Genetic and Serological Evidence for the Recognition of Four Pentachlorophenol-Degrading Bacterial Strains as a Species of the Genus Sphingomonas

System. Appl. / ... licrobiol. I H, 539-548 (1995) © Gustav Fischer Verlag· Scungan . Jena . New York

Genetic and Serological Evidence for the Recognition of Four Pentachlorophenol-Degrading Bacterial Strains as a Species of the Genus Sphingomonas

ULRICH KARLSON 1, FERNANDO RO]02, JAN DIRK VAN ELSAS3 , and EDWARD MOORE4

I ~Jtional Environmental Research Insticute, Frederiksborgvej 399, P.O. Box 358, DK-4000 Roskilde, Denmark Centro Nacional de Biotecnologia, CSIC, Universidad Autonoma, Canto Blanco, E-28049 Madrid, Spain

1 IPO-DLO, Binnenhaven 5, P.O. Box 9060, NL-6700 GW Wageningcl1, The Netherlands 4 Division of ~licrobiology, National Research Centre for Biotechnology, Mascheroder Weg I, 0-38124 Braunschweig, Germany

Received July 10, 1995

Summary

Four pentachlorophenol (PCP)-degrading bacterial strains: Arthrobacter sp. ATCC 33790, Pseudomonas sp. 5R3, Flavobacterium sp_ ATCC 39723 and Pseudomonas sp. RA2; were analyzed by 165 rRNA gene sequence comparisons, REPIERIC PCR fingerprinting of genomic DNA and serological typing by reaction to antisera prepared against each strain. The results of the analyses suggest that a close phylogenetic relationship exists between these four PCP-degrading strains, all of which were determined to cluster within the existing genus Sphi'lgomonas, and supporr the recognition of Arthrobacter sp. A TCC 33790 and Pseudomonas sp. 5R3, Flavobacterium sp. ATCC 39723 and Pseudomonas sp. RA2 as a separate Sphingomonas species. A gene probe, prepared by PCR-amplification of the PCP-4-monooxygenase gene (pcpB) of Flavobacterium sp. ATCC 39723, was used to group PCP-degrading strains on the basis of hybridization ro the gene probe: all strains of the Gram-negative, PCP-degrading Sphingomonas spp. hybridized to the gene probe, whereas Gram-positive PCP-degrading strains analyzed did not hybridize to the gene probe.

Key words: Pentachlorophenol-degrading bacteria - Sphingomonas - 16S rRNA gene sequence - REP/ ERIC DNA fingerprinting - Serological typing - pcpB gene hybridization - Taxonomy - Phylogeny -Arthrobacter - Flavobacterium - Pseudomonas

Introduction

Pentachlorophenol (PCP) has been used worldwide as a fungicidal wood preservative and has caused serious con­tamination of soil and water due to its persistence in the environment (Crosb..,., 1981). Nevertheless, PCP has been shown to be biodeg~adable and several PCP-mineralizing microorganisms have been isolated. These include Artl"o­bacter sp. strain ATCC 33790, isolated from wood preser­vative-contaminated soil (Edgehill and Finn, 1982), Flavo­bacterium sp. strain ATCC 39723, isolated from sediment in PCP-contaminated artificial channels (Pignatel/o et aI., 1983; Saber and Crawford, 1985), PseudomOllas sp. strain SR3, isolated from contaminated soil at a wood­preserving facility (Resnick and Chapman, 1994), Pseudomonas sp. strain RA2 (DSM 8671), isolated from soil contaminated with wood preservative waste (Rade-

haus and Schmidt, 1992), Mycobacterium chloropheno/­icum PCPl (DSM 43826), isolated from lake sediment (Apa;alahti and Salkino;a-Salonen, 1986; Briglia et aI., 1994; Haggblom et aI., 1994), and Mycobacterium for­tuitum CG2, isolated from saw mill soil (Haggblom et aI., 1988; Nohynek et aI., 1993).

The initial steps in the metabolism of PCP are similar in all of the PCP-degrading strains used in this study (Table 1), although differences exist among some of them regard­ing the dehalogenation mechanism of the enzymes in­volved (Orser and Lange, 1994; Uotila et aI., 1991, 1992). The same dehalogenase observed in the Gram-negative Flavobacterium sp. ATCC 39723 was reported also to be present in the Gram-positive Arthrobacter sp. ATCC 33790 (Orser et aI., 1993), suggesting that the PCP de-

540 U.K.Hlslln, 1'. Rojo, J. D. \".111 Ek1S, and E. \Ioorc

halogenase gene, pcpB, evolved early and was conserved over long periods of bacterial evolution. Since this was of fundamental interest for understanding the ecophysiology of biodegradation, we attempted to gain insight into the ecologicll and genetic features of PCP degradation through a detailed characterization and estimation of the phylogenetic relationships of several of these bacterial strains using 16S rDNA sequence analysis, REP and ERIC­peR typing and by serological assay. The results of these analyses suggest the recognition of the strains Arthro/Jac­fer sp. ATCC .B790, Pseudomollas sp. SR3, Fld/lobac­ferilll11 sp. ATCC: 39723 ;lI1d Pseudomollas sp. RA2 as strains of a separate SphillgOlllc>IIl.1s species. In light of the pending recLlssification of these four PCP-degrading strain~, it was important also to reevaluate the similarity of the pcpB genes with respect to the phylogentic relation­ships of the bacterial strains.

Materials and Methods

B.lctcrial straills ,md ntlth',ltion. The hacterial strain, used in this srudy, and their ori~ins, arc listed in Tahle I. All straim were maintained and cultivated on the media recommended in the rcspectiVl" rderences. Mineraliz,uion of PCP was confirmed for • 111 PCP-degraders by incubatin~ ~ure cultures in minimal med,um amended w,th !l mg l.' PCI' ta~ged w,th unl\'ersally labelled "C-PCI' Jlld collecting 14C02 in 0.4 IllL of I l\i[ KOH. 14e W.1S measured in a liquid scintillation counter (BeckmJll)

T'lble 1. Strains used ill this srudy

after addition of 1.5 mL of ReadyGcI (Becknun; ,lnd 1 mL of H20 to the KOH trap. Non-chlorophenol-degrading straim were (ultured in LB medium (S'I/l/brook et aI., 1989).

Detcrmination ,wd ,1II,1Iysis 0/ If .. S rRNA gene sequellces. C;enomic DNA was prepared from approximately 0.1 g (wet wt.) cells after the method of Wilson i 1987). The 16S rRNA genes were amplified bv peR (AII/llis and Fallooll<l, 1987; Saiki et aI., 1988) using a Perkin-Elmer Cetlls GencAmp peR System 9600. The PCR reaction conditions and primers were the ,amI' as de­scribed in detail previously (Karlsoll, et aI., 1993). PCR products were purified using Cemricon-l on micrlKonccmrarors (Amicon GmbH, Witren, Germany) and the .lInplified 16S rDNA was se­quenced directly using an Applied Biosyqems, Inc. ]7~A DNA Sequencer and the protocols of the manufacturer (Perkin-Elmer, Applied Biosystems GmbH, Weiterstadt, Germany) for "'[:1'1 cy­cle-sequencing" with tluorescent dye·labdled dideoxynucleo­tides. The se<.juencing primers used have been described previous­ly (Lilli', 1991).

Sequence data were aligned with known 16S rRNA (.1I1d rRNA gene) sequences (De Rijk et aI., 1994; Olsell et aI., 1991), using conserved regions and secondary stnKtural characteristics .15 reference (Woese et aI., 198]; Glaefl et al.. 1985). Sequence similarity values and evolutionary distances, incorporating a cor­rection factor UI/kes and Call tor, 1969) for reverse mutations, were calculated for masked (Lme, 1991) and unmasked se­quence-pair comparisons using unambiguous nucleotide posi­tions. Dendrograms were produced using a weighted. pairwise, least-squares distance method (Olsell. 19!17) .

Strain typing by REP/ERIC PCR. Colonies of Arthro/Jacter sp. ATCC 33790, PseUdOm01lt1S sp. SR3, FLwobactaillH/ sp. ATCC 39723, PSCl/dOIl/OfWS sp. RA2, .1I1d Bllrkholdcria cepacia

Strain Obtained from Reference

Pentachlorophenol-degraders:

Arthrobacter sp., ATCC 33790 F/,lI'obacterilllll sp., ATCC 39723

M)'cobacterilllll fortllitll11l, CG2 Alycohacterillm ch/orophenoliclIH/, CG I MY<,(l/hlcterilllll chiorophenoliollll, CP2 Mycobacterilllll ch/oro(JhI'1I0IiwlII. PCI'I

(= DSM 43826)' PS<'lIdcJIIIOII,lS sp., SR3 PSl'IIdol1l0llilS sp., RA2 (= DS1\1 8671)

T rich lorophenol-dcgrader:

Illlrkl}()ldcri,1 (Pselldomonas) c<'pacia, ACt 100

Non-chlorophenol-degraders:

Brt'I'lIIldil1lollas dill/illl/fa, LMG 2089·' I's,."ut/OI1l01l<lS t7l1orcsce1ls. R2f Sphill?,Oll/()1/,IS sp., HH69 (= D5M 7135) Sphingo/llO/Ws sp., RW1 (= DSM 6014) Sphill?,O/l/OII'IS sp., S53 (= DSM 6432) Sphingollloll,ls ,ldlhlt'sit'd, IFO 15099T

SpiJ;lIgolllonds ,'apslIl,lta, DS!vl 30196' SpiJillgolllonds paw:imohi/is, DS1\1 1098T

ATCC R.Crawford

M. Salkinoja-Saloncn M. Salkinoja-Salonen 1\1. Salkinoja-5alonen M. Salkinoja-Salonen

P. Chapman DSM

A. M. Chakrabarty

LMG (own collection) R.-M. Wittich R.-M. Wittich R.-M. Wittich <- S. Schmidt H.-J. Busse <- IFO DSM DSM

Edgehill and Finn, 1982 Pigllatello et aI., 1983; Saber and

Crawford, 1985; BrowlI et aI., 1986 ll.1ggblom et aI., 19&8 Hdggblom et aI., 1988 Hdggb/ol1l et aI., 1988 Ap,ljalahti and Sdlki1lo;a-S<l10f1l'n. 1986

Resllick and Ch,lpmml, 1994 Radehalls and Schmidt, 1992

Kilba1lt' et aI., 1982

Lei/s(lI! and Hugh, 1955 Vall Elsas et aI., 1988 Harms et aI., 1991 Wittich et aI., 1992 Schmidt et aI., 1992 Yabuuchi et aI., 1990 YabllUchi et aI., 1990 Yablwchi et aI., 1990

Abbreviations: ATCC, American Type Culture Collection, Rockville, \<Iaryland; D51\1, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany; IFO, Institute for Fermentation, Osaka, Japan; LMG, Laboratorium voor Microbiologie, Rijhuniversitcit Gem. Belgium; T, t"pestrain for the species

ACII0() were t,\ken dirc.:!1y from agar plales and resllspended in 400 ~ll of Ivsi~ buii .. r 150 m1\1 Tris-HCI pH 8.0, 50 ml\l EDTA, 5 mg mL- 1 Iysoz\·mc. 100 units mVI mutanolysin, 3 mg mL- 1

fairY acid-free bovine ~erum albumin). Ailer 60 min incubation at 37 'c. the slIspensions were trt~ated -' times for 5 sec, with reposi­tioning in between. in a microwave oven (Moulinex, model FM430) at 1300 \1(,'. Cdl fragments were removed by centrifuga­tion at 15000 x g for 15 min. Genomic DNA was recovered by centrifugation at ISOOO x g for 20 min after precipitation by the addition of l\:aCI to a fiml concentration of 100 mM and 2.5 I'olumes of 96% EtOH.

REP (repetitive extragenic palindromic) and ERIC (entero· bacterial repetititive intragenic consensus) peR fingerprinting methods were c.uried out using AmpliTaq (perkin Elmer/Cetus) DNA polymerase and the primers and conditions as described by Versa/ovic et a!. (1994). Positive controls included F.scherichia mli or Pseudomollas (IllOrescells strain R2f cells as targets. whereas negative controls contained water instead of targeted DNA. The type strain of Sphillgommws p'lIIcimobilis (DSM 10981

) was included in the analysis as an our-group. The PCR products were separated by electrophoresis in 1.5% agarose gels and sized by comparison to a l-kb molecular size marker. Eucli­dean distances between the four strains, based on the presence (scored as + 1.0) or absence (scored as 0.0) of any of the bands were calculated using STATISnCNMac (StatSoft, Tulsa, Ok­lahoma). Weak bands were entered as +0.5.

Serological typing. Polyclonal antibodies were taised in rabbits (Harboe and Ingild, 1983) by intramuscular injection of heat­killed whole cells (Arthrobacter sp. ATCC 33790), or purified (Chart and ROIl·e. 1992) cell membrane lipids (Flavobacterium sp. A TCC 39723. Pselldomonas sp. SR3, and Pseudomollas sp. RA2l. Crude sera were used as obtained for Arthrobacter sp. ATCC .>3790, PselldomOl/as sp. SR3, and Pseudomonas sp. RA2. Gamma-globulins against Fiat'obacteriu11/ sp. A TCC 39723 wert: isolated and concentrated after the methods of Har­boe and ingild (1983). Dot Elisa was performed, modified after Arredolldo and Jerez (1989) and Hawkes et a!. (1982), using rabbit antisera as primary antibody, and alkaline phosphatase­conjugated antirabhit swine immunoglobulins (DAKO, Glosrrup, Denmark) as secondary antibodies. The specificity of the im­munoassay was optimized by decreasing the concentration of primary antiserum in the reaction buffer. Alkaline phosphatase activity was assayed hy suspending the nitrocellulose filters in a fresh mixture of 15 mL of alkaline phosphate buffer 02.1 g Tris, 5.84g l\:aCI. IO.2g MgCll . 6H20 per L, pH 9.5), 66 ilL of NBT­solution (75 mg nitroblue terrazolium, 700 ~IL dimethyl form­amide, ,00 ~Il H20) and 50 III of BClP solution (50 mg 5-brolllo-4-chloro·3-indolylphosphate. 1 ml dimethylformamide). The reaction was stopped using Tris-EDTA-buffer (2.42 g Tris, 1.86 g EDT A per 1000 ml, pH 7.5). Intensity of dot blue-colora­tion was assessed by visual examination and scored qualitatively.

SOllthern blot hybridizatioll for pcpB gene. DNA frOIll Arthro­bacter sp. ATCC 33790, Pseudomonas sp. SR3, Flavobacterium sp. ATCC 39723. Burkholderia cepacia ACll 00, and Pseudo­mOllas sp. RA2 was prepared as described above for peR-finger­printing. DNA from the Gram-positive PCP·degrading strains, PCPl, CP2, CGl, and CG2, was extracted directlv from colonies as described above, except that after the microwa~e treatment 0.8 m1. of acid-washed glass beads (100-150 11m diameter) were added to the suspension, along with 50 \-ll of 20% 5DS and 700 ~Il of phenol. The mixture, in ice-cold condition, was treated with a Mini-Beadbeater (Biospec Products, Bartlesville, Oklaho­ma) for 45 sec. After sedimentation of the glass beads, 400 ~Il of of supernatant were brought to 100 mM NaCl and mixed with 400 \-ll of lysis buffer-saturated chloroform through vigorous shaking. After brief centrifugation, the water phase was removed

Reco!:llItion of 4 PCI'·lk!:r,1ding a~ SphillgOIllOlhlS spp. 541

and extracted twil'e with 200 !ll of chloroform:isoamyblcohol (24: I. v/l'). The DNA was precipitated and washed as above.

An 832-hp hybridization probe for the F/,ll'obacteriuIII sp. ATCC 39723 PCP dehalogenase gene (pcpB) was obtained by PCR amplification of chromosomal DNA, targeting the region hetween nucleotides 37 to 869 of the pcpB gene (Orser et aI., 1993), covering most of the S' half of the pcpB gene. The am­plified DNA was purified and labelled with digoxigenin as indi­cated by rhe supplier (Boehringer, Mannheim, Germany).

The probe was lIsed for Southern blot hybridization (Southall, 1975) of total DNA (chromosomal DNA plus plasmids) of each of the hacterial strains analyzed. The DNA (1 Ilg) was digested with endonuclease FcoRI, and the fragments generated were separated in ,1 0.8% agarose gel along side adequate size st.md­:lrds. The DNA was subsequently denatured and transferred to a nylon membrane as instructed by the supplier (GeneScreen, NEt-:). Hybridization with the DNA probe was performed essen­tiall\' a, described bv Sambrook et 31. (1989). Hybridization was carr'ied out for 16 hours at 42°C, with subseq~ent washing of membranes under stringent conditions (2 xiS min at 68°C, in 0.1 x SSe, 0.1 % 5DS).

Results and Discussion

PCR-amplification of the 165 rRNA genes between nu­cleotide positions 28 and 1488 (E. coli numbering) of the four PCP-degrading strains allowed the determination of 1441 nucleotide positions, which comprises approximate­ly 94% of the complete gene sequence (estimated by com­parison with the 16S rRNA gene sequence of E. coli).

Masked sequence comparisons (1349 nucleotide posi­tions) of the 165 rDNA indicated that a close phylogenetic relationship exists among the four PCP-degraders, all of which clustered within the rRNA group IV (Pal/erolli et aI., 1973) or alpha subgroup (Woese et aI., 1984) of the Proteobacteria (Stackebrandt et aI., 1988), most similar to

Sphingomonas yalloikuyaeT (Figure 1). In unmasked sequence comparisons between Arthro­

bacter sp. ATCC 33790 and Pseudomm1L1s sp. 5R3, their 165 rRNA gene sequences were 100% identical. This was also the case with the comparison between Flavobacterium sp. ATCC 39723 and Pseudomonas sp. RA2_ The se­quence similarity between the two pairs: Arthrobacter sp. ATCC 33790lPseudomonas sp. 5R3; and Flavobacterium sp. ATCC 39723lPseudomonas sp. RA2; was 97.7%. This suggests that Arthrobacter sp. A TCC 33790 and Pseudo­monas sp. SR3 belong to one species and Flavobacterium sp. ATCC 39723 and Pseudomonas sp. RA2 possibly be­long to another. However, it should be emphasized that identical 165 rONA sequences are nor conclusive proof that two strains belong to the same species, as the resolu­tion of the 16S rRNA genes may be insufficient to definite­ly distinguish closely related species (Ash et aI., 1991; Fox et a!., 1992; Martinez-Murcia et aI., 1992). The only mor­phological differences observed between Flavobacterium sp. ATCC 39723 and Pseudomonas sp. RA2 was that strain RA2 was motile by microscopic observation, while strain ATCC 39723 was non-motile. Arthrohacter sp. ATCC 33790 and Pseudomonas sp. SR3 were distinguish­able by colony color, in addition to motility (strain SR3 being motile).

542 U. Karlson, F. Rojo, J. D. van Elsas, and E. Moore

0.02 0.04 0.06

S. paucifTlObj/ls-T

S. parapaucimobJlit>-T

S. adhusiv&-T

ATCC33790

SR3

ATCC39723

S. ya"",kuyae-T

SS3

01

S. macrogoltabi<lus-T

S. tefT/Je-T

S. cap$Ullit. T

PtJrphy_er fl6USlonensls-T

Erythrobacter longus

Agrobactenum turoofaci6ns

Bnwundimonas dim/nuts-T

Burld'!okJeria cepacia-T Escherichia coli

Pseudomonas 8efIJginosa

Fig. I. Unrooted dendrogram derived from a least-squares analy­sis of evolutionary distance determinations of pairwise compari­sons of 165 rRNA gene sequences from the PCP-degrading strains Arthrobacter sp. ATCC 33790, Pseudomonas sp. 5R3, Flavobacterium sp. ATCC 39723 and Pseudomonas sp. RA2 with Sphingomonas spp. reference strains and other Sphingo­mondS spp. able to degrade xenobiotic compounds. Accession numbers for the EMBL nucleotide sequence database: A TCC 3]790 = X87161; 5R3 = X87162; ATCC 39723 = X87163; RA2 = X87164; S. paueimobilis = X72722; F. deuormlS from Olsen et aI., 1991; S. Pdrapaueimobilis = X72721; S. sanguis = D13726; S. adhaesiva = X72720; RW1 = X72723; HH69 = X87166; R. suberifdeiens = D13737; C7 = L22759; S. Yd'lOikuYde = X72725; 553 = X87165; Q1 = X87167; S. »!d­crogoltabidlls = DI3723; S. terrae = D13727; S. capsulata = M59296; P. neustonensis = LOI785; E. longus = D12699; A. tumefaciens = M 11223; B. diminuta = M59064; B. cepacia = M22518; E. coli = J01695; P. aeruginoasa = M34133.

Phenotypic and chemotaxonomic analyses (Nohynek, et aI., 1995) demonstrated few characteristics differentiating Arthrobacter sp. ATCC 33790 and Pseudomollas sp. SR3 from Flavohacterium sp. ATCC 39723 and Pseudomonas sp. RA2. However, the 16S rONA sequence difference of approximately 2% noted between the two pairs may be too large [0 justify placing all four strains within one species. Although no officially-recognized standard exists relating 16S rRNA gene sequence similarity to taxonomic hierarchy, Stackebrandt and Goebel (1994) have noted that it is unlikely that bacterial strains possessing 16S rRNA gene sequence similarities below approximately 97.5% will have more than 60 to 70% DNA similarity. This is a conservative approximation providing confidence that strains with sequence differences> 2.5% are not re-

lated at the species level. However, experience (unpub­lished data) has shown that the 16S rRNA gene sequences of strains of the same species are unlikely to differ by more than 1 % (approximately 15 nucleotide differences). How­ever, in the absence of DNA-DNA hybridization data, and considering the low degree of phenotypic differentiation between the four PCP-degrading strains (Nohynek et aI., 1995), the issue whether one or two (or perhaps more) different species exist among the four PCP-degrading strains, becomes moot. In any case, the sequence data ag­reed with the chemotaxonomic data (Noh),nek et aI., 1995) in that the four PCP-degrading strains cluster within the existing species of the genus Sphillgomonas (Yabuuchi et aI., 1990). The sequence similarity values determined between Arthrobacter sp. A TCC 33790 and Flavobac­terium sp. ATCC 39723 and reference strains of Sphing­omonas spp. are presented in Table 2. Sequence similarities between the four PCP-degraders and S. yanoikuyaeT range from 95.8-96.8%, demonstrating that these isolates are related to, but clearly a species separate from, S. yanoikuyaeT . However, the PCP-degrading strains possessed 16S rDNA sequence similarities with other species of Sphingomonas as low as 90.1 % (i. e., with S. paucimobilis, the type species of Sphingomonas). Such a range of sequence similarities is relatively broad for species within one genus and supports the previous suggestion (van Bruggen, et aI., 1993) that Sphingomonas, as it cur­rently exists, should be reorganized into more than one genus. It is noted that S. )'anoiku)'aeT and related strains, including the four PCP-degrading strains, cluster in the vicinity of Rhizomonas suberifaciens (Fig. 1) and may eventually be reclassified as species of Rhizomollas. How­ever, until the reorganization of Sphingomonas is formally adopted, and due to their close phylogenetic affiliation to what is currently recognized as S. yanoikuyae T, Arthro­bacter sp. ATCC 33790, Pseudomonas sp. SR3, flavo­bacterium sp. ATCC 39723 and Pseudomonas sp. RA2 should be recognized as a separate species of Sphingo­monas.

Genome fingerprinting methods such as REP and ERIC­PCR typing protocols provide the means to detect geneti­cally different organisms at the species and strain levels (Versalovic et aI., 1991). Whereas the 16S rRl\;A gene sequence analysis is derived from a relatively small, highly conserved portion of the genome, REP and ERIC PCR fingerprint analyses target the entire genome and elucidate genotypic differences between strains, hence supplement, to a higher resolution, the information obtained by 16S rONA sequencing. The results from the REP and ERIC­PCR analyses are visualized in the dendogram presented in Figure 2. A total of 39 bands ranging from approximately 0.2 to 5.6 kb were obtained with the twO methods. A Euclidean distance of 3.24 between Arthrobaeter sp. ATCC 33790 and Pseudomonas sp. SR3, and a smaller distance of 1.73 between Flavobacterium sp. ATCC 39723 and Pseudomonas sp. RA2, was determined. The Euclidean distance between the two pairs and S. pauci­mobilis T was determined to be 4.58. These results confirm the close taxonomic relationships between the four PCP­degrading strains, as well as the sub-clustering into two

Table 2. Unma,ked 165 rDNA sequence similarity value, between Arthrobdcter sp. ATCC .B790 and FI<IU(>iJdcterilll11 sp. ATCC 3972.' with Sphingollloll<ls spp. and other reference strains

o

RA2 ----------

ATee 39723

Alee 33790

SR3---

S. paucimob llis T

Recognition of 4 PCP-Degrading as Sphingomollas spp. 543

Organism

Arthrobacter sp. PSeUdOlllOlIilS sp. Fiauobacteriu/1I sp. Pst'lIdo1ll011<IS sp. Sphillgomonas adhaesiua Sphillgo/llollas capsulata Sphingo/llonas macrogo/tabidlls Sphing()/llonas par<lp<llIcimo/Jilis Sphingomoll<ls palleimobilis Sphillgomonas sangllis SphillgomOll<ls terrde Sphillgolllollas ydl/Oikllyae Rhizomollas slIberi(aeiells Sp/Jillgolllon<ls sp. Sphillgomollas sp. Sphingomonas sp. Sphillgomonas sp. Breulllldimonas diminllta AgrobaeteriunI tUnlet'leiens Burkholderia cepacia Escherichia coli

Strain

ATCC 33790 SR3 ATCC 39723 RA2 1CM 7370T

ATCC 14666T

IFO 150B' 1C1\·1 75 lOT DS1\! 10981

IFO 13937T

IFO 150981

1CM 7370T

IFO 15211 T

C7 HH69 RWI S53 ATCC 11568T

ATCC 4720 ATCC 25416T

Sequence Similarities with

ATCC 33790

100.0 97.7 97.6 92.4 91.0 92.2 91.5 90.1 92.1 91.2 96_8 93.8 95.0 94.4 93.3 97.0 86.5 86.5 76.5 77.5

ATCC 39723

97.7 97.7

100.0 92.5 91.3 91.6 91.1 90.3 91.9 90.4 95.8 92.5 94.4 94.4 92.9 96.0 86.3 86.3 76.6 78.0

Sequence data for: S. adhaesiua, S. parapaueimobilis, S. pat/cimobilis, S. yalloikll)'ae and Sphillgomollas sp. RWI were taken from Aloore et aI., 1993; S. capsulata, B. diminllta, A. tllllle(aciells, B. apacia and E. coli from OlsclI et aI., 1991; S. macrogoltabidlls, S. sanguis and S. terrae from Takeuchi et aI., 1993; R. slIberifaciens from Va/I Bruggen et aI., 1993; Sphillgomonas sp. C7 from Godlldasu'ami et aI., 1993; Sphingomonas sp. HH69 and Sphillgomollils S53 from manuscripts in preparation Abbreviations: see Table 1

Euclidean Distance

2 3 4 5

l-

I i i I

I I I

Fig. 2. Euclidean distances between srrains Fiauobacterium sp. ATCC 39723, Arthrob,uter 'p. ATCC 33790, Pseudomonas sp. RA2 and Pseudomon<ls sp. 5R3 based on REP and ERIc-peR (Tahle 2).

closely ~elated pairs, noted from 16S rRNA gene sequence compansons.

It was not possible to distinguish between Arthrobacter sp. A Tee 33790, Flavobacterium sp. A Tee 39723,

Pseudomonas sp_ RA2, and Pseudomonas sp. SR3 using the polyclonal antihodies generated in this study, as the four antisera cross-reacted with all four strains (Tahle 3). Furthermore, strains HH69 and SS3, which, based on 16S

544 U. Karlson, F. Rojo, J. D. van Elsas, and E. !\loore

Strain (T = typesrrain for the species) Antibody raised against Table 3. Serological diffcrcntiation be­tween stfains lIsed in this ~[Udy

Fl.wobacterilllll sp., ATCC 39723 Arthrobacter sp., ATCC 33790 Pselldomonas sp., SR3 Pselldon!OIwS sp., RA2, D5t .. l 8671 SphillgOIllOlIllS sp., 553 Sphingolllontls sp., 1-11-169 Sphillgomonas 'p., RW1 Sphingolllonas ,/(ihaesil.'a, IFO 15099T

Sphingom(mas pal/cilllobilis, DSM 109ifi

Sphingomollas fapsl/lat". D5M 30196T

Bret'lmdill101l<ls dil1li1llIt", LMG 2089'1 ,I"lycobacterilllll chloropheno/icu/ll PCP-J,

DSM 43826 1

Escherichia coli

RA2 ATCC 3972]

+ + + + + + + + + + + +

5R3 ATCC 33790

+ + + + + + + + + + + +

(+)

Scores: + = clear response, - = no response, (+) = weak response

rONA sequence comparisons (Fig. 1), are phylogenetically dose to the four PCP-degraders, also reacted with the four antibodies, and strain RWI reacted weakly with one anti­body. Beginning with S. adhaesivaT , and 'with increasing evolutionary distance from the PCP-degrading strains (based on 16S rONA sequence comparison), no cross­reactions were observed. Strain PCPI and E. coli tested negative with all four antisera.

The genus Sphingomonas is characterized by the ab­sence of O-antigens (Iipopolysaccharides) and, instead, by the presence of glycosphingolipids, whose glycosyl por­tions consist of a tetrasaccharide (Kawahara et aI., 1991). The antigenic properties of the cell surface should be minor with such a short glycosyl chain length. Indeed, the antibody titer of the sera from rabbits that had been im­munized with a purified lipid fraction dose recommended by Chart and Rowe (1992), was so low that the dose had to be raised 25-fold to receive satisfactory titers. With only four saccharides to be varied, the immunological specifici­ty of the glycosyl chains should be low. This could explain the relatively low specificity of the antisera. Nevertheless, the results of the immunoassav confirm the close relation­ship of the Arthrobacter sp. ATCC 33790, Pseudomonas sp. SR3, Flavobacterium sp. ATCC 39723 and Pseudo­monas sp. RA2 with each other and with other xenobiotic­degrading Sphillgomonas species, suggested by the 16S rONA sequence comparisons.

The environmental source of Arthrobacter sp. ATCC 33790 was reported as wood preservative-contaminated soil from the base of a treated pole. The strain was isolated by chemostat enrichment using PCP as the sole source of carbon (Edgehill and Fiml, 1982) and has been shown to degrade PCP after inoculation into soil and sand (Edgehill and Finn, 198.1; Edgehill, 1994). This strain was originally characterized as an Arthrobacter sp. based on cell mor­phology, physiology and cultivation characteristics, and the presence of diaminopimelic acid (DAPA) (Edgehil/ and Fi1lll, 1982). However, none of the criteria originally used

to identify Arthrobacter sp. A TCC 33790, including the presence of DAPA, is definitive for the identification of Arthrobacter spp. In light of current bacterial taxonomy, the original characterization of Arthrobacter sp. A TCe 33790 is consistent with the description of Sphillgomollas (Yabuuchi et aI., 1990).

Pseudomonas sp. SR3 was isolated from PCP-contami­nated soil in Pensacola, Florida (Resllick and Chapmall, 1994). It was reported to grow with PCP as a sole source of carbon at concentrations as high as 175 mg L -I, with maximum growth rates at PCP concentrations of 100 mg L -1. The strain has been used as a bioremediation tool in contained bioreactors, where it removed PCP from creo­sote- and PCP-contaminated groundwater (Mueller et aI., 1993). Originally identified as a Pseudomonas sp., the strain has been further characterized as Pseudomonas (Brevundimonas) vesicularis (personal communication, S. M. Resnick, Univ. Iowa). It was observed to produce opa­que, off-white colonies on LB media. While Sphingomonas spp. typically produce bright yellow colonies when grown on complex agar medium, strains of S. yalloiku)'ae 1 are known to produce distinctive white colonies. Carbon source utilization by strain SR3 correlated closely with that of Brevundimonas vesicularis (like Sphingomonas spp., a rRNA group IV species), although, fatty acid methyl ester (FAME) analyses from two separate studies (Resnick and Chapman, 1994; Nohynek et aI., 1995) pro­duced profiles more typical of species of the rRNA group III.

Flavobacterium sp. A TCC 39723 was isolated from PCP-contaminated sediments of man-made channels in Minnesota (Pignatel/o et aI., 1983), and has been shown to utilize PCP at high concentrations (100-200 mg L -1) as a sole source of carbon. The pathways of PCP degradation in this strain have been well elucidated, key enzymes have been isolated and characterized, and their genetic regula­tion has been clarified to a large extent (Orser and Lange, 1994). The characterization of this strain as a Flavobac-

terillnl sp. was reported separately (Saber and Cr<lw(ord, 1985; Browl1 et aI., 1986) and was based primarily on morphological characteristics, i. e., Gram-negative, non­motile cells, exhibiting a nondiffusible yellow pigment. Other strains, e. g., S. cLzpsulata and" Flat'obacterillm" de­VorallS, also were classified initially as Flavobacterium by essentially the same criteria, but were later recognized to

belong to the genus Sphingomonas, based on chemotax­anomic and 16S rRNA gene sequence characterization (Yabu/lchi et aI., 1990). The original Flavobacterium sp. strain 39723 deposited with the ATCC lost its capacity to degrade PCP during reculturing and was replaced by a redeposit of the same strain under the designation ATCC 53874 (official communication, ATCC). Aside from the capability to degrade PCP, the two strains should be iden­tical. The strain used in this study was obrained directly from the depositor (Table 1).

Pseudomonas sp. RA2 was isolated from PCP-contami­nated soil at the Broderick Wood Products site near Den­ver, Colorado (Radehaus and Schmidt, 1992) and was reported to grow on PCP, as a sole source of carbon, at concentrations as high as 200 mg L -I. It was originally identified as a Pseudomonas sp. based on carbon source utilization and fatty acid profile characterization. interest­ingly, Radehaus and Schmidt (1992) recognized certain similarities between their strain RA2 and Flavobacterium sp. ATCC 39723, although they were able to easily distin­guish strain RA2 by a polar flagellum and the ability to store polY-I:~-hydroxybutyrate. More recently, strain RA2 has been recognized as a Sphingomonas sp. (personal com­munication, S. K. Schmidt, Univ. Colorado).

Sphingomonas spp. are readily isolated from soil, water and sediment, including deep subsurface sediment samples (Fredrickson et aI., 1995). Sphingomonas spp. are being recognized to be capable of metabolizing a wide range of xenobiotic compounds (Fredrickson et aI., 1995, Moore et aI., 1993; Takeuchi et aI., 1993), and the list of xenobio­tics which are known to be degraded by Sphillgomollas spp. is expanding. In the present study, the reclassification of the four PCP-degrading strains is presented within the context of the validly-described species of the genus Sphin­gomonas (Yabuuchi et aI., 1990; Takeuchi et aI., 1993), and also in comparison with different xenobiotic-degrad­ing strains determined to belong also to Sphingomonas (Figure 1). These strains include: C7, capable of the aerobic degradation of azo dyes (Govindaswami et aI., 1993); RW1, able to mineralize dibenzo-p-dioxin, diben­zofuran, biphenyl, and various substituted mono-aromatic compounds (Wittich et aI., 1992); HH69, able to utilize dibenzofuran, biphenyl and substituted mono-aromatics (Harms et aI., 1991); S53, able to degrade diphenylether, phenol, catechol and their mono-chlorinated derivatives (Schmidt et aI., 1991); and Q1, able to grow using bi­phenyl and substituted mono-aromatic compounds (Furu­kawa et aI., 1983).

Since the results obtained thus far suggested that the four PCP-degrading strains analyzed are genetically very similar, the similarity between the genes coding for chlorophenol degradation enzymes of these and several other chlorophenol-degrading strains was investigated. in-

Recogniuon oi 4 PCl'-Degr.lJing as Sphillgolllonas spp. 545

deed, it has been reported that the gene coding for the PCP-dehalogenase of Flavobacterium sp. ATCC 39723, which has been cloned and sequenced, may also be present in Arthrobacter sp. A TCC 33790 and Pseudomonas sp. SR3, bur not in strain PCP1 (Orser et aI., 1993). We have extended this analysis to all the strains analyzed in this work. On the basis of the results from Southern blot hy­bridizations using a probe prepared from the PCP-de­halogenase gene of Flavobacterium sp. ATCC 39723 (Fi­gure 3), the strains may be clustered into two groups. One group includes Arthrobacter sp. A TCC 33790, Pseudo­monas sp. SR3, Flavobacterium sp. ATCC 39723 and Pseudomonas sp. RA2. These strains probably contain the same genes for PCP degradation, since the probe strongly hybridized in all four cases with a DNA fragment of simi­lar or identical size (approximately 3.0 Kbp, Fig. 3). The second group of strains includes the Mycobacterium spp. strains (PCPl, CG1, CG2 and CP2) containing a PCP de­halogenase gene (or genes) which did not hybridize with the probe generated from the dehalogenase gene of strain Flavobacterium sp. ATCC 39723 (Figure 3). Burkholderia cepacia AC1100 was reponed to consume small quantities of O2 upon exposure to PCP (Kilbane et aI., 1982), and to harbour PCP-degrading enzymes that are biochemically similar to those of Flavobacterium sp. ATCC 39723 (To­masi et aI., 1995). Our hybridization results suggest that the genes coding for these enzymes may be different, or at least have a sequence divergence high enough to give a negative result in the hybridization assay, which was per­formed under stringent conditions. Thus, our findings ag­ree with earlier reports (Orser and Lange, 1994), and ex­tend the number of bacterial strains assayed to cover all chlorophenol-degraders available to us.

Orser and Lange (1994) demonstrated that the PCP de­halogenase of Flavobacterium sp. A TCC 39723 bears structural similarities to a number of other bacterial mono-oxygenases, oxidizing different xenobiotics, and suggested that the pcpB gene of this strain may be a useful character for analysis of genetic similarities among aerobic dechlorinating microbes. Similarities were found in a vari­ety of very different microorganisms, ranging from Gram­negatives to Gram-positives, including the actinomycetes. Such observations may help to elucidate the evolution of degradative pathways for xenobiotics. As long as the strain Arthrobacter sp. ATCC 33790 was considered to be an Arthrobacter sp., i. e., a Gram-positive species within the "Actinomycete line of descent" (Embley and Stacke­brandt, 1994), the pcpB gene appeared to be a somewhat universal gene, i. e., common to Gram-positive and Gram­negative strains. The fact that the pcpB probe did not hybridize to M. chlorophenolicum PCP1, could have been considered an exception. However, the results presented here, demonstrate that (1) there are at least two distinct, important families of chlorophenol-dechlorinase genes, and (2) that these different gene classes are not represented across the whole range of the bacterial taxonomy, but at least one border is drawn between the Gram-positive My­cobacterium group ("Actinomycete line of descent") and the Gram-negative Sphingomonas group.

546 U. Karlson, F. Rojo. J. D. van Elsas. and E. !\.\oore

A B c o E F G H J K

-.- ...

Fig. 3. Southern blot of fragments of total genomic DNA from the strains indicated below. using a 832-nt DNA probe for the pcpB gene from strain ATCC 39723. The DNA was digested with endonuclease EcoRI, resolved by electrophoresis in an agarose gel, and blotted onto a nylon membrane. The probe strongly hybridized with a 3 Kbp Dl'A fragment present in four of the nine strains analY7.ed. (A) and (K), unlabelled DNA probe included in rhe gel as hybridization control; (B) F1avobacterillm sp. ATCC 39723; (C) Pseudomonas sp. RA2; (D) ArtiJrobacter sp. ATCC 33790, (E) Pseudomonas sp. SR3, (F) Mycobacterium chlorophenolicum PCP 1; (G) Mycobacterium chloropheno/icum CG I, (H) Mycobacterium (ortllitll111 CG2 (I) Mycobacterium chlorophenolicum CP2 (jJ Bllrkho/deri.z cep,1cia AC1100.

Conclusions

Our data demonstrate that the Arthrobacter sp. ATCC 33790/Pseudomonas sp. SR3 pair, and the Fl.1Vobacterium sp. ATCC 39723lPseudomonas sp. RA2 pair, together comprise at least one separate species within the presently described genus Sphingomonas.

Acknowledgements. We thank Brian Tindall, Norbert Weifl. Mir;a Salkino;a-Salonen, Ole Nybroe and Bjarne M. Hansen for valuable advice, and Anneke Wolters, Heidi Andersen, Angelika Arnscheidt and Annette Krjiger for excellent technical assistance. Bacterial strains were generously provided by R. Crawford, M. Salkinoja-Salonen, P. Chapman, A. M. Chakrabarty, R.-M. Wit­tich, and H.-J. Busse. This work was supported by the European Commissions's R&D Programme Em/ironment (DG XIIID-l), contract nr. EV5V-CT93-0250. E.M. was supported, in part, by the European Community (Biotechnology Project BI02-(T94-3098) and by the German Ministry of Research and Technology (BEO Project FKZ 0319433 B).

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Ulrich Karlsoll, National Environmental Research Institute, Frederiksborgvej ,399, P.O. Box 358, DK-4000 Roskilde. Denmark, Te!. +4S-4630US7, fax +45-46.301114, e-mail: [email protected]