘‘)1’’ valuewas given for less than 50% DOD546 without dye colorchange. Soluble and volatile diaryl and PAH compounds(e. g., biphenyls and naphthalenes) were tested in PBMmedium as described above, while insoluble and non-volatile compounds were tested using PBM agar plates(+15 g Oxoid Nr.l Bacto-agar in 1 L PBM medium), ofwhich the surface was coated with a thin layer of 2–5 mMsubstrate dissolved in diethyl ether. Diethyl ether was air-dried for 24 h under sterile conditions before use. Theagar plates were incubated at 30�C for 3–4 weeks inparaffin-sealed glass jars to retain the moisture. Testcontrol plates without diaryl/PAH compound were in-cluded under the same conditions. The results of the agarplate assays were scored with the same numerical system{-1,0, 1}, according to colony size and formation of clear

zone and colored by-product, which accumulated in thecell or was released to the medium: 1, significant differ-ence in colony size relative to the colony size on controlplates; 0, formation of a clear zone and/or coloredbyproduct without a significant difference in colony size;)1, no difference between the cultures on test and controlplates.

For numerical taxonomy, Rhodococcus rhodochrousstrains 116 [19] and 117 (kindly provided by Dr. U.Karlson), and Janibacter spp. DPO360 and DPO1361[27] were used as reference strains.

Isolation and PCR Amplification of DNA. Myco-bacterium spp. strains PYR100, PYR-1, and PYR GCKwere used for extraction of genomic DNA, according tothe cetyltrimethylammonium bromide (CTAB) miniprepprotocol for bacterial genomic DNA preparations [46].The final DNA was suspended in 100 ll of 1 · TE bufferand stored at )20�C. The extracted DNAs of strainsPYR100, PYR-1, and PYR GCK were targeted foramplification of 16S rDNA and 16S-23S IGS. The tem-plate DNA was amplified with a forward primer (16F27;5¢-AGAGTTTGATCCTGGCTCAG-3¢) annealed at 16SrDNA positions 8 to 27 (Escherichia coli numbering) anda reverse primer (23R23; 5¢-TCGCCAAGG CATCCACC-3¢) annealed at the complement of 23S rDNA positions39 to 23. The PCR reaction mixture (50 lL) consisted of200 ng of genomic DNA, 0.2 mM each of four deoxy-nucleoside triphosphates, 50 pmol each of the twoprimers, 1 mM MgCl2, 5ll of 10 · PCR buffer, and 2.5 Uof rTaq DNA polymerase (Amersham Biosciences, Pis-

Table 1. Chemicals used in numerical tests for the assays of hydrolases and sole sources of carbon, nitrogen, and energy (C/N/E)

Hydrolase assays casein, cellulose, chitin, gelatin, pectin, poly)b)hydroxybutrate, starch (soluble), Tween 20/80 (1:1)

C/E sourcesAlcohols/glycols/

alkanes/ketones/estersmethanol, ethanol, 1)propanol, 1)butanol, 1)pentanol, 2)propanol, 2)butanol,

2)methyl)1)butanol, 3)methyl)1)butanol, benzyl alcohol, ethylene glycol,glycerol, 1,4)butandiol, hexadecane, acetone, ethyl acetate

Carbohydrates erythritol, D)arabinose, L)arabinose, ribose, D)xylose, L)xylose, adonitol, D)arabitol,L)arabitol, xylitol, D)glucose, galactose, D)fructose, D)mannose, L)sorbose,rhamnose, dulcitol, inositol, mannitol, sorbitol, a)methyl)D)glucoside,a)methyl)D)mannoside, salicin, cellobiose, maltose, lactose, melibiose,saccharose, trehalose, melezitose, D)raffinose, b)gentiobiose, glycogen

Organic acids lactate, pyruvate, citrate, DL)isocitrate, 2)oxoglutarate, succinate, fumarate,L)malate, maleic acid, malonate, oxalacetate, glyconate, adipate, gluconate

Phenols/benzoates benzoate, 2)methoxyphenol, 3)methoxyphenol, 4)methoxyphenol, phenol, salicylatediaryls/PAHs biphenyl, dibenzyl ether, diphenyl ether, dibenzofuran, 2)methoxybiphenyl,

4)methoxybiphenyl, naphthalene, 1)methoxynaphthalene, 2)methoxynaphthalene,1)methylnaphthalene, 2)methylnaphthalene, bibenzyl, biphenylene, acenaphthene,phenanthrene, anthracene, fluorene, carbazole, dibenzothiophene,xanthene, acridine, acridone, phenoxazine, iminostilbene, fluoranthene, pyrene,benzo[a]pyrene

C/N)sourcesAmino acids D)alanine, L)alanine, arginine, aspartate, asparagine, cysteine, cystine,

S)methyl)L)cysteine, glutamate, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, DL)tryptophan, tyrosine, valine

Aminobenzoates 2)aminobenzoate, 4)aminobenzoate

112 Y-H. KIM ET AL.: NUMERIC AND GENETIC ANALYSIS

cataway, NJ). The PCR conditions were a hot start at95�C for 2 min, followed by 33 cycles of 94�C of 1 min,52�C for 1 min, 72�C for 2 min 30 s, and extension of thefinal 72�C segment to 4 min 30 s before cooling to 4�C.Approx. 1.9-kb DNA bands were excised from agarosegels and purified using a QIAEX II gel extraction kit(Qiagen Inc., Mississauga, ON). The extracted DNA wasre-suspended in 20 lL of deionized water and stored at -20�C prior to DNA cycle sequencing.

Sequencing of PCR-Amplified DNA. With PCR-amplified template DNA, the DNA sequence was deter-mined by ABI PRISM 377 DNA Sequencer (PerkinElmer,Missisauga, ON). The 30 · cycle sequencing reaction wasperformed using primers 16F27, 16R519 (5¢-GTATTACCGCGGCTGCTG-3¢), 16F806 (5¢-TTAGATACCCTGGTAGTCC-3¢), 16R1098 (5¢-GGGTTGCGCTCGTTGCG-3¢), 16R1492 (5¢-TACGGYTACCTTGTTACGACTT-3¢), 16F1511 (5¢-AAGTCGTAACAAGGTARCCG-3¢), and 23R23, according to the standard protocol ofBigDye Terminator v 3.1 cycle sequencing kit (AppliedBiosystems, Foster City, CA).

Phylogenetic Analysis. The primary and second-ary structure of 16S rRNA were analyzed manually usingtemplates published in the Ribosomal Database Project(Ribosomal Database Project (RDP), ref. [30]) to com-pare the variable and homologous sequence positions.Sequences were aligned using ClustalW [41], and phy-logenetic analysis was performed using the PHYLIPpackage [9]. Distance analyses were performed as de-scribed by De Soete [8]. Reference 16S rDNA sequenceswere obtained from Genbank and the RDP.

Nucleotide Sequence Accession Numbers. The16S ribosomal RNA gene and 16S-23S intergenic spacer(IGS) sequences of strains PYR100, PYR-1, and PYR

GCK are available from GenBank (accession numbersAY636002, AY636003, and AY694989).

Results and Discussion

Morphological and Physiological Properties. Cells ofPAH-degrading mycobacteria were Gram positive, acid-alcohol fast, non-motile, and rod shaped (length, 2–2.5lm; diameter, 0.8–1 lm). They often formed V-shapedcells and thick cells in exponential and late exponentialperiods. All strains produced yellow pigments of scoto-chromogenic colonies on tryptic soy agar plates. Thecolony morphology varied, even for a single strain fromsmooth to rough. Optimal growth was observed at 0%NaCl and 30�C in modified R2A broth and on tryptic soyagar plates. Optimal pH conditions varied from 6.5(strain PYR300) to 9.5 (strain PYR210), but most strainsgrew within a range of pH 7.0 to 8.5. Below pH 6.5, no ormarginal growth was observed.

Numerical Analysis. Fourteen PAH-degradingMycobacterium strains, two Rhodococcus strains, and twoJanibacter strains were analyzed with a total of 131numerical data. The full data are available on-line athttp://elib.uni-stuttgart.de/opus/volltexte/200l/754/pdf/Kim_diss.pdf from the Library of Universitat Stuttgart.The numerical data were useful for calculation of thecorrelation coefficients, r, to determine similarity be-tween strains. By ranking the correlation coefficient inthe highest order of the r-values, the analyzed clusters areshown in Figure 1. The genera of strains were clearlyseparated at the points less than the mean, rav = 0.548, ofthe total r-values: i.e., the breaking points with an r-valueof less than the total rav distinguished among the generaMycobacterium, Rhodococcus, and Janibacter. PAH-degrading Mycobacterium strains were divided into twosubgroups with an internal breaking point at which the

Figure 1. Cluster analysis of correlation coefficients (r) for numerical data among PAH-degrading mycobacteria and reference strains.Black arrows indicate the breaking points less than the average of total r-values (total rav = 0.584) for discriminating among the generaMycobacterium, Rhodococcus, and Janibacter, and white arrow indicates a breaking point less than the subtotal average of the genusMycobacterium cluster (subtotal rav = 0.734) for dividing 14 PAH-degrading strains into two subgroups, I and II.

r-value was less than the mean (subtotal rav = 0.734) ofthe subtotal r-values in Mycobacterium-cluster. By thiscriterion, three strains PYR-1 (=DSM 7251T), S10, andPYR400, which all originated from heavily contaminatedsites, comprised subgroup I. Subgroup II consisted ofhumus soil-originated strains. Table 2 shows the selecteddata from the cluster analysis to briefly summarize thecharacteristics that determine the strain-specific activitiesand the taxonomically related phenotypes of PAH-degrading Mycobacterium strains in subgroups I and II.

The strain-specific activities for hydrocarbon degra-dation were similar to each other. The PAH-degradingMycobacterium strains were, in general, able to utilizeprimary alcohols from ethanol to pentanol, 1,4-butane-diol, benzyl alcohol, hexadecane, ethyl acetate, fluoranth-ene, phenanthrene, and pyrene as the sole C/E sources.Most strains did not grow on methanol, secondary alco-hols, ethylene glycol, acetone, phenols, anthracene, naph-thalenes, diaryl compounds, or benzo[a]pyrene. It wasnotable that strain PYR-1 degraded biphenyl and anthra-cene, and that isolate S10 was capable of utilizing anthra-cene. The degradation activities for fluoranthene,phenanthrene, and pyrene were considered signatures forPAH-degrading Mycobacterium strains.

Separation of the two mycobacterial subgroups I and IIseemed to be related to contamination of the isolation siteswith oil spills and PAHs. Summarized in Table 2, themembers of subgroup I utilized inositol, but not adonitol,D-arabitol, L-arabitol, erythritol, rhamnose, sorbitol, orxylitol. In contrast, the members of subgroup II exhibited areversed pattern of the utilization of these polyalcoholicsugars. It has been known that rtl-atl/gat alternation ofalleles in Escherichia coli C strain changes the substrateutilization patterns for D-arabitol, ribitol, and galacitol [28,48]. However, it is not yet certain whether similar chro-mosomal rearrangements occur in Mycobacterium species,and whether the prolonged exposure to high concentra-tions of industrial pollutants is related to the changes in theutilization of polyalcoholic sugars.

Members of the genera Mycobacterium are consid-ered potential degraders for hazardous industrial pollu-tants, such as aliphatic hydrocarbons, chlorinated

hydrocarbons, and polycyclic aromatic hydrocarbons [3,14, 16, 22, 26, 45]. Ever since strain PYR-1 was firstisolated for degradation of high molecular weight PAHsfrom an oil-contaminated site [17], the mycobacterialPAH degradation has been intensively studied with avariety of PAHs [18, 20, 21, 33–35]. These activities havebeen found in various Mycobacterium strains, includingstrain PAH135 (= RJGII-135) [13]; strain BB1 [2]; strainPYR GCK [7]; M. austroafricanum [26]; M. hodleri [26];strain CH1 [4]; strains LB208, LB 307T, and LB501T [3];M. frederiksbergense [45]; strain 1B [6]; and strains JLS,KMS, and MCS [31]. In addition, there are several directsubmissions of the 16S rDNA sequences of isolates to theGenBank database exhibiting the typical traits for PAH-degrading mycobacteria.

Analysis of 16S rDNA and 16S–23S Intergenic

Spacer. Because strains PYR-1 and PYR 100 resultedin the greatest distance between strains from the clusteranalysis (Fig. 1), they represented subgroups I and II,respectively, and their partial 16S rDNA and 16S–23Sintergenic spacer (IGS) sequences were further analyzedindependently three times.

Surprisingly, alignment of the 16S rDNA sequencesresulted in 100% similarity between the two strains. Sincethe 16S–23S IGS had a greater discriminatory power than16S rDNA [15], alignment of the IGS sequences distin-guished with 98% similarity between closely related strainsPYR100 and PYR-1 (Fig. 2). The comparative analysis of16S rDNA and 16S–23S IGS indicated that strain PYR100was an IGS sequevar variant of M. vanbaalenii.Comparison of almost complete 16S rDNA sequencesshowed that strain PYR 100 was a member of the fast-growing mycobacteria. The 16S rDNA sequences of strainsPYR100 and PYR-1 were 99.9% similar to M. austroafri-canum ATCC 33464T. A dendrogram of relatedness, gen-erated by the distance analysis of Kimura�s two-parametercorrection of similarity to compensate for multiplenucleotide exchanges [9], is shown in Figure 3. The max-imum-likelihood and maximum parsimony also recoveredthe M. austroafricanum–M. vanbaalenii cluster (trees notshown).

Figure 2. Sequence alignment of 16S–23S intergenicspacer regions of strains PYR100 and PYR-1. Identicalsequences are shown with dots, and gap positions areshown with hyphens. The similarity in the overlap regionis 98%. M = A or C.

By this study, former M. flavescens strain PYR GCKwas reclassified as M. gilvum strain PYR GCK, becausethe 16S rDNA sequence was very similar to type strainM. gilvum ATCC 43909.

The primary and secondary structures of mycobac-terial 16S rRNA are not divergent, but certain regions arehighly variable [40]. Figure 4 shows the most variableregions, V2 and V3, in members of fast-growing myco-bacterial species, including published and unpublished(directly submitted to GenBank) PAH-degrading Myco-

bacterium strains. The V2 region was the most variableamong species of fast-growing mycobacteria. In this re-gion, there was no difference between strains PYR-1 andPYR 100, and the high similarity was seen withM. austroafricanum ATCC 33464T and M. vaccae ATCC15438T, consistent with the phylogenetic analysis of the16S rDNA.

Besides this V2 region, there was another variable V3region, corresponding to 16S rRNA of Escherichia coli atpositions 1005 to 1023. The 7 base pairs comprised ashort helix structure. Despite nucleotide sequence varia-tion, the alternative stem-and-loop structures were con-served in a group of mycobacteria. The ribosomal RNAsequence, 5¢-GCCGGCAGAGAUGUCGGU-3¢, wasbroadly found from the type strains of M. parafortuitum,M. chlorophenolicum, M. aichiense, M. doricum and M.gilvum. The sequence, 5¢-GUGCCUAGAGAUAGGUAU-3¢, was found in the M. vanbaalenii–M. austroafricanum–M. aurum cluster. However, the closely related M. vaccaehad a different sequence, 5¢-GCUGGUAGAGAUAUC-AGU-3¢, which was identical with the M. hodleri–M.neoaurum–M. diernhoferi cluster and the M. monacensecluster. As another case, M. frederiksbergense had two basepair differences in the V3 region sequence, 5¢-GAC-GGCAGAGAUGUCGUU-3¢, compared to the closelyrelated strains LB501T and CH1.

Even though strains have a similar pattern in one ofthe two variable regions, there could be significant dif-ferences in the other variable region. The patterns of thetwo variable regions were used for dividing 10 subgroupsof fast-growing PAH-degrading Mycobacterium strainswith known type strains. Among members of the sub-group, there were no more than 3 base pair differences inthe two variable regions to be more or less consistentwith previous findings [1, 6, 12, 22, 26, 29, 31, 44, 45].Accordingly, strain PYR100 belonged to subgroup I withtype strains M. vanbaalenii DSM 7251T (= strain PYR-1)and M. austroafricanum ATCC 33464T. It was remarkablethat subgroup I did not include M. aurum ATCC 23366T

and M. vaccae ATCC 15438T, because more than 3 basepair differences existed in either V2 or V3 region. StrainsJLS, KMS, and MCS (subgroup II) were similar to M.monacense strain B9-21-178. Mycobacterium sp. 15 andM. doricum (subgroup III) had a similar V2 region withmembers of subgroup II, but they displayed a differentpattern in the V3 region. M. hodleri EMI2T was yet theonly member of subgroup IV that showed a different V2region sequence compared to the closely related M.neoaurum ATCC 25795T and M. diernhoferi ATCC19340T. In contrast, M. diernhoferi ATCC 19340T showedsimilar V2 and V3 patterns with M. anthracenicum(subgroup V), although they formed different subcladesin the phylogenetic tree. The V3 region sequence of strainS65 (subgroup VI) was very different from those of strainPAH 135 and M. aichiense ATCC 27280T (subgroup VII),

Figure 3. A dendrogram of relatedness obtained from the distancematrix analysis of 16S ribosomal RNA sequences within fast-growing Mycobacterium species. Positions of strain PYR100 andother PAH-degrading strains are shown in shadowed boxes. Thenumbers are bootstrap values (expressed as percentage) greaterthan 50%. Bar, 1 nucleotide substitution per 100 positions.

whereas there were no more than 3 base pair differencesin the V2 regions among all members of the subgroupsVI and VII. Subgroup VIII consisted of closely relatedstrains CH1, LB501T and M. frederiksbergense DSM44346T. M. gilvum ATCC 43909 was constituted repre-sentative of the largest subgroup IX including a broadrange of strains PYR GCK, IB clone2, RF002, LB 208,LB307T, C2-3, and BB1 (=DSM 9487). However, strain1B clone1 was not included in this subgroup because ofsignificant differences in the V2 region. It was reportedthat the 16S rDNA sequencing clone 1 and clone2 wereobtained from strain 1B isolated from a PAH-degradingmixed culture [6], but it seemed that strain 1B was amixed culture of at least two different Mycobacteriumstrains. Strain WF2 was yet the only member of subgroupX with a unique V2 region sequence. Apart from thesesubgroups of fast-growing PAH-degrading Mycobacte-rium strains, different patterns of the two variable regionswere found from other fast-and slow-growing mycobac-terial species (data not shown).

In conclusion, this study demonstrates that PAH-degrading strains are diverse from fast-growing andthermosensitive mycobacteria. They could be responsiblefor the degradation of a variety of PAHs, including flu-oranthene, phenanthrene, and pyrene, in various envi-ronments. We first reported that uncontaminated humussoil-originated PAH-degrading mycobacteria show not

only the typical traits for PAH-degrading mycobacteriaisolated from heavily contaminated sites, but also thatone isolate PYR100 among them has 100% similarity in16S rDNA sequence with M. vanbaalenii strain PYR-1originated from a different location with heavy contam-ination of oil spills and PAHs. Although those strainsexhibit inverted phenotypes for the utilization of poly-alcoholic sugars, such as adonitol, D-arabitol, L-arabitol,erythritol, inositol, rhamnose, sorbitol, and xylitol, theyare phylogenetically related to each other. In addition tothe numerical analysis (e.g., biochemical fingerprints),the 16S–23S IGS sequencing is used to differentiate clo-sely related species and subspecies. Patterns of the vari-able V2 and V3 regions in 16S rDNA sequence are usefulfor the classification of PAH-degrading mycobacteriaconsistent with the phylogenetic positions.

Acknowledgments

Y.-H Kim thanks Dr. Peter C.K. Lau and colleagues forfriendly help since he wrote a part of this study during avisit to Biotechnology Research Institute, National Re-search Council Canada, Montreal, Quebec. This studywas supported in part under fellowship programs of theDeutscher Akademischer Austauschdienst (DAAD) andthe Oak Ridge Institute of Science and Education (OR-ISE).

Figure 4. Patterns of the variable V2 and V3 regions in 16S ribosomal RNA gene for dividing 10 subgroups among fast-growingPAH-degrading mycobacteria. The V2 region corresponds to Escherichia coli positions 179 to 197, and the V3 region corresponds tothe positions 1006 to 1023. Identical sequences are shown with dots and gap positions are shown with hyphens. Seven base pairs forming ashort helix of the V3 region are shown with shadowed boxes.

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Numerical and Genetic Analysis of Polycyclic Aromatic Hydrocarbon-Degrading Mycobacteria

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