Desulfomonile Limimaris Sp. Nov., an Anaerobic Dehalogenating Bacterium from Marine Sediments

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International Journal of Systematic and Evolutionary Microbiology (2001), 51, 365–371 Printed in Great Britain

Desulfomonile limimaris sp. nov., an anaerobic dehalogenating bacterium from marine sediments

Baolin Sun, James R. Cole and James M. Tiedje

Author for correspondence: James M. Tiedje. Tel: j1 517 353 9021. Fax: j1 517 353 2917. e-mail: tiedjej!pilot.msu.edu

Center for Microbial Ecology Strains DCB-MT and DCB-F were isolated from anaerobic 3-chlorobenzoate and Department of Crop (3CB)-mineralizing cultures enriched from marine sediments. The isolates are and Soil Sciences, Plant and Soil Sciences Building, large, Gram-negative rods with a collar girdling each cell. The isolates are Michigan State University, obligate anaerobes capable of reductive dechlorination of 3CB to benzoate. East Lansing, MI Growth by chlororespiration in strain DCB-MT yielded 17 g protein molN1 3CB 48824-1325, USA dechlorinated with lactate as the electron donor. Strain DCB-MT also used fumarate, sulfate, sulﬁte, thiosulfate and nitrate as physiological electron acceptors for growth, but grew poorly on sulfate and nitrate. Reductive dechlorination was inhibited completely by sulﬁte and thiosulfate but not by sulfate. Both strains were incapable of growth at NaCl concentrations below 032% (w/v). They grew well at sea-water salt concentrations; however, the optimum growth rate was achieved at a NaCl concentration half that of sea water. The 16S rDNA sequence analysis shows strains DCB-MT and DCB-F to be 99% similar to each other and 93% similar to their closest relative, Desulfomonile tiedjei strain DCB-1T. Strain DCB-MT can also be distinguished from strain DCB-1T by its inability to use acetate for growth on 3CB and by its requirement for NaCl. The morphology, physiology and 16S rDNA sequences of DCB-MT and DCB-F suggest that these strains represent a new, marine-adapted species of the genus Desulfomonile, designated Desulfomonile limimaris sp. nov. The type strain is strain DCB-MT (l ATCC 700979T).

Keywords: Desulfomonile limimaris, reductive dehalogenation, marine sediments

INTRODUCTION are frequently oxygen-limited and accumulate pollutants, reductive dehalogenation can be a key initial An understanding of the fate of natural and man-made step in achieving the biodegradation of chlorinated halogenated aromatic compounds in the environment compounds in these environments (Lee et al., 1998). has become more important, as many of the most toxic In order to clarify and optimize reductive dehalo- and environmentally persistent pollutants are in this genation, major efforts have been made to isolate chemical class. Reductive dehalogenation is the only dehalogenating micro-organisms. Whilst these known biodegradation mechanism for certain signifi- organisms were very difficult and tedious to isolate in cant pollutants, including the highly chlorinated the initial studies, several have now been isolated and polychlorinated biphenyls, perchloroethene and characterized (Shelton & Tiedje, 1984; Madsen & chlorobenzenes (Mohn & Tiedje, 1992). Reductive Licht, 1992; Cole et al., 1994; Utkin et al., 1994; dehalogenation usually makes xenobiotic compounds Steward et al., 1995; Bouchard et al., 1996; less toxic and more readily degradable by aerobes. Christiansen & Ahring, 1996; Sanford et al., 1996; Since groundwaters and sediment microenvironments Boyle et al., 1999; Sun et al., 2000). Desulfomonile tiedjei strain DCB-1T, the first such strain, was isolated ...... from sewage sludge in which 3-chlorobenzoate (3CB) Abbreviation: 3CB, 3-chlorobenzoate. was being used as the electron acceptor (Shelton & The GenBank accession numbers for the 16S rDNA sequences of Desulfo- Tiedje, 1984). This strain is a large, Gram-negative, monile limimaris strains DCB-MT and DCB-F are AF230531 and AF282177. obligately anaerobic rod with a unique morphological

01529 # 2001 IUMS 365 中国科技论文在线 http://www.paper.edu.cn B. Sun, J. R. Cole and J. M. Tiedje

feature, a collar surrounding the cell. The organism benzoate and the visual increase in culture turbidity over conserves energy for growth from the reductive three successive feedings. dechlorination of 3CB (Dolfing & Tiedje, 1987; In order to determine the range of electron acceptors, Dolfing, 1990; Mohn & Tiedje, 1991) and hence is a halobenzoates at 1 mM and chlorophenols at 250 µM were halorespirer (also termed a dehalorespirer). The 16S T tested in duplicate in the sea-water medium with lactate as rDNA sequence of strain DCB-1 indicates that the the electron donor. Fumarate, sulfate, sulfite, thiosulfate organism is a member of the δ-Proteobacteria. and nitrate were also tested as potential electron acceptors at DeWeerd et al. (1990) described this strain as a new 5 mM, with lactate as the electron donor. Growth was genus among the sulfate-reducing bacteria. determined by monitoring the depletion of substrates, the accumulation of products and the visual increase in culture Although most of the natural haloaromatic com- turbidity. pounds are produced in the marine environment (Gribble, 1992), only marine debrominating isolates In order to test the relationship between reductive dechlorination and potential competitive electron acceptors, have been reported until recently (Steward et al., 1995; sulfate, sulfite, thiosulfate and nitrate at 5 mM were added Boyle et al., 1999). The first marine dechlorinating T separately to the sea-water medium containing 1 mM 3CB isolate, Desulfovibrio dechloracetivorans strain SF3 , and 5 mM lactate. Dechlorination activity was determined dechlorinates 2-chlorophenol reductively to phenol in duplicate cultures by measuring the depletion of 3CB and and grows at concentrations of NaCl ranging from the appearance of benzoate by HPLC. 0n16 to 2n5% (w\v) (Sun et al., 2000). The effect of NaCl on reductive dechlorination was tested by In this paper, we report the isolation of two additional including various concentrations of NaCl in the medium. marine dehalogenating strains, DCB-MT and DCB-F, Growth was measured by monitoring the depletion of 3CB from marine sediments from Florida, and we describe and the appearance of benzoate. a new species, Desulfomonile limimaris sp. nov. Growth rate and protein yield. The rate of growth on lactate and 3CB was estimated by monitoring the production of benzoate from reductive dechlorination of 3CB. Samples METHODS were taken from duplicate cultures every 24 h and analysed by HPLC. To measure protein yield, replica cultures were Media, enrichment and isolation. A sulfate-free, anaerobic, grown in 100 ml sea-water medium supplemented with synthetic sea-water medium (Sun et al., 2000) with 2n5% 5 mM lactate and with or without 1 mM 3CB. After (w\v) NaCl was used to favour dechlorinators rather than approximately 1 mM 3CB had been consumed, cultures sulfate-reducers. Shallow marine sediments were collected were harvested and analysed for substrate transformation from Gulf Breeze (north-west Florida, USA), Big Pine Key and protein yield. (south Florida, USA), King Salmon (Alaska, USA), San Francisco Bay (California, USA), Pearl Harbour and Chemical analysis. Benzoate and halogenated aromatic Kanehoe Bay (Hawaii, USA), Johnston Island (Pacific compounds were analysed by reverse-phase HPLC with a " Ocean) and Mayaguez (Puerto Rico). Sediment samples Hibar RP-18 (10 µm) column, a flow rate of 1n5 ml min− for (5–10 g) were mixed with 100 ml anaerobic sea-water H#O\CH$CN\H$PO% (66:33:0n1) and a UV detector set to medium supplemented with 1 mM 3CB and incubated in 230 nm. The appearance of products and the disappearance 160-ml serum bottles in the dark, first at 25 mC and of substrates were verified by comparison with authentic subsequently at 30 and 37 mC. No additional electron donors standards and culture samples taken at time zero. Organic were provided in microcosms and enrichment cultures. After acids such as acetate, formate, fumarate, lactate, pyruvate further serial transfers, the active dechlorinating culture was and succinate were analysed by ion-exclusion HPLC. H# was diluted into deep agarose shake cultures containing 10 ml analysed by using a gas chromatograph equipped with a sea-water medium solidified with 1% low-gelling-tempera- reduction gas detector. To measure protein yield, cells were ture agarose and supplemented with 3CB and pyruvate or collected and analysed by the method of Lowry after alkaline lactate to final concentrations of 1 and 5 mM, respectively. hydrolysis (Hanson & Phillips, 1981). Cultures were incubated at 37 mC. Bacterial colonies were picked and transferred to the same medium without agarose 16S rRNA gene sequencing and analysis. DNA was extracted in order to test for dechlorination and growth. from 50 ml culture by using a method developed for diverse bacteria (Visuvanathan et al., 1989). The 16S rRNA gene Gram staining and microscopy. Gram staining was done by was amplified by using primers FD1 and RD1 (Weisburg et the method of Hucker (Doetsch, 1981). Phase-contrast al., 1991) and the protocol described previously (Cole et al., photomicroscopy was carried out with a Zeiss photo- 1994). The PCR product was purified by using the Wizard microscope. Scanning electron microscopy was performed purification system (Promega) and sequenced in both as described previously (Klomparens et al., 1986). directions by automated fluorescent dye-terminator sequencing. Primers corresponding to conserved regions of Characterization of reductive dechlorination. Duplicate 20 the 16S rRNA gene sequence (Woese, 1987) were used for ml cultures of sea-water medium were supplemented with sequencing. The resulting sequences were analysed and the 1 mM 3CB and different electron donors (at 2n5 mM). The phylogenetic placement was obtained by using the Ribo- cultures were inoculated with a 1% transfer from an active somal Database Project (RDP-ΙΙ, release 7.1) (Maidak et al., dehalogenating culture grown on pyruvate and 3CB. Acet- 2000). A maximum-likelihood phylogenetic tree was created ate, benzoate, butyrate, formate, fumarate, H#, lactate, with the program fastDNAml (Olsen et al., 1994). propionate and pyruvate were tested as potential electron donors for reductive dechlorination. Growth was deter- Nucleotide accession numbers. The 16S rDNA sequences for mined by measuring the depletion of 3CB, the production of the following organisms were obtained, in aligned format,

366 International Journal of Systematic and Evolutionary Microbiology 51 中国科技论文在线 http://www.paper.edu.cn Desulfomonile limimaris sp. nov.

from RDP-ΙΙ (Maidak et al., 2000) (RDP-ΙΙ and GenBank accession numbers are given in parentheses): Bacillus subtilis (K00637), Desulfovibrio desulfuricans subsp. desulfuricans (M34113), Desulfuromonas acetoxidans DSM 684T (M26634), Desulfomonile tiedjei DCB-1T (M26635). Environ- mental clone Btol was isolated from sulfate-reducing, toluene- oxidizing enrichment culture (Beller et al., 1992; Cascarelli, 1995). Its 16S rDNA sequence is available under GenBank accession number AF282178.

RESULTS Enrichment and isolation Sediment microcosms were incubated at 25 mC for 7 months with no evidence of 3CB dechlorination. When cultures were transferred to 30 mC, dechlorination was observed after 1 month for sediments from Gulf Breeze and 2 months for sediments from Pearl Harbour and ...... San Francisco Bay. 3CB-dechlorinating populations Fig. 2. Growth of strain DCB-MT on 3-chlorobenzoate (3CB) plus lactate at 37 C. Data are means from duplicate cultures. from the active cultures were enriched further at 37 mC m

with repeated 3CB feedings, since a temperature of 37 mC gave a faster rate of dechlorination. A 10% transfer was made into fresh sea-water medium with 3CB and no additional electron donors. A dilution " series of cultures (5% inoculum, dilutions of 10− to ( 10− ) was made after approximately 4 mM 3CB was metabolized in the second transfer. Dechlorination activity was obtained within 6 weeks in the cultures ( diluted 10 -fold and was sustained by successive feedings with 3CB. Benzoate accumulated in these enrichment cultures. Isolation was attempted from ( dilutions (10− ) of the ﬁfth serial transfer. After approximately 2 weeks, small brown colonies became visible in the deep agarose shake cultures from the Gulf Breeze source only. Eight individual bacterial ' ( colonies were picked from dilutions (10− and 10− ) and tested for dechlorination activity in sea-water medium containing pyruvate and 3CB. After 4 months, 3CB had disappeared from three of the eight cultures, with the concomitant appearance of approximately equal amounts of benzoate. One culture was selected for the second round of puriﬁcation by dilution and colony selection from deep agarose shake cultures. Dechlorination activity was obtained from these clones. One clone was designated strain DCB-MT (to indicate a dechlorinating bacterium from a marine environment) and was used for further study. The purity of this clone was also demonstrated by homo- geneous colony and cell morphology and by the recovery of dechlorination activity from cultures grown on fumarate plus pyruvate. Strain DCB-MT is a Gram-negative, non-motile, large rod (6–8i0n4–0n6 µm) with a collar girdling each cell (Fig. 1). The strain is a strict anaerobe, since no growth

...... was observed when oxygen was introduced into the Fig. 1. Phase-contrast (a) and scanning electron (b) micrographs culture. The organism has a generation time of of cells of strain DCB-MT. The arrow points to the collar approximately 3n5 d in sea-water medium supple- structure of the cell. Bars: 5 µm (a) and 1 µm (b). mented with 3CB and lactate at 37 mC (Fig. 2). The

International Journal of Systematic and Evolutionary Microbiology 51 367 中国科技论文在线 http://www.paper.edu.cn B. Sun, J. R. Cole and J. M. Tiedje

Table 1 Growth yield for strain DCB-MT grown on 3CB plus lactate

Electron acceptor 3CB consumed (µmol) Benzoate produced (µmol) Protein (µg)* Protein/benzoate (g mol−1)

3CB (culture 1) 988 976 3300 1n91 3CB (culture 2) 994 983 2790 1n40 None – – 1390† –

* The protein yield was calculated after subtracting protein measured in control cultures with lactate only. † Mean from duplicate control cultures with lactate only.

...... Fig. 3. Maximum-likelihood phylogenetic tree based on the 16S rDNA sequences of strains DCB-MT and DCB-F and repre- sentative bacteria. Numbers at internal nodes are the percentages of 100 bootstrap samples in which the groups to the right of the nodes were monophyletic. The scale bar gives the expected number of substitutions per position.

growth yield with 3CB as the electron acceptor and 0·4 −" lactate as the electron donor was 1n7 g protein mol 3CB dechlorinated (Table 1). DCB-MT )

Strain DCB-F was isolated previously from a distant –1 0·3 (1000 km) Florida sediment by using a similar en-

richment and isolation procedure. This strain was T T DCB-1 morphologically identical to DCB-M and also DCB-F dechlorinated 3CB reductively to benzoate. However, 0·2 strain DCB-F had a slower growth rate, which made it diﬃcult to study compared with strain DCB-MT; hence, more eﬀort was focused on strain DCB-MT. 0·1 Growth rate (doublings d

Phylogeny of isolates Comparison of the 16S rDNA sequences of strains T 0·08 0·16 0·32 0·63 1·25 2·5 5·0 10 DCB-M and DCB-F with available 16S rDNA [NaCl] (% w/v) sequences indicated that these strains belong to the sulfate-reducing group of the δ-Proteobacteria. The ...... nearly full-length 16S rDNA sequences of these two Fig. 4. Effect of NaCl on the growth of strains DCB-MT, DCB-F strains are 99% similar to each other and only 93% and DCB-1T. Growth was estimated from the rate of reductive similar to their closest relative, strain DCB-1T.A dechlorination of 3CB. Data are means from duplicate cultures. fourth 3CB-dechlorinating bacterium, strain DCB-O, isolated from sewage sludge in Columbus, OH, had a 16S rDNA sequence identical to that of strain DCB-1T at 1n25% NaCl (Fig. 4). The proﬁle of the eﬀect of (J. R. Cole, M. Fettig & J. M. Tiedje, unpublished T T T NaCl on growth rate for strain DCB-M was very results). Strains DCB-M , DCB-F, DCB-1 and DCB- similar to that for strain DCB-F, whereas the fresh- O form a monophyletic group (Fig. 3). water (sewage sludge) strain DCB-1T grew better at much lower salt concentrations (Fig. 4). Effect of NaCl on dechlorination and growth Range of electron donors and acceptors Strain DCB-MT was enriched and isolated in medium with 2n5% NaCl. Reductive dechlorination and In addition to oxidizing pyruvate and lactate, strain T growth occurred at concentrations of NaCl ranging DCB-M oxidized formate, H#, butyrate, benzoate from 0n32 to 2n5%, with the growth optimum occurring and propionate for reductive dechlorination and

368 International Journal of Systematic and Evolutionary Microbiology 51 中国科技论文在线 http://www.paper.edu.cn Desulfomonile limimaris sp. nov.

Table 2 Electron acceptors tested for growth of strain DCB-MT in lactate medium ...... Growth is scored as positive (growth) or negative (no growth) as deﬁned in Methods. , Not determined.

Electron acceptor Growth Product(s)

2-Chlorobenzoate k 3-Chlorobenzoate j Benzoate 3-Bromobenzoate j Benzoate 3-Chloro-4-hydroxybenzoate k 2-Chlorophenol k 3-Chlorophenol k 2,3-Dichlorophenol k 2,3-Dichlorobenzoate j 2-Chlorobenzoate 2,5-Dichlorobenzoate j 2-Chlorobenzoate 3,4-Dichlorobenzoate k 3,5-Dichlorobenzoate j 3-Chlorobenzoate; benzoate 2,3,5-Trichlorobenzoate j 2,3-Dichlorobenzoate; 2,5-dichlorobenzoate; 2-chlorobenzoate 2,4,6-Trichlorobenzoate k Fumarate j Succinate Nitrate j  Sulfate j  Sulﬁte j  Thiosulfate j 

growth, but did not oxidize acetate or fumarate. Strain polar growth and cell division (Mohn et al., 1990). DCB-MT also used a variety of electron acceptors for Strain DCB-MT gains energy from the reductive growth, including fumarate, sulfate, sulfite, thio- dechlorination reaction and hence is a halorespirer. sulfate, nitrate and halobenzoates, but only used those This was shown by growth with 3CB as the only with chlorine in the meta position (Table 2). substrate, by growth with 3CB on substrates (H#, Substitutions by chlorine or a hydroxyl group at the formate) that provide no substrate-level phosphoryl- −" para position blocked meta dechlorination. Fumarate ation, and by a growth yield of 1n7 g protein mol 3CB was reduced to succinate. Although sulfate and nitrate dechlorinated. This yield compares favourably with T −" were reduced by strain DCB-M , the growth rate was values of 1n9 g protein mol 3CB dechlorinated for much slower than with sulfite or thiosulfate. strain DCB-1T (Dolfing & Tiedje, 1987) and 2 9g " n protein mol− 2-chlorophenol dechlorinated for strain When sulfate, sulfite or thiosulfate were added to the 2CP-1 (Cole et al., 1994). medium as potential competitive electron acceptors, sulfate did not show any inhibition of reductive Although it is phylogenetically, morphologically and dechlorination (data not shown). Sulfite and thio- physiologically similar to strain DCB-1T, strain DCB- sulfate inhibited reductive dechlorination almost com- MT has several features that distinguish it from strain pletely at the concentration tested (5 mM). DCB-1T. Strain DCB-1T was capable of growth in the absence of NaCl, unlike strain DCB-MT (and strain T DISCUSSION DCB-F). Propionate was used by strain DCB-M but not by strain DCB-1T, whilst strain DCB-1T used Strains DCB-MT and DCB-F, along with the pre- acetate whereas strain DCB-MT did not. Strain DCB- viously isolated strains DCB-1T (Shelton & Tiedje, 1T grew on pyruvate alone but strain DCB-MT (and 1984; DeWeerd et al., 1990) and DCB-O, represent strain DCB-F) did not. Finally, the 16S rDNA a coherent group of obligately anaerobic, 3CB- sequences of strains DCB-MT and DCB-1T differed by dechlorinating, sulfidogenic bacteria according to 7%. It has been suggested that strains with differences physiological, morphological and phylogenetic cri- of more than 2n5–3n0% in their 16S rDNA sequences teria. All of these strains grow by respiring sulfate, belong to different species (Stackebrandt & Goebel, sulfite, thiosulfate and 3CB and use similar (though 1994). The morphology, physiology and 16S rDNA not identical) electron donors. All of these strains are sequences suggest that strains DCB-MT and DCB-F long rods exhibiting a unique morphological feature, are marine relatives of strain DCB-1T but that they are a collar girdling each cell. Studies have shown that sufficiently different to be regarded as representing the collar structure of strain DCB-1T is involved in another species.

International Journal of Systematic and Evolutionary Microbiology 51 369 中国科技论文在线 http://www.paper.edu.cn B. Sun, J. R. Cole and J. M. Tiedje

Strain DCB-MT used a reasonably broad range of each cell. Growth is obligately anaerobic. 3CB is electron donors and acceptors for anaerobic growth. reductively dechlorinated to benzoate. Benzoate, lac- Few halogenated substrates supported growth, how- tate, formate, H#, butyrate, propionate and pyruvate ever. Only the meta positions of halobenzoates were are used as electron donors. Sulfate, sulfite, thiosulfate, dehalogenated, and substitution by chlorine or a nitrate, fumarate and 3CB serve as electron acceptors hydroxyl group at the para position blocked meta for growth. The organism grows at concentrations of dechlorination. The additional substituents could NaCl ranging from 0n32 to 2n5%, with optimum affect the chemical reactivity of the substrates, their growth occurring at 1n25% NaCl. The optimum T uptake into the cell or their affinity for the active growth temperature is 37 mC. Strain DCB-M is the enzyme. The specificity that Desulfomonile strains type strain and has been deposited in the American exhibit for the removal of meta-substituted halogens Type Culture Collection as strain ATCC 700979T. The and the coupling to respiratory growth suggest that organism was isolated from marine sediment at Gulf this capacity is not fortuitous but may have been the Breeze, FL, USA. product of selection for a particular chemical resource. Many haloaromatic compounds occur in natural ACKNOWLEDGEMENTS environments as a consequence of animal, fungal and algal activities (Ashworth & Cormier, 1967; Craigie & We thank Klaus Nuesslein, Todd Stevens and Mark Ingoglia for collecting the sediment samples. We also thank Hans Gruenig, 1967; King, 1986; Gribble, 1992; de Jong et Tru$ per for advice on naming the organism and Frank Dazzo al., 1994) and some of them can be dehalogenated by and Ewa Danielewicz for microscopy. This research was undescribed marine micro-organisms (King, 1988). supported by US Office of Naval Research grant N00014- Reductive dehalogenation may be inhibited by other 95-1-0115 and by the Center for Microbial Ecology, through National Science Foundation grant DEB 9120006. competitive electron acceptors, which is a particularly important consideration in sulfate-rich marine environments. The relationship between reductive REFERENCES dehalogenation and potential electron acceptors seems Ashworth, R. B. & Cormier, M. J. (1967). Isolation of 2,6- to be complex. For strain DCB-1T (Stevens et al., 1988) dibromophenol from the marine hemichordate, Balanoglossus and DCB-MT, sulfate was a much less favourable biminiensis. Science 155, 1558–1559. electron acceptor than sulfite or thiosulfate. Inhibition Beller, H. R., Reinhard, M. & Grbic-Galic, D. (1992). 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