Four pentachlorophenol (PCP)-degrading bacterial strains: Arthrobacter sp. ATCC 33790, Pseudomonas sp. 5R3, Flavobacterium sp_ ATCC 39723 and ...
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 ELSAS 3 , and EDWARD MOORE 4 I
1 4
~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 IPO-DLO, Binnenhaven 5, P.O. Box 9060, NL-6700 GW Wageningcl1, The Netherlands 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. ATCC 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-
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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)
after addition of 1.5 mL of ReadyGcI (Becknun; ,lnd 1 mL of H 20 to the KOH trap. Non-chlorophenol-degrading straim were (ultured in LB medium (S'I/l/brook et aI., 1989).
Detcrmination ,wd ,1II,1Iysis
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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
