Bacterium Degrade Fluoranthene

Mycobacterium fluoranthenivorans sp. nov.,a Fluoranthene and Aflatoxin B1 Degrading Bacteriumfrom Contaminated Soil of a Former Coal Gas Plant

D. Hormisch1,4, I. Brost2, G.-W. Kohring1, F. Giffhorn1, R. M. Kroppenstedt3, E. Stackebrandt3, P. Färber2, andW. H. Holzapfel2

1 Saarland University, Applied Microbiology, Saarbruecken, Germany2 Federal Research Center for Nutrition and Food, Institute of Hygiene and Toxicology, Karlsruhe, Germany3 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany4 LUFA – Landwirtschaftliche Untersuchungs- und Forschungsanstalt, Speyer, Germany

Received: May 21, 2004

Summary

Mycobacterium strain FA4T was isolated with fluoranthene as the single carbon source from soil of a for-mer coal gas plant, polluted with polycyclic aromatic hydrocarbons. The physiological properties, fattyacid pattern, and the 16S ribosomal RNA gene sequence indicated membership to the genus Mycobac-terium, but were different from all type strains of Mycobacterium species. Based on comparative 16SrRNA gene sequence analyses strain FA4T could be assigned to the Mycobacterium neoaurum taxonshowing 98% sequence similarity to M. diernhoferi as its closest neighbour. The occurrence of epoxymy-colate in the cell wall differentiates FA4 from all members of this taxon which synthesize wax-ester my-colates in addition to alpha-mycolates. Strain FA4T is able to degrade aflatoxin B1. This biological at-tribute might be useful in biological detoxification processes of foods and feeds. From the investigatedcharacteristics it is concluded that strain FA4T represents a new species, for which we propose the nameMycobacterium fluoranthenivorans sp. nov. The type strain of Mycobacterium fluoranthenivorans isFA4T (DSM 44556T = CIP 108203T).

Key words: Mycobacterium fluoranthenivorans sp. nov. – polycyclic aromatic hydrocarbons – fluoran-thene – aflatoxin B1 – degradation

Introduction

Among toxic waste product degrading bacteria, nu-merous strains from the genera Rhodococcus and My-cobacterium have been isolated from the environmentand were described in detail [6, 7, 12, 13, 16, 27]. Someof these organisms are able to degrade chlorinated com-pounds like vinyl chloride [11] and pentachlorophenol[9], or compounds from the chemically diverse group ofpolycyclic aromatic hydrocarbons (PAH’s) [2, 5, 12, 28,34, 36]. These bacterial strains can either utilise a widevariety of xenobiotics as growth substrates or they are re-stricted to only a few or a single hazardous compound.Growth of Mycobacterium vanbaalenii [20], for example,is described with naphthalene, phenanthrene, anthracene,fluoranthene, pyrene, 1-nitropyrene, 3-methylcholan-thene, 6-nitrocrysene and benzo[a]pyrene. Mycobacteri-um frederiksbergense [41] mineralises phenanthrene, flu-oranthene or pyrene and Mycobacterium hodleri [21]

grows with fluoranthene as the only carbon source, butco-oxidizes other PAH’s during growth with fluoran-thene.

Aflatoxins with the naturally occurring subgroups B1,B2, G1, G2, M1 and M2 are bisfurano-coumarin deriva-tives. They are structurally closely related to the abovementioned chemical compounds. Aflatoxins are pro-duced during the secondary metabolism of filamentousfungi. Especially aflatoxin B1 (AFB1) belongs to the mostdangerous naturally occurring mycotoxins because of itshepatotoxic, hepatocarcinogenic, mutagenic and terato-genic effects, both on humans and on animals. AFB1 isproduced both under moderate and under subtropic andtropic climatic conditions. Because of its hazardous nature,

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System. Appl. Microbiol. 27, 653–660 (2004) http://www.elsevier.de/syapm

Genbank accession number: AJ617741

different physical and physico-chemical techniques havebeen developed to detoxify and to degrade AFB1 [40].AFB1 is a physically and physico-chemically stablemolecule, e.g. temperatures >250 °C are necessary for ef-fective destruction. Foods treated for physical degrada-tion of AFB1 are not acceptable for human consumption.Therefore, biological degradation of AFB1 without sig-nificant alteration of the sensory characteristics of afood, would be most advantageous, particularly in situa-tions of food scarcity. During the search for bacterial de-graders of AFB1, strains from different habitats, includ-ing soils from a former coal gas plant, were screened fortheir ability to degrade AFB1. Among the few positivestrains, one was found to belong to the genus Mycobac-terium but could not be allocated to any known speciesof this genus, thus representing a putative new species.This paper describes Mycobacterium strain FA4T whichis able to degrade AFB1 most effectively.

Material and Methods

IsolationPAH polluted soils from a former coal gas plant in Saar-

bruecken-Burbach, Germany, were screened for potential PAHdegraders; in this process, strain FA4T was isolated by the fol-lowing procedure: The sampling comprised 3 g of soil whichwere added to 25 ml of liquid medium containing in 1000 mlH2O: 0.9 g KH2PO4, 2.5 g Na2HPO4

·H2O, 0.2 g MgCl2·H2O,

0.5 g KNO3, 0.1 g (NH4)2SO4, 0.2 g NH4Cl, 3.59 mgCaCl2

·H2O, 1.66 mg FeCl3·H2O, 10 ml SL4 [30], and 10 ml ofvitamin solution [31], adjusted to a final pH of 7.2. As a carbonsource 2.5 ml of fluoranthene (10 mg/ml) dissolved in hep-tamethylnonane (ICN Biochemicals) were loaded on top of themedium, which provided the organisms with a constant concen-tration of fluoranthene in the water phase, determined by thepartition coefficient. The cultures were incubated at 28 °C on arotary shaker. After visible growth, the enrichment cultures weretransferred to fresh media for at least three times and then sepa-rated on R2A agar plates (Difco Lab.). Single colonies weregrown again in the described liquid medium and finally testedfor purity on R2A agar plates.

Characterisation and biochemical testsAll tests followed methods indicated [41] and were per-

formed in duplicate. As a control, all tests were also carried outwith M. hodleri which exhibited the same results as reported inthe literature [21]. Morphological analysis, Gram staining andacid-alcohol fastness were investigated by phase contrast andbright field microscopy. The ability to grow at different temper-atures, pigment production and photoreactivity were deter-mined after 2 weeks of growth in Middelbrook medium [23].Tests for urease [10], catalase [22] and nitrate reductase activityor Tween 80 hydrolysis [1] were performed as described in thecitations. Growth investigations with sugars and sugar alcohols[40], and other physiological tests were performed as described[19, 41].

Growth with different PAH’s as single carbon sources was in-vestigated in the same medium described under “isolation”, sup-plemented with 10% (w/v) of each PAH dissolved in hep-tamethylnonane (10 mg/ml). Degradation of the PAH’s was de-termined by reverse phase HPLC (Nucleosil 120-5-C-18 col-umn, 48.5% methanol and 3% acetic acid in water as the mo-bile phase, UV detection, Beckman System Gold) after diluting

samples of the heptamethylnonane phase 1:350 with methanolprior to injection.

Analytical methodsAs described [37], 40 mg wet weight of cells were saponified,

methylated and extracted. The derived fatty acid methyl esters(FAMEs) were analysed by GC (model 5898A, Hewlett Packard,controlled by MIS software, Microbial ID) and the “MicrobialIdentification” standard software package [35]. Thin layer chro-matography investigation of mycolic acids followed describedmethods [24, 26]. Mycolic acids were extracted from saponifiedcells and transferred to their bromophenacyl esters [3, 25]. Afteraddition of low and high molecular mass standards (Ribi Im-munoChem Research) the mixture was separated and analysedby HPLC (C18 Ultrasphere XL cartridge column, 35 °C,Hewlett Packard Series 1050 operated with the Sherlock Systemsoftware, MIDI Inc.). For the analysis of the mycolic acid cleav-age products (MACP) the cells were treated as described abovefor fatty acids. To enhance the pyrolysis of the mycolic acids 10µl of 0.2 M methanolic TMSH was added to the fatty acid/my-colic acid methyl esters extract and analysed by GC [29].

16S rRNA gene sequence determinationExtraction of the DNA, PCR-mediated amplification of the

16S rDNA and purification of the PCR products were done asdescribed [33]. The purified amplificates were analysed with anApplied Biosystems 373A DNA Sequencer. The 16S rRNA genesequence was aligned manually with published sequences fromrepresentatives of the actinomycete sublines of descent includedin the DSMZ database of sequences. Neighbour joining analysisand calculation of bootstrap values were done according to thePHYLIP program [8].

AFB1 degradation experimentsThe method for the quantitative determination of AFB1 is

based on the CEN/DIN method prEN 14123 (“Foodstuffs – De-termination of aflatoxin B1 and the sum of aflatoxin B1, B2, G1

and G2 in peanuts, pistachios, figs and paprika powder”). Sim-plification was possible by using a miniaturised in vitro test sys-tem, whereby there was no need for the use of an immuno-affin-ity column clean-up of the test samples. In contrast to complexfoodstuff like peanuts, pistachios or maize, which are rich inprotein and fat/oil, the complete medium used in these tests didnot require a clean-up of samples. The test cultures of strain FA4have been regularly subcultured on an AFB1-containing mediumat least three times for 12 hours at 30 °C for a possible induc-tion of enzymes involved in AFB1 degradation. Media used forthe precultivation of the cells and used in the degradation exper-iments itself, were supplemented with AFB1 to an initial concen-tration of 2.5 ppm. The degradation experiments were per-formed on a rotary shaker at 30 °C in the dark. Samples foranalysis of the AFB1 concentration were withdrawn at 12 h, 36h and 72 h after inoculation. The remaining AFB1 concentrationwas quantitatively determined by HPLC (stationary phase:LiChroCart 250-4 Hypersil ODS [5 µm] [MERCK, Germany];mobile phase: CH3CN:CH3O:H2O [25:25:50/vol:vol :vol];detection: DAD at 365 nm).

Results and Discussion

Morphological and physiological properties

The organism FA4T was rod shaped (2 µm length,1 µm diameter), Gram-positive, acid-alcohol-fast and notmotile. The strain was nonchromogenic and grew at

654 D. Hormisch et al.

20 °C, 28 °C and 37 °C but not at 42 °C. In contrast to awell expressed catalase activity, no urease or nitrate re-duction activity was observed, and Tween 80 was not hy-drolysed (Table 1).

From the group of polycyclic aromatic hydrocarbons,only the isolation substrate fluoranthene was metabolisedby strain FA4T. No growth was detected with naphtha-lene, anthracene, phenanthrene, acenaphthene, acenaph-thylene, fluorene, chrysene, or pyrene as the single carbonsource (Table 2), and the strain could not utilise biphenyl.However, phenylacetic acid, benzoate, benzoate deriva-

tives and p-nitrophenylphosphoryl-choline were utilisedas aromatic substrates (Table 1).

Table 1 summarises the physiological characteristics ofstrain FA4T, including its utilisation of a broad spectrumof carbon sources such as sugars, sugar alcohols, aminoacids, organic acids and mono aromatic compounds.

Degradation of AFB1

AFB1 degradation by Mycobacterium strain FA4 wasrelatively rapid and effective, leaving no detectable AFB1

Mycobacterium fluoranthenivorans sp. nov. 655

Table 1. Physiological properties of M. fluoranthenivorans DSM 44556T, M. diernhoferi, M. hodleri and M. vanbaalenii.

Mycobacterium Mycobacterium Mycobacterium Mycobacterium diernhoferid fluoranthenivorans hodleri d vanbaalenii e

Formation of pigment a N N S SGram stain + + + +Acid fast stain + + + +Growth at 20 °C/28 °C/37 °C + + + +Growth at 42 °C – – – –Tween 80 hydrolysis – – – (+)c +Nitrate reduction + – – +Urease activity n.a. – +c +Catalase activity – + + +

Growth on:Xylose + + + –Trehalose – + + (–)c –Sorbitol – + + –

Utilisationb of:N-acetyl-d-glucoseamine – + + n.a.D-Glucosaminic acid – + – n.a.Gluconate + + + n.a.D-Sucrose – + – –D-Turanose – + – n.a.L-Rhamnose – + + –D-Ribose – + + n.a.D-Arabitol – + + n.a.I-Inositol + + + n.a.Mannitol + + + n.a.Citrate – + – –2-Oxoglutarate + + + n.a.Glutarate n.a. + – n.a.Succinate – + + n.a.L-Alanine + + + n.a.L-Aspartate – + + n.a.L-Leucine + + + n.a.L-Proline + + + n.a.L-Valine – + + n.a.Putrescine + + + n.a.Acetamide – + – n.a.Phenyl acetic acid – + – n.a.Benzoate – + – n.a.4-Amino-benzoate – + – n.a.4-Hydroxy-benzoate – + – n.a.Quinate – + + n.a.P-nitrophenylphosphoryl-choline + + – n.a.

a S, scotochromogenic, N, nonchromogenic.b Assimilation of auxanographic substrates was detected photometrically by means of reduction of the redox dye MTT (Kirchhofet al. 1992). + = E540(test) – E540 (control) > 0.129; – = E540 (test) – E540 (control) <0.129.c Data from Kleespies et al. [21], d Data from Willumsen et al. [41], e Data from Khan et al. [20] n.a. data not available

after 72 h. The AFB1 concentration was reduced toamounts of 70% to 80% of the initial concentrationwithin 36 hours. In further experiments, cell-free extractsof Mycobacterium FA4 have been used in the same way,both to confirm these observations and the enzymatic na-ture of the degradation activity (data not shown).

Chemotaxonomic properties

The analysis of the fatty acids revealed a pattern com-posed of straight chain saturated and unsaturated fattyacids with an even number of carbon atoms. The diag-nostic 10-methyl branched fatty acids were found in ad-

dition (Table 3). The pyrolysis esters of the mycolic acidsshowed a chain length of 22, 24 and 26 carbon atoms(Table 4). The mycolic acid pattern separated by TLCwas composed of alpha-mycolates and epoxy mycolates(Table 4). Analyses of mycolic acids by HPLC revealed acharacteristic UV-HPLC chromatogram with a two-peakcluster that emerged late and close together after 6–8minutes (Fig. 1). It seems that there is a correlation be-tween this HPLC-chromatogram and the occurrence ofalpha-mycolate and epoxy-mycolates because this two-peak cluster is always present in mycobacteria specieswhich synthesise alpha-mycolates and epoxy-mycolatesi.e. M. agri, M. chelonae, M. chitae, M. farcinogenes,


Table 2. Growth of M. fluoranthenivorans DSM 44556T, M. hodleri, M. vanbaalenii and M. frederiksbergense with biphenyl or sev-eral polycyclic aromatic hydrocarbons as single carbon sources.

Mycobacterium Mycobacterium Mycobacterium Mycobacterium fluoranthenivorans hodleri a vanbaalenii b frederiksbergense c

Biphenyl – n.a. + n.a.Naphthalene – n.a. + n.a.Anthracene – – + –Phenanthrene – – + +Acenaphthene – n.a. n.a. n.a.Acenaphthylene – n.a. n.a. n.a.Fluorene – – n.a. –Fluoranthene + + + +Chrysene – n.a. n.a. n.a.Pyrene – – + +

a Data from Kleespies et al. [21], b Data from Khan et al. [20], c Data from Willumsen et al. [41] n.a. data not available

Table 3. Composition of FAMEs derived from whole-cell hydrolysates of strain FA4T and members of the Mycobacterium neoaurumcluster.

Fatty acid (%) FA4T DSM 43524T DSM 44346T DSM 44077T DSM 44183T DSM 44074T

10:0 0.66 – – – – –12:0 0.60 – – – – –14:0 10.02 6.23 5.45 4.73 5.74 5.7215:0 – – 0.66 0.35 1.05 – 16:1 cis-7 – 2.17 6.30 0.54 1.49 3.4816:1cis-9 13.46 0.77 – 0.83 2.42 5.2316:1cis-10 4.60 5.65 3.41 3.56 6.07 –16:0 42.11 25.27 31.44 26.87 25.17 28.3116:0 10-methyl 2.17 – – – 0.76 –17:0 – – – 0.42 – –18:2 – – – 0.45 0.63 –18:1cis-9 16.80 20.57 34.83 14.55 20.27 30.4618:0 3.37 3.98 2.27 3.00 1.24 1.9618:0 10-methyl 5.09 11.15 1.36 9.33 18.46 5.5720:0 1.11 – 1.36 0.46 – –18:0 alcohol – 18.00 10.78 16.77 13.93 15.0720:0 alcohol – 4.57 1.71 16.32 2.77 1.74

Values are percentages of total fatty acids. Secondary alcohols that are released from wax esters are included. Examples of abbrevia-tions: 16:0, hexadecanoic acid (palmitic acid);18:1 cis-9, cis-9-octadecenoic acid (oleic acid); 18:0 10 methyl, 10-methyloctadecanoic acid (tuberculostearic acid); 20:0 alcohol, 2-eicosanol.

M. chitae DSM 43633, M. farcinogenes DSM 43637, M. fortuitum DSM 46621, M. peregrinum DSM 43271, M. porcinum DSM44242, M. senegalensis DSM 43656, M. smegmatis DSM 43756.

Fig. 1. Mycolic acid HPLCelution profile of FA4T

(= DSM 44556T). LIS, low-molecular-mass internal stan-dard; HIS, high-molecular-mass internal standard.

members of the M. fortuitum., M. goodie, M. pere-grinum, M. smegmatis, M. wolinskyi, M. porcinum andM. senegalense [4, 15].

Phylogenetic analysis

Determination of the 16S rRNA gene sequence and thecomparison with other sequences of members of thegenus Mycobacterium revealed that strain FA4T belongsto M. neoaurum subgroup of fast-growing mycobacteria.The closest relationship obtained with the type of a de-

scribed species was M. diernhoferi ATCC 25795T (98%sequence similarity). Strain ATCC BAA-823, proposed as“Mycobacterium hackensackense” [17] (AY266138)shows identity with the homologous sequence of strainFA4T.

Differentiation of strain FA4T from other mycobacteria

Strain FA4T could clearly be separated from the othermembers of the M. neoaurum complex by its fatty acidpattern. FA4T lacks the two secondary alcohols C18:0


Table 4. Mycolic acids and pyrolysis esters of strain FA4T and members of the M. neoaurum groupa.

Mycolic acidsa FA4T DSM 43524T DSM 44346T DSM 44077T DSM 44183T DSM 44074T

alpha-mycolates + + + + + +alpha′-mycolates (+) – – – – –Methoxy-mycolates – – – – – –Keto-mycolates – + + + + +Epoxy-mycolates + – – – – –Waxester-mycolates – + + + + +w-1-Methoxy-mycolates – – – – – –

Pyrolysis estersC22:0 + + + n.d. + +C24:0 + + – n.d. + –C26:0 + – – n.d. – –

a Data from Willumsen et al., 2001; b +, present; –, absent; (+), small amounts present; n.d., no data.M. diernhoferi DSM 43524T, M. frederiksbergense DSM 44346T, M. gadium DSM 44077T, M. hodleri DSM 44183T, M. neoaurumDSM 44074T.

and C20:0 which are always present in members of theM. neoaurum complex. This taxon includes M. diern-hoferi, M. frederiksbergense, M. gadium, M. hodleri andM. neoaurum [32, 41]. Instead of the secondary alcoholsa C26:0 mycolic acid pyrolysis ester is found in the ex-tract of FA4T. This ester is missing in the other species ofthis taxon (Table 3). FA4T synthesized alpha-mycolatesand epoxymycolates. This mycolic acid pattern is sharedby M. chitae, M. farcinogenes, M. fortuitum, M. pere-grinum, M. porcinum, M. senegalense and M. smegmatis[15] (Table 5) which are not related to FA4T by 16S rRNAgene sequences. In members of the M. neoaurum complex

waxester mycolates instead of epoxy-mycolates are foundin addition to alpha-mycolates which are present in allmycobacteria [15, 41].

In combination, the results of this investigationdemonstrate that the described strain FA4T represents anew species of the genus Mycobacterium. Compared toother PAH metabolising mycobacteria, there are pro-found differences to bacteria with a broad PAH substratespectrum such as M. frederiksbergense or M. vanbaalenii(Table 2). M. hodleri, which could also only use fluoran-thene, exhibited many differences in the physiologicaldata as shown in Table 1. In contrast to M. fluoran-thenivorans sp. nov., M. hodleri was scotochromogenic,expressed urease activity and could not utilise most of themono aromatic substrates. The strain with the highestsimilarity with respect to 16S rRNA gene sequence analy-sis, M. diernhoferi, expressed also significant differencesin the utilisation of sugars, some amino acids and themono aromatic compounds (Table 1).


Table 5. Mycolic acids of Mycobacterium sp. FA4T and other species showing alpha- and epoxy-mycolates in their mycolic acid pat-terns.

Mycolic acidsa

alpha-mycolates + + + + + + + +alpha′-mycolates (+) + (+) + (+) + (+) +methoxy-mycolates – – – – – – – –keto-mycolates – – – – – – – –epoxy-mycolates + + + + + + + +waxester-mycolates – – – – – – – –omega-1-methoxy-mycolates – – – (+) (+) (+) (+) –

a Data from Vincent Levy-Frebault & Portales, 1992; Hinrikson & Pfyffer, 1994.M. chitae DSM 43633T, M. farcinogenes DSM 43637T, M. fortuitum DSM 46621T, M. peregrinum DSM 43271T, M. porcinum DSM 44242T, M. senegalensis DSM 43656T, M. smegmatis DSM 43756T.

FA4T

DSM

436

33T

DSM

436

37T

DSM

466

21T

DSM

432

71T

DSM

442

42T

DSM

436

56T

DSM

437

56T

Fig. 3. AFB1 degradation by viable cells of Nocardia corynebac-terioides DSM 44601 (former Flavobacterium aurantiacum,DSM 12676) and Nocardia corynebacterioides DSM 20151 incomparison to Mycobacterium fluoranthenivorans DSM 44556T

(FA4T). All strains were cultivated in Standard I medium con-taining 2.5 ppm AFB1, at 30 °C under shaking conditions for12 h, 36 h and 72 h.

Fig. 2. Dendrogram of 16S rRNA gene sequence relatednessshowing the phylogenetic position of strain DSM 44556T withinthe radiation of some fast growing species of the genus My-cobacterium. Numbers at branching points refer to bootstrapvalues (500 re-samplings). Scale bar, 5 inferred nucleotide sub-stitutions per 100 nucleotides.

Phylogenetic and chemotaxonomic data strongly sup-ported the description of a new species, because the simi-larity to M. diernhoferi is only 98%. This value is signifi-cantly below 99.0%, the borderline of species differentia-tion in the genus Mycobacterium which could be shownin the recent description of new Mycobacterium species.The publication of Hong et al. [17] gives no indicationthat the proposed species “Mycobacterium hackensack-ense” will be validly described. Though similarities in themycolic acid patterns are obvious, identical 16S rRNAgene sequence similarities do not exclude affiliation todifferent species. Also, while strain FA4T was isolatedfrom a contaminated soil sample, strain ATCC BAA-823originated from a 6-year old female patient with relapsedpre-B-cell acute lymphocytic leukaemia.

Description of Mycobacterium fluoranthenivoranssp. nov.

Mycobacterium fluoranthenivorans (flu.or.an.the.ni-vo´rans, N.L. n. fluoranthene; connecting vowel, -i-; L.v.vorare; to devour; L. part. adj vorans, devouring, digest-ing; N.L. part. adj. fluoranthene, digesting fluoranthene).Gram-positive, acid-alcohol-fast, non motile rod shaped(length 2 µm, diameter 1 µm) bacterium. It is nonchro-mogenic, catalase positive and grows between 20 °C and37 °C. The strain utilises xylose, trehalose, sorbitol, N-acetyl-D-glucoseamine, D-glucosaminic acid, gluconate,D-sucrose, D-turanose, L-rhamnose, D-ribose, D-ara-bitol, I-inositol, mannitol, citrate, 2-oxoglutarate, glu-tarate, succinate, L-alanine, L-aspartate, L-leucine, L-pro-line, L-valine, putrescine, acetamide, phenyl acetic acid,benzoate, 4-amino-benzoate, 4-hydroxy-benzoate,quinate, p-nitrophenyl-phosphoryl-choline. Mineralisesfluoranthene. The fatty acid pattern from whole-cellmethanolysates is composed of tetredecanoic acid (10%),cis-9-hexadecenoic acid (14%), cis-10-hexadecenoic acid(5%), hexadecanoic acid (42%), 10-methyl-hexadecanoicacid, cis-9-octadecenoic acid (17%), octadecanoic acid(3%), 10-methyl-octadecanoic acid (5%) and eicosanoicacid (1%). TLC of mycolic acid methanolysates revealsalpha-mycolates and epoxy-mycolates. The mycolic acidUV-HPLC elution profile shows two-peak clusters thatemerge late and close together.

Isolated from PAH contaminated soils from the site ofa former coal gas plant in Saarbruecken-Burbach, Ger-many.

The type strain is FA4T (= DSM 44556T = CIP 108203).

AcknowledgementsThe support by (1) the EU Commission within the INCO-DC

project “Biological degradation of aflatoxins in fermented maizeand sorghum products” (contract no. ERBIC18 CT980315),and (2) a grant of the Saarberg AG, Saarbruecken Germany, isgratefully acknowledged. Views expressed in this paper do notnecessarily reflect those of the European Commission. The skill-ful technical assistance by Birgit Hasper, Gabriele Pötter, Jolan-tha Swiderski and Ina Kramer is gratefully acknowledged.

References

1. Boenike, R.: Die Bedeutung der Acrylamidasen fuer dieIdentifizierung und Differenzierung der verschiedenenArten der Gattung Mycobacterium. Jahresber. Borstel. 5,7–87 (1961).

2. Boldrin, B., Tiehm, A., Fritzsche, C.: Degradation ofphenanthrene, fluorene, fluoranthene, and pyrene by a My-cobacterium sp. Appl. Environ. Microbiol. 59, 1927–1930(1993).

3. Butler, W. R., Guthertz, L. S.: Mycolic acid analysis byhigh-performance liquid chromatography for identificationof Mycobacterium species. Clin. Microbiol. Rev. 14,704–726 (2001).

4. Butler, W. R., Thibert, L., Kilburn, J. O.: Identification ofMycobacterium avium complex strains and some similarspecies by high-performance liquid chromatography. J.Clin. Microbiol. 30, 2698–2704 (1992).

5. Cerniglia, C. E., Heitkamp, M. A.: Polycyclic aromatic hy-drocarbon degradation by Mycobacterium. Methods Enzy-mol. 188, 148–153 (1990).

6. Cheung, P. Y., Kinkle, B. K.: Mycobacterium diversity andpyrene mineralization in petroleum-contaminated soils.Appl. Environ. Microbiol. 67, 2222–2229 (2001).

7. Churchill, S. A., Harper, J. P., Churchill, P. F.: Isolation andcharacterization of a Mycobacterium species capable of de-grading three- and four-ring aromatic and aliphatic hydro-carbons. Appl. Environ. Microbiol 65, 549–552 (1999).

8. Felsenstein, J.: PHYLIP (phylogenetic inference package)version 3.5.1. Department of Genetics, University of Wash-ington, Seattle, WA, USA (1993).

9. Häggblom, M. M., Nohynek, L. J., Palleroni, N. J., Kron-qvist, K., Nurmiaho-Lassila, E. L., Salkinoja-Salonen, M.S., Klatte, S., Kroppenstedt, R. M.: Transfer of poly-chlorophenol-degrading Rhodococcus chlorophenolicus(Apajalahti et al. 1986) to the genus Mycobacterium as My-cobacterium chlorophenolicum comb. nov. Int. J. Syst. Bac-teriol. 44, 485–493 (1994).

10. Hamilton-Miller, J. M., Gargan, R. A.: Rapid screening forurease inhibitors. Invest. Urol. 16, 204–208 (1979).

11. Hartmans, S., de Bont, J. A.: Aerobic vinyl chloridemetabolism in Mycobacterium aurum L1. Appl. Environ.Microbiol. 58, 1220–1226 (1992).

12. Heitkamp, M. A., Cerniglia, C. E.: Mineralization of poly-cyclic aromatic hydrocarbons by a bacterium isolated fromsediment below an oil field. Appl. Environ. Microbiol. 54,1612–1614 (1988).

13. Heitkamp, M. A., Cerniglia, C. E.: Polycyclic aromatic hy-drocarbon degradation by a Mycobacterium sp. in micro-cosms containing sediment and water from a pristine ecosys-tem. Appl. Environ. Microbiol. 55, 1968–1973 (1989).

14. Heitkamp, M. A., Freeman, J. P., Miller, D. W., Cerniglia,C. E.: Pyrene degradation by a Mycobacterium sp.: identifi-cation of ring oxidation and ring fission products. Appl.Environ. Microbiol. 54, 2556–2565 (1988).

15. Hinrikson, H. P., Pfyffer, G. E.: Mini-review: mycobacterialmycolic acids. Med Microbiol Lett 3, 49–57, 97–106(1994).

16. Ho, Y., Jackson, M., Yang, Y., Mueller, J. G., Pritchard, P.H.: Characterization of fluoranthene- and pyrene-degrad-ing bacteria isolated from PAH-contaminated soils and sed-iments. J. Ind. Microbiol. Biotechnol. 24, 100–112 (2000).

17. Hong, T., Butler, W. R., Hollis, F., Floyd, M. M., Toney, S.R., Tang, Y. W., Steele, C., Leggiadro, R. J.: Characterizationof a novel rapidly growing Mycobacterium species associatedwith sepsis. J. Clin. Microbiol. 41, 5650–5653 (2003).


18. Holzapfel, W. H., Färber, P.: Final report of the INCO-DCproject “Biological degradation of aflatoxins in fermentedmaize and sorghum products.” (INCO-DC contract no.ERBIC18 CT980315) (2003).

19. Kämpfer, P., Dott, W., Kroppenstedt, R. M.: Numericalclassification and numerical identification of some nocar-dioform bacteria. J. Gen. Appl. Microbiol. 36, 309–331(1990).

20. Khan, A. A., Kim, S. J., Paine, D. D., Cerniglia, C. E.: Clas-sification of a polycyclic aromatic hydrocarbon-metaboliz-ing bacterium, Mycobacterium sp. strain PYR-1, as My-cobacterium vanbaalenii sp. nov. Int. J. Syst. Evol. Microbi-ol. 52, 1997–2002 (2002).

21. Kleespies, M., Kroppenstedt, R. M., Rainey, F. A., Webb, L.E., Stackebrandt, E.: Mycobacterium hodleri sp. nov., anew member of the fast-growing mycobacteria capable ofdegrading polycyclic aromatic hydrocarbons. Int. J. Syst.Bacteriol. 46, 683–687 (1996).

22. Kubica, G., Pool, G. L.: Studies on the catalase activity offast acid bacilli. I. An attempt to subgroup these organsismson the bases of their catalase activities at different tempera-tures and pH. Am. Rev. Respir. Dis. 81, 387–391 (1960).

23. Levy-Frebault, V. V., Portales, F.: Proposed minimal stan-dards for the genus Mycobacterium and for description ofnew slowly growing Mycobacterium species. Int. J. Syst.Bacteriol. 42, 315–323 (1992).

24. Luquin, M., Ausina, V., Lopez Calahora, F., Belda, F., Gar-cia Barcelo, M., Celma, C., Prats, G.: Evaluation of practi-cal chromatographic procedures for identification of clini-cal isolates of mycobacteria. J. Clin. Microbiol. 29,120–130 (1991).

25. Miller, J. L.: Mycobacterial identification by high perfor-mance liquid chromatography. A training manual. Newark,DE: MIDI Inc. (1997).

26. Minnikin, D. E., Hutchinson, I. G., Caldicott, A. B., Good-fellow, M.: Thin-layer chromatography of methanolysatesof mycolic acid-containing bacteria. J. Chromatogr. 188,221–233 (1980).

27. Molina, M., Araujo, R., Hodson, R. E.: Cross-induction ofpyrene and phenanthrene in a Mycobacterium sp. isolatedfrom polycyclic aromatic hydrocarbon contaminated riversediments. Can. J. Microbiol. 45, 520–529 (1999).

28. Moody, J. D., Fu, P. P., Freeman, J. P., Cerniglia, C. E.:Regio- and stereoselective metabolism of 7,12-dimethyl-benz[a]anthracene by Mycobacterium vanbaalenii PYR-1.Appl. Environ. Microbiol. 69, 3924–3931 (2003).

29. Müller, K., Schmid, E. N., Kroppenstedt, R. M.: Improvedidentification of mycobacteria by using the microbial iden-tification system in combination with additional trimethyl-sulfonium hydroxide pyrolysis. J. Clin. Microbiol. 36,2477–2480 (1998).

30. Pfennig, N.: Anreicherungskulturen fuer rote und grueneSchwefelbakterien. In: H. G. Schlegel (ed.) Zentralbl. Bak-

teriol. Parasitenkd. Infektionskr. Hyg., Abt I (Anreicherungund Mutantenauslese) 179–189 (1966).

31. Pfennig, N., Lippert, K.: Ueber das Vitamin B12-Beduerfnisphototropher Schwefelbakterien. Arch. Microbiol. 55,245–256 (1966).

32. Pitulle, C., Dorsch, M., Kazda, J., Wolters, J., Stackebrandt,E.: Phylogeny of rapidly growing members of the genusMycobacteria. Int. J. Syst. Bacteriol. 42, 337–343 (1992).

33. Rainey, F. A., Ward-Rainey, N., Kroppenstedt, R. M.,Stackebrandt, E.: The genus Nocardiopsis represents a phy-logenetically coherent taxon and a distinct actinomycetelineage: proposal of Nocardiopsaceae fam. nov. Int. J. Syst.Bacteriol. 46, 1088–1092 (1996).

34. Rehmann, K., Noll, H. P., Steinberg, C. E., Kettrup, A. A.:Pyrene degradation by Mycobacterium sp. strain KR2.Chemosphere 36, 2977–2992 (1998).

35. Sasser, M.: Identification of bacteria by gas chromatogra-phy of cellular fatty acids. MIDI Technical Note no. 101.Newark, DE: MIDI Inc. (1990).

36. Schneider, J., Grosser, R., Jayasimhulu, K., Xue, W., War-shawsky, D.: Degradation of pyrene, benz[a]anthracene,and benzo[a]pyrene by Mycobacterium sp. strain RJGII-135, isolated from a former coal gasification site. Appl. En-viron. Microbiol. 62, 13–19 (1996).

37. Schroeder, K. H., Naumann, L., Kroppenstedt, R. M.,Reischl, U.: Mycobacterium hassiacum sp. nov., a newrapidly growing thermophilic mycobacterium. Int. J. Syst.Bacteriol. 47, 86–91 (1997).

38. Silcox, V. A., Good, R. C., Floyd, M. M.: Identification ofclinically significant Mycobacterium fortuitum complexisolates. J. Clin. Microbiol. 14, 686–691 (1981).

39. Vincent Lévy-Frebault, V. V., Portaels, F.: Proposed minimalstandards for the genus Mycobacterium and for descriptionof new slowly growing Mycobacterium species. Int. J. Syst.Bacteriol. 42, 315–323 (1992).

40. Weidenbörner, M.: Encyclopedia of food mycotoxins.Springer Verlag Berlin Heidelberg (ISBN 3540675566)(2001).

41. Willumsen, P., Karlson, U., Stackebrandt, E., Kroppenstedt,R. M.: Mycobacterium frederiksbergense sp. nov., a novelpolycyclic aromatic hydrocarbon-degrading Mycobacteri-um species. Int. J. Syst. Evol. Microbiol. 51, 1715–1722(2001).

Corresponding author:W. H. Holzapfel, Federal Research Center for Nutrition and Food,Institute of Hygiene and Toxicology, Haid-und-Neu-Str. 9,76131 Karlsruhe, GermanyTel.: ++(0)721-6625-450; Fax: ++(0)721-6625-453;e-mail: [email protected]


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