Reclassification of Desulfobacterium Phenolicum As Desulfobacula

International Journal of Systematic and Evolutionary Microbiology (2001), 51, 171–177 Printed in Great Britain

Reclassiﬁcation of Desulfobacterium phenolicum as Desulfobacula phenolica comb. nov. and description of strain SaxT as Desulfotignum balticum gen. nov., sp. nov.

Jan Kuever,1 Martin Ko$ nneke,1 Alexander Galushko2 and Oliver Drzyzga3

Author for correspondence: Jan Kuever. Tel: j49 421 2028 734. Fax: j49 421 2028 580. e-mail: jkuever!mpi-bremen.de

1 Max-Planck-Institute for A mesophilic, sulfate-reducing bacterium (strain SaxT) was isolated from Marine Microbiology, marine coastal sediment in the Baltic Sea and originally described as a Department of Microbiology, ‘Desulfoarculus’ sp. It used a large variety of substrates, ranging from simple Celsiusstrasse 1, D-28359 organic compounds and fatty acids to aromatic compounds as electron donors. Bremen, Germany Autotrophic growth was possible with H2,CO2 and formate in the presence of 2 Fakulta$ tfu$ r Biologie, sulfate. Sulfate, thiosulfate and sulﬁte were used as electron acceptors. Sulfur Universita$ t Konstanz, and nitrate were not reduced. Fermentative growth was obtained with Postfach 5560, D-78457 Konstanz, Germany pyruvate, but not with fumarate or malate. Substrate oxidation was usually complete leading to CO , but at high substrate concentrations acetate 3 University of Bremen, 2 Center for Environmental accumulated. CO dehydrogenase activity was observed, indicating the

Research and Technology operation of the CO dehydrogenase pathway (reverse Wood pathway) for CO2 (UFT), Department of ﬁxation and complete oxidation of acetyl-CoA. The rod-shaped cells were Marine Microbiology, Leobener Strasse, D-28359 08–10 µm wide and 15–25 µm long. Spores were not produced and cells Bremen, Germany stained Gram-negative. The temperature limits for growth were between 10 and 42 SC (optimum growth at 28–32 SC). Growth was observed at salinities ranging from 5 to 110 g NaCl lV1, with an optimum at 10–25 g NaCl lV1. The GMC content of the DNA was 624 mol%. Vitamins were required for growth. Based on the 16S rRNA gene sequence, strain SaxT represents a new genus within the δ-subclass of the Proteobacteria. The name Desulfotignum balticum gen. nov., sp. nov. is proposed. After the 16S rDNA sequences of all members of the genus Desulfobacterium were published (GenBank accession nos. AJ237601–AJ237604, AJ237606, AJ237607), the need to reclassify most members of the genus Desulfobacterium became obvious due to their strong phylogenetic afﬁliation to other genera. Here, we propose to reclassify Desulfobacterium phenolicum as Desulfobacula phenolica comb. nov. Desulfotignum balticum, Desulfobacterium phenolicum and Desulfobacula toluolica contain cellular fatty acids which have so far only been found in members of the genus Desulfobacter.

Keywords: Desulfobacula, Desulfotignum, marine sulfate-reducing bacterium, complete oxidation, FAME

INTRODUCTION to grow on a large variety of aromatic compounds, including phenolic compounds and toluene. They were Among the sulfate-reducing bacteria of the δ-subclass originally classiﬁed as members of the genera Desulfo- of the Proteobacteria several marine isolates are known bacterium and Desulfobacula (Bak & Widdel, 1986a, b;

...... Brysch et al., 1987; Rabus et al., 1993, 2000). Besides Abbreviations: FAME, fatty acid methyl ester; HMN, 2,2,4,4,6,8,8- the use of various aromatic compounds, members of heptamethylnonane. the genus Desulfobacula are characteristically restric- The GenBank accession number for the nearly complete 16S rRNA ted to the utilization of short-chain fatty acids sequences of strain SaxT (DSM 7044T) is AF233370. and simple organic compounds as electron donors;

01545 # 2001 IUMS 171 J. Kuever and others whereas members of the genus Desulfobacterium can 7044T. Desulfobacula toluolica (DSM 7467T), Desulfo- T also grow chemoautotrophically on H# and CO# (Bak bacterium phenolicum (DSM 3384 ), Desulfobacterium auto- & Widdel, 1986a, b; Brysch et al., 1987; Rabus et al., trophicum (DSM 3382) and Desulfobacter postgatei (DSM 1993; Schnell et al., 1989). In contrast, members of the 2034) were obtained from the DSMZ. genera Desulfococcus, Desulfonema and Desulfosarcina Media and culture conditions. For enrichment and cul- only use a limited number of aromatic compounds, tivation the medium was prepared as described previously mainly benzoic acid derivatives, but also long-chain using benzoate (2n5 mM) as electron donor (Drzyzga et al., fatty acids as their sole electron donor and carbon 1993). Pure cultures were obtained by repeated use of source (Fukui et al., 1999; Widdel, 1980, 1988; Widdel deep agar dilution series (Widdel & Bak, 1992). Substrate & Bak, 1992; Widdel & Hansen, 1992; Widdel et al., utilization was determined by adding the carbon and energy 1983). Recently obtained isolates with interesting sources from sterile stock solutions; the cultures were incubated for about 3 weeks. To avoid possible toxic effects degradation capabilities might indicate that in general of the substances, toluene and xylene isomers were supplied marine sulfate-reducing bacteria are more versatile to cultures following adsorption in the deaerated organic than isolates obtained from freshwater habitats solvent 2,2,4,4,6,8,8-heptamethylnonane (HMN; 2%, v\v) (Galushko et al., 1999; Harms et al., 1999; Rueter et (Rabus et al., 1993). A check was made to ensure that HMN al., 1994). However, there are many other sulfate- did not inhibit growth of strain SaxT on benzoate. reducing bacteria (including spore-forming ones) iso- To test the autotrophic growth capability, cultures were lated from freshwater habitats which use a large variety grown under a headspace of H#\CO# (80\20%, v\v) at of organic compounds as electron donor and are able an overpressure of 101n3 kPa. The temperature range for to grow in marine media (Kuever et al., 1999). Another growth was determined by incubation in a temperature marine isolate, which was isolated with benzoate and gradient block from 35 to 80 mC in increments of 2–4 mC. The tentatively classified as a ‘Desulfoarculus’ (correct pH range of growth was determined in mineral medium at spelling should be ‘Desulfarculus’) species, can grow pH values from 5 to 9. The dependence of growth on the concentration of NaCl was determined in mineral medium slowly on fatty acids with a chain length higher than " with NaCl concentrations from 0 to 130 g NaCl lV . Strain butyrate (Drzyzga et al., 1993). Therefore, it resembles T Desulfobacterium and Desulfobacula spp. In this paper Sax was routinely cultivated in a carbonate-buffered, we describe this species as a new genus within the δ- sulfide-reduced medium for marine sulfate-reducing bacteria Proteobacteria supplied with 2n5 mM benzoate as a growth substrate. Salt subclass of the . composition of the medium and incubation conditions were The genus Desulfobacterium was established by using as described by Widdel & Bak (1992). mainly physiological properties (Brysch et al., 1987). Chemical and biochemical characterization. The presence of After the 16S rDNA sequences of all members of this desulfoviridin was tested as described by Postgate (1959). genus were published by E. Stackebrandt (GenBank The GjC content was determined by thermal denaturation. accession nos. AJ237601–AJ237604, AJ237606, Analysis of aromatic compounds, identification of cyto- AJ237607), the need to reclassify most members of the chromes and measurement of sulfide production was as genus Desulfobacterium became obvious due to their described previously (Drzyzga et al., 1993). strong phylogenetic affiliation to other genera. A For fatty acid methyl ester (FAME) analysis strain SaxT, comparative analysis using the sequences published by Desulfobacula toluolica and Desulfobacterium phenolicum Stackebrandt (see above) indicates that the genus were grown in marine mineral medium with benzoate comprises only the type species of the genus, Desulfo- (2n5 mM) as the only electron donor and carbon source and bacterium autotrophicum, and two other species: the sulfate (28 mM) as electron acceptor. Desulfobacterium non-validly published and as yet undescribed ‘Desulfo- autotrophicum and Desulfobacter postgatei were grown with bacterium vacuolatum’ (only listed by Widdel, 1988) butyrate (10 mM) and acetate (20 mM), respectively. Cells and the species originally named Desulfococcus niacini from the late exponential growth phase were centrifuged and (Imhoff & Pfennig, 1983), described as ‘Desulfo- used for a comparative FAME analysis. The whole-cell fatty bacterium niacini acid composition was determined by means of capillary GC ’. That both species are members of and MS analysis. FAME preparation was carried out using the genus Desulfobacterium had been demonstrated by a method as described by Sasser (1997). several phylogenetic analyses (Rabus et al., 1993) and T by use of the genus-specific oligonucleotide probe 221 Preparation of cell extract. Strain Sax was grown in 1200 ml (Devereux et al., 1992; Manz et al., 1998). All other bottles containing 1000 ml mineral medium supplied with members of the genus have to be reclassified because 2n5 mM benzoate under a gas phase of N#\CO# (80\20%, v\v). Cells from the late exponential growth phase were they belong to other genera or represent new genera. harvested by centrifugation and washed once in anoxic Here we suggest that the former Desulfobacterium saline buffer (50 mM potassium phosphate, pH 7 3, con- " " n phenolicum be incorporated within the genus Desulfo- taining 26 g NaCl lV \11n2 g MgCl#;6H#OlV ). Collected bacula (Rabus et al., 1993, 2000). cells were resuspended in anoxic potassium phosphate buffer (50 mM, pH 7n3) supplied with 2n5 mM MgCl#;6H#O and METHODS 2n5 mM dithiothreitol and were passed 2–3 times through an anoxic French press cell at 137 MPa. Cell debris and intact Source of organism. Strain SaxT was isolated from a benzoate cells were removed from the homogenate by centrifugation enrichment culture inoculated with anoxic marine mud from (30000 g, 20 min). The extract was placed in a small glass Saxild, Denmark (Drzyzga et al., 1993). Strain SaxT was vial under N# gas phase and stored on ice. Measurements of deposited in the DSMZ under the accession number DSM enzyme activities were done on the day of preparation.

172 International Journal of Systematic and Evolutionary Microbiology 51 Desulfotignum balticum

Table 1. Major cellular fatty acids of strain SaxT and some phylogenetically related species ...... Percentage of total fatty acids is shown. Fatty acids present in all strains at less than 1% are not listed. c, cis; i, iso; cyc, cyclopropane; OH, hydroxy; Me, methyl.

Fatty acid SaxT Desulfobacula Desulfobacula Desulfospira Desulfobacterium Desulfobacter phenolica toluolica joergensenii* autotrophicum postgatei

14:1 – 1n4–– – – 14:0 3n78n78n313n92n815n4 i15:0 – – – 1n8– 0n7 15:1c90n8– – – 6n5– 15:0 0n22n01n61n35n13n1 3-OH 14:0 – 1n3–1n6– – 16:1c98n516n5† 19n738n930n710n0 16:0 24n220n431n228n413n220n9 10-Me 16:0 14n017n217n6– 7n411n2 17:1c11 – – – – 10n5– 17:0cyc 19n53n92n62n4– 31n5 17:0 0n40n70n70n62n92n3 3-OH 16:0 – – – 2n30n5– 18:1c93n7– – – – – 18:1c11 7n43n96n05n32n71n2 18:0 9n82n13n10n71n10n5 i19:0 1n7– 2n5– – – 19:0cyc 1n1– – – – –

* Data obtained from Finster et al. (1997). † In addition, 5.6% of unidentiﬁed 16:1 isomers were found.

Enzyme assays. All enzyme assays were done under strictly described by Buchholz-Cleven et al. (1997). The sequence anoxic conditions at 30 mCin1n5 ml glass cuvettes sealed reaction mixtures were electrophoresed on an Applied with butyl rubber stoppers. Assays were performed in 1 ml Biosystems 373S DNA sequencer. potassium phosphate buffer (50 mM, pH 7n3) which was Phylogenetic analyses of 16S rRNA gene sequence data. The slightly reduced by addition of several microlitres of 0n05% sequences were loaded into the 16S rRNA sequence database dithionite solution. All additions were done from anoxic of the Technical University of Munich, Germany, using the stock solutions with microlitre syringes. One unit of enzyme ARB program package (http:\\www.mikro.biologie.tu- V" V" activity was defined as 1 µmol min (mg protein) . muenchen.de). The tool I was used for sequence The presence of 2-oxoglutarate:electron acceptor oxido- alignment. The alignment was visually inspected and reductase was checked by following the reduction of benzyl corrected manually. Tree topologies were evaluated by viologen (2 mM) at 578 nm in the presence of 2-oxoglutarate performing maximum-parsimony, neighbour-joining and (3 mM) and CoA (0n2mM). maximum-likelihood analyses with different sets of filters. Activity of CO dehydrogenase was determined by following Only sequences with at least 1200 nt were used for the calculation of different trees. The partial sequence of strain the reduction of benzyl viologen (5 mM) in the presence of T CO. To perform the assay, cuvettes were flushed with CO Sax (1462 nt) was added to the reconstructed tree by until the assay buffer was saturated with CO. applying parsimony criteria without allowing changes in the overall tree topology. The strain designations and nucleotide Protein content in cell-free extract was determined with sequence accession numbers which were not included in the bichinchoninic acid as a reagent by following a standard ARB database are as follows: Desulfobacula toluolica DSM protocol assay (BCA protein assay kit; Pierce) and with 7467T, X70593; Desulfobacterium phenolicum DSM 3384T, bovine serum albumin fraction V (Pierce) as a standard for AJ237606 (submitted by E. Stackebrandt); Desulfospira calibration. Gases were purchased from Messer-Griesheim joergensenii, DSM 10085T ; X99637; Desulfotignum balticum and Sauerstoffwerke Friedrichshafen. (strain SaxT), DSM 7044T, AF 233370; clone SB-9, PCR amplification and sequencing of the 16S rRNA gene. To AF029042; clone Sva0605, AF230098. amplify the almost complete 16S rRNA-encoding gene T (1500 bp) of strain Sax , primers GM3F and GM4R were RESULTS used in a 35-cycle PCR with an annealing temperature of 40 mC (Muyzer et al., 1995). PCR products were purified by FAME analysis using the QIAquick Spin PCR purification kit (Qiagen) as T described by the manufacturer. The Taq DyeDeoxy Ter- The fatty acid composition of strain Sax and its minator Cycle Sequencing kit (Applied Biosystems) was phylogenetically closest relatives are listed in Table 1. used to directly sequence the PCR products, according to Significant amounts of unidentified fatty acids the manufacturer’s protocol. The sequencing primers were (ECL16.07, ECL18.09) were found in Desulfobacula

International Journal of Systematic and Evolutionary Microbiology 51 173 J. Kuever and others

Table 2. Comparison of selected characteristics of Desulfobacula toluolica, Desulfobacula phenolica, Desulfospira joergensenii and strain SaxT ...... , Not reported; j, good growth; (j), poor growth; k, no growth; a, autotrophic growth; sp, single polar ﬂagellum. Data obtained from Bak & Widdel (1986a), Drzyzga et al. (1993), Finster et al. (1997) and Rabus et al. (1993). All strains were negative for desulfoviridin and for growth on isobutyrate, 2-methylbutyrate and 3-methylbutyrate and could use sulfate as electron acceptor. Substrate oxidation was complete for all strains.

Characteristic Desulfobacula Desulfobacula Desulfospira SaxT toluolica phenolica joergensenii

Morphology Oval Oval to curved rod Vibrioid Rod Widthilength (µm) 1n2–1n4i1n2–2n0 1–1n5i2–3 0n7–0n8i1–2 0n5–0n7i1n5–3n0 Motility j* j(sp) kj(sp) GjC content (mol%) 42 41 50 62 Major menaquinone  MK-7(H#) MK-7 and MK-7(H#)  " Optimum salinity (g NaCl lV ) 20 20 12–20 20 Optimum temperature (mC) 28 28 26–30 28–32 Compounds used as electron donor and carbon source: a H# j CO# kk j (j ) Formate k (j) ja (ja) Acetate k (j) k (j) Fatty acid (no. of C atoms) 4 (4) 4, 8, 12, 14 4 (10, 12, 16, 18) Ethanol j (j) kk Lactate kk j j Pyruvate jj j j Fumarate j (j) jj Succinate j (j) j (j) Malate j (j) kj Benzoate jj k j 4-Hydroxybenzoate jj k j Phenol kj k (j) Phenylacetate kj k (j) Toluene j (j) kk Others Propanol, butanol, Propanol, butanol, Crotonate, glutarate, Crotonate, maleinate p-cresol, glutarate 2-hydroxybenzoate, maleinate, glycolate, p-cresol, glutarate glycerol, betaine, proline, yeast extract Fermentative growth   kj(on pyruvate) Electron acceptors: Sulﬁte  kj j Sulfur  kj  Thiosulfate  jj j Nitrate  kk k Growth factor requirement Vitamins k Biotin Vitamins

* Cells are motile during early exponential growth phase, but motility can rapidly decline during growth.

toluolica (7n5%) and Desulfobacula phenolica (16n6%), of the citric acid cycle) was not detected. This ﬁnding and also in trace amounts in strain SaxT. The FAME indicated the presence of the CO dehydrogenase analysis for Desulfobacula toluolica grown on ethanol pathway for the oxidation of acetyl-CoA (Thauer, (van der Maarel et al., 1996) was very similar to our 1988). results.

Enzyme activities Physiological and morphological properties In cell-free extracts of strain SaxT active CO de- All physiological and morphological properties of " T hydrogenase was found [2n6 U (mg proteinV )], strain Sax , Desulfobacterium phenolicum and Desulfo- whereas 2-oxoglutarate dehydrogenase (a key enzyme bacula toluolica are listed in Table 2 and compared to

174 International Journal of Systematic and Evolutionary Microbiology 51 Desulfotignum balticum

Bdellovibrio stolpii ‘Desulfobotulus sapovorans’ Desulfonema/Desulfococcus Desulfovibrio Desulfocella halophila Desulfosarcina

Desulfofrigus/Desulfofaba

Desulfurella acetivorans

Desulfobacterium Geobacter

Desulfobacter Syntrophus Desulfospira joergensenii Clone SB-9 Desulfomonile tiedjei Desulfotignum balticum

Clone Sva0605 Desulfobacca acetoxidans Desulfobacula toluolica ‘Desulfarculus baarsii’ Desulfobacterium phenolicum 10 % Desulforhabdus/Desulfacinum Desulfobulbus

...... Fig. 1. Phylogenetic tree showing the afﬁliations of 16S rDNA sequences from Desulfobacula phenolica and Desulfotignum balticum to selected reference sequences of the δ-subclass of the Proteobacteria. The tree was calculated by neighbour-joining analysis and corrected with ﬁlters which considered only 50% conserved regions of the 16S rRNA of δ-Proteobacteria. The sequence of Desulfurella acetivorans was used as outgroup. The bar represents 10% estimated sequence divergence.

the closely related species Desulfospira joergensenii. with any of the other genera and was therefore For a more detailed description the original publi- proposed as a new genus in its own right. cations should be consulted (Bak & Widdel, 1986a; Drzyzga et al., 1993; Finster et al., 1997; Rabus et al., DISCUSSION 1993). T The physiological and morphological properties Strain Sax did not grow on toluene, nor on o-, m- and (Table 2) in combination with the comparative 16S p-xylenes supplied adsorbed in HMN; nor did this rDNA and FAME analyses (Fig. 1 and Table 1) organic solvent inhibit growth of the bacterium on clearly demonstrate that Desulfobacterium phenolicum benzoate. is a member of the genus Desulfobacula. It can be clearly distinguished from Desulfobacula toluolica by its motility, morphology, missing vitamin requirement Phylogenetic analyses and use of different electron donors for sulfate reduction. Therefore, we propose to rename it as The 16S rRNA gene sequence of strain SaxT shares Desulfobacula phenolica. less than 94n1% identity with the 16S rRNA gene sequences of other sulfate reducers of the δ-subclass of In view of the 16S rRNA gene sequence analyses the Proteobacteria (data not shown). The phylogenetic presented in this study strain SaxT should be regarded position of strain SaxT and its closest relatives is shown as belonging to a new genus, since it was not closely in Fig. 1. The closest affiliation was found to clone SB- related to other genera (a maximum of 94n1% 16S 9 (Phelps et al., 1998) with a similarity value of 96n6%, rRNA sequence identity). The distinct morphological followed by Desulfospira joergensenii (94n1%) (Finster and physiological properties (Table 2) in accordance et al., 1997) and Desulfobacterium phenolicum (93n9). with the FAME analysis (Table 1) argue in the same As can be seen in Fig. 1 the sequence of clone Sva0605, direction. Both Desulfobacula species and strain SaxT obtained from permanently cold sediments at contain relatively high amounts of the fatty acid 10-Me Svalbard, is in close proximity to these organisms 16:0, whereas this fatty acid is completely absent in (Ravenschlag et al., 1999). Clearly, they are all Desulfospira joergensenii (Finster et al., 1997). The members of the δ-subclass of the Proteobacteria. finding of this fatty acid in Desulfobacula toluolica is Furthermore, the comparative phylogenetic analyses consistent with the results obtained by van der Maarel indicated Desulfobacterium phenolicum to be a member et al. (1996). Strain SaxT can be distinguished from of the genus Desulfobacula. It was closely related to both Desulfobacula species by the high amount of the Desulfobacula toluolica (98n8% similarity of their 16S cyclopropane fatty acid 17:0cyc, which is also a rRNA sequence), which is the only species of this dominant fatty acid in Desulfobacter spp. Therefore, genus described so far (Fig. 1). Based on the sequence the designation Desulfotignum balticum gen. nov., sp. of the 16S rRNA, strain SaxT could not be affiliated nov. is proposed for strain SaxT.

International Journal of Systematic and Evolutionary Microbiology 51 175 J. Kuever and others

Ecological relevance of members of the genera acetate can accumulate. Electron acceptors used are Desulfobacula and Desulfotignum sulfate, sulfite and thiosulfate. Nitrate and nitrite are not used. Slow fermentative growth on pyruvate. T " The cellular fatty acid composition of strain Sax Addition of at least 10 g NaCl lV is necessary; op- Desulfobacter " shows the highest similarity to that of timum NaCl concentration for growth is 20 g lV , but postgatei " . It is distinguished from the composition of NaCl is tolerated up to 110 g lV . Vitamins are required Desulfobacula toluolica and Desulfobacula phenolica T for growth. Temperature requirements: Tmin,10mC; by the presence of 17:0cyc. Nevertheless, strain Sax , T , 28–32 C; T ,42 C. The pH range for growth Desulfobacula phenolica and Desulfobacula toluolica opt m max m is 6n5–8n2; optimum 7n3. The GjC content of the contain significant amounts of 10-Me 16:0 which was DNA is 62 4mol% (T ). The type strain is SaxT Desulfobacter n m previously described as a biomarker for ( DSM 7044T). spp. (Dowling et al., 1986) and was also found in lesser l amounts (! 10%) in Desulfobacterium autotrophicum (Vainshtein et al., 1992). Therefore, the use of this fatty ACKNOWLEDGEMENTS acid as a specific biomarker for micro-organisms belonging to the genus Desulfobacter should no longer We thank Ingrid Kunze for technical assistance, Hans- be considered reliable. The presence of this fatty acid Georg Tru$ per, Karl-Heinz Blotevogel for advice and help, and Geoff Mattison for linguistic improvements to the in marine sediment could also account for sulfate- manuscript. This work was in part funded by the Max- reducing bacteria belonging to the aforementioned Planck Society, Munich, Germany. genera.

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176 International Journal of Systematic and Evolutionary Microbiology 51 Desulfotignum balticum

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