International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1587–1592
DOI 10.1099/ijs.0.057281-0
Sulfurisoma sediminicola gen. nov., sp. nov., a facultative autotroph isolated from a freshwater lake Hisaya Kojima and Manabu Fukui Correspondence
The Institute of Low Temperature Science, Hokkaido University. Kita-19, Nishi-8, Kita-ku, Sapporo 060-0819, Japan
Hisaya Kojima [email protected]. jp
A novel facultatively autotrophic bacterium, strain BSN1T was isolated from sediment of a freshwater lake in Japan. The cells were rod-shaped, motile and Gram-stain-negative. As sole energy sources for autotrophic growth, the strain oxidized thiosulfate, elemental sulfur and hydrogen. Strain BSN1T was a facultative anaerobe utilizing nitrate as an electron acceptor. Growth was observed at temperatures lower than 34 6C, and the optimum growth was observed at 30–32 6C. The range of pH for growth was pH 6.8–8.8, and the optimum pH was pH 7.8–8.1. The optimum growth of the isolate occurred at concentrations of NaCl less than 50 mM. The G+C content of genomic DNA was 67 mol%. The major component in the fatty acid profile of strain BSN1T grown on fumarate was summed feature 3 (C16 : 1v7c and/or iso-C15 : 0 2-OH). Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain was a member of the class Betaproteobacteria, and it showed the highest sequence similarity with Georgfuchsia toluolica G5G6T (96.2 %). Phylogenetic analyses were also performed on genes involved in sulfur oxidation. On the basis of its phylogenetic and phenotypic properties, strain BSN1T (5DSM 26916T5NBRC 109412T) is proposed as the type strain of a novel species of a novel genus, Sulfurisoma sediminicola gen. nov., sp. nov.
Members of the family Rhodocyclaceae within the class Betaproteobacteria are diverse in physiological properties. Recently, a facultatively autotrophic member of this family was isolated and characterized, as Sulfuritalea hydrogenivorans sk43HT (Kojima & Fukui, 2011). This strain is capable of autotrophic growth on reduced sulfur species and hydrogen under anoxic conditions and utilizes organic acids as growth substrates. Such physiological properties are largely different from those of its closest relative, Georgfuchsia toluolica G5G6T, a strict anaerobe that degrades aromatic compounds by reducing Fe(III), Mn(IV) or nitrate (Weelink et al. 2009). In the present study, a novel sulfur oxidizer related to these organisms was isolated and characterized. Strain BSN1T was isolated from sediment of a freshwater lake in Japan, Lake Biwa. The sediment sample was obtained from a site with 90 m water depth. The basal medium used for enrichment and isolation was bicarbonate-buffered low-salt defined medium (Kojima & Fukui, 2011). The composition of the medium was as follows (l21): 0.2 g MgCl2 . 6H2O, 0.1 g CaCl2 . 2H2O, 0.1 g NH4Cl, 0.1 g KH2PO4, 0.1 g KCl, 1 ml trace element solution, 1 ml selenite–tungstate solution, 1 ml vitamin mixture solution, The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain is AB842427. The accession numbers for the soxB, dsrA, aprA and sqr genes are AB842428, AB842429, AB842430 and AB842431, respectively.
057281 G 2014 IUMS
Printed in Great Britain
1 ml vitamin B12 solution, 1 ml thiamine solution, 30 ml NaHCO3 solution and 1.5 ml Na2S2O3 solution. All stock solutions were prepared as described previously (Widdel & Bak, 1992). To establish the first enrichment, 0.5 ml sediment was inoculated into 70 ml medium. Into the culture, anoxic stock slurry of poorly crystalline Fe(III) oxide was added to obtain a final concentrations of 10 mM, as a scavenger of sulfide generated by thiosulfate disproportionation. The slurry was prepared by neutralizing FeCl3 solution with NaOH and repeated washing with pure water (Lovley, 2013). The head space of the bottle was filled with N2/CO2 (80 : 20 v/v), and incubation was performed at 15– 18 uC. After progression of disproportionation was confirmed by the change in colour of the precipitate in the culture (from reddish brown to black), a small portion of culture (1–2 % volume of fresh medium) was transferred to the medium of the same composition to obtain subsequent cultures. From the fifth culture of disproportionation, sulfur oxidizers were enriched by changing culture conditions. To enhance growth sulfur-oxidizing bacteria, elemental sulfur (approximately 0.5 g l21) and nitrate (20 mM) were added in the basal medium instead of Fe(III) oxide. After several transfers of the culture growing on elemental sulfur, the electron donor was further changed to 20 mM thiosulfate. Finally, the isolate was obtained by agar shake dilution (Widdel & Bak, 1992). Purity of the isolate was checked by phase-contrast light microscopy and sequencing of the 16S rRNA gene 1587
H. Kojima and M. Fukui
fragments amplified with several universal PCR primer pairs. The tests of Gram-staining, catalase activity and oxidase activity were performed as described previously (Kojima & Fukui, 2011). The G+C content of the genomic DNA was determined using HPLC methods as descried previously (Katayama-Fujimura et al., 1984). For the characterization of the strain, medium modified for aerobic culture was used unless otherwise specified. The composition of the modified medium was almost the same as that of the basal medium used for isolation, but it was buffered with 20 mM MOPS–NaOH and contained no NaHCO3. The aerobic cultures were incubated at 25 uC without shaking, and each experiment was performed in duplicate. Effects of salt concentration (0, 20, 50, 100, 150, 180 and 200 mM NaCl) and temperature (5, 8, 10, 15, 18, 22, 25, 28, 30, 32, 34, 36 and 37 uC) on growth of the strain were tested under aerobic conditions by using the MOPSbuffered medium (pH 7.5) amended with 2.5 mM sodium fumarate. Sensitivity to kanamycin (100 mg ml21) was tested in the same medium at 32 uC. The effect of pH on growth was tested with modified media containing 5 mM sodium fumarate as substrate, buffered with 20 mM MES (pH 5.7, 6.1, 6.2, 6.4, 6.6, 6.8 and 7.0), PIPES (pH 6.4, 6.8 and 7.1), MOPS (pH 6.6, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0 and 8.2) or Tricine (pH 7.7, 8.1, 8.2, 8.4, 8.7, 8.8 and 9.0). Utilization of organic substrates was tested in MOPS-buffered media, each containing one of the substances listed later. For comparison with the closest relative, growth on organic substrates was also tested under nitrate-reducing conditions, using bicarbonate-buffered basal medium. The capability of autotrophic growth on inorganic compounds was assessed with the same medium under a gas mixture (N2/CO2, 80 : 20). The utilization of electron acceptors was tested in the basal medium amended with 10 mM sodium acetate. Stock slurry of MnO2 was prepared by mixing solutions of KMnO4 and MnCl2 (Lovley, 2013). Growth in ordinary complex liquid media was tested for R2A, NB, LB and TSB at 32 uC without shaking. Growth in defined medium for the closest relative (Weelink et al., 2009) was tested with succinate and pyruvate as substrates.
by PCR and then sequenced directly. The fragments of soxB, dsrA and sqr genes were amplified with the primer pairs soxB 704F/1199R, dsrA 625F/877R and sqr 473F/ 982R, respectively (Luo et al., 2011). The fragments of aprA genes were amplified with Apr-1-FW/Apr-5-RV (Meyer & Kuever, 2007). The PCR conditions for these primer sets were as follows: initial denaturation for 2 min at 94 uC; 34 cycles of denaturation (30 s at 94 uC), annealing (30 s at 55 uC), and extension (45 s at 72 uC)); and the final extension for 10 min at 72 uC. All these primer sets were also tested with genomic DNA of the most closely related species, Sulfuritalea hydrogenivorans sk43HT and G. toluolica G5G6T. The genomic DNA of strain G5G6T was purchased from DSMZ and that of strain sk43HT was obtained from a culture maintained in our laboratory. With the genomic DNA samples of three strains, a primer set targeting bssA, bssA_f/bssA_r was also tested under the PCR conditions previously described (Botton et al., 2007). The gene encodes benzylsuccinate synthase, a key enzyme for anaerobic degradation of toluene and xylenes. Phylogenetic analyses of 16S rRNA gene and functional genes were performed as described previously (Kojima & Fukui, 2011). The cells of the obtained isolate BSN1T were motile Gramstain-negative rods (1.0–2.6 mm long and 0.4–0.6 mm wide). Spore formation was not observed. The tests for catalase and oxidase both gave positive results. Growth of the isolate was inhibited by kanamycin. The G+C content of the genomic DNA of the isolate was 67 mol%. The fatty acid profile of strain BSN1T grown on fumarate was characterized by outstanding abundance of summed feature 3 (C16 : 1v7c and/or iso-C15 : 0 2-OH; 70.5 %). The other fatty acids detected were C16 : 0 (14.3 %), C10 : 0 3-OH (6.9 %), C18 : 1v7c (4.5 %), C18 : 3v6c (1.7 %), C16 : 1v5c (1.1 %), C10 : 0 (0.7 %) and C14 : 0 (0.3 %).
A fragment of the 16S rRNA gene was amplified with the primer pair 27F and 1492R (Lane, 1991), and the resulting PCR product was directly sequenced. The PCR amplification was initiated with 2 min of denaturation at 94 uC, followed by 25 cycle of 30 s at 94 uC, 30 s at 55 uC, and 90 s at 72 uC. The final extension was carried out for 10 min at 72 uC. Partial fragments of four genes for sulfur oxidation (soxB encoding sulfate thioesterase/sulfate thiohydrolase, dsrA encoding dissimilatory sulfite reductase, aprA encoding adenosine-59-phosphosulfate reductase and sqr encoding sulfide–quinone oxidoreductase) were also amplified
The isolate could grow heterotrophically on the following substrates in the presence of oxygen or nitrate: pyruvate (5 mM), lactate (5 mM), acetate (5 mM), propionate (2.5 mM), succinate (2.5 mM), fumarate (2.5 mM) and malate (2.5 mM). The following substrates could not support aerobic or anaerobic growth of the novel strain: benzoate (2.5 mM), butyrate (2.5 mM), isobutyrate (2.5 mM), methanol (5 mM), ethanol (2.5 mM), formate (5 mM), citrate (5 mM), lactose (2.5 mM), glucose (2.5 mM), xylose (2.5 mM), phenol (2 mM), o-cresol (1 mM), m-cresol (1 mM) and toluene (250 mM). Under nitrate-reducing conditions, the isolate could grow chemolithotrophically on H2 (H2/N2/CO2 50 : 40 : 10, v/v; 200 kPa total pressure), thiosulfate (20 mM) and S0 (0.5 g l21). The end product of sulfur oxidation was sulfate. Sulfide (2 mM), sulfite (5 mM), or FeSO4 (20 mM) did not support autotrophic growth of the novel strain. The strain could utilize oxygen and nitrate (20 mM) as electron acceptors. During growth with nitrate reduction, gas production was observed and nitrite did not accumulate. Sulfite (5 mM), poorly crystalline Fe(III) oxide (10 mM), Fe(III)
1588
International Journal of Systematic and Evolutionary Microbiology 64
The fatty acid profile of the strain was analysed with cells aerobically grown on fumarate, harvested at stationary phase. The fatty acid analysis was performed at Techno Suruga (Shizuoka, Japan), by using the Sherlock Microbial Identification System (Version 6.0; database, TSBA40; MIDI).
Sulfurisoma sediminicola gen. nov., sp. nov.
87 53 53
0.02
Sulfurisoma sediminicola BSN1T (AB842427) Georgfuchsia toluolica G5G6T (EF219370) Sulfuritalea hydrogenivorans sk43HT (AB552842)
51
Denitratisoma oestradiolicum AcBE2-1T (AY879297)
94
Sterolibacterium denitrificans Chol-1ST (AJ306683) Methyloversatilis universalis FAM5T (DQ442273)
78
Rhodocyclus purpureus 6770T (M34132) Azospira oryzae N1 (DQ863512)
92
‘Dechloromonas aromatica’ RCB (AY032610) Thauera aromatica K172T (X77118) Escherichia coli ATCC 11775T (X80725)
Fig. 1. Phylogenetic position of BSN1T within the family Rhodocyclaceae, based on 16S rRNA gene sequence analysis. Escherichia coli is included as an outgroup. The tree was reconstructed with the minimum-evolution method using 1405 sites, and an identical tree was obtained with the neighbour-joining method. Numbers at nodes represent percentage values for 1000 bootstrap resamplings (values larger than 50 % are shown). Bar, 0.02 substitutions per nucleotide position.
nitrilotriacetic acid (10 mM) or MnO2 (20 mM) could not support anaerobic growth of the strain as electron acceptors. The strain exhibited no growth on R2A, NB, LB and TSB. The growth of strain BSN1T was observed over a temperature range between 34 uC and 8 uC, with an optimum at 30– 32 uC. The range of pH for growth was pH 6.8–8.8, and the optimum pH was pH 7.8–8.1. Growth of the isolate was observed in medium containing 100 mM or lower NaCl, and the optimum growth was observed with 0–50 mM NaCl. The 16S rRNA gene analysis revealed that strain BSN1T represented a member of the family Rhodocyclaceae within
57
0.1
94
the class Betaproteobacteria (Fig. 1). The characterized strain which showed highest sequence similarity (96.2 %) to strain BSN1T was G. toluolica G5G6T (Weelink et al., 2009). It has been reported that G. toluolica could not utilize carboxylic acids or hydrogen as electron donor. The next closest cultivated relative was Sulfuritalea hydrogenivorans sk43HT, a facultative autotroph (Kojima & Fukui, 2011). Similarity to Sulfuritalea hydrogenivorans was consistently observed in phylogenetic analyses of genes involved in sulfur oxidation, but novelty of the isolate was also shown by these analyses (Figs 2, 3, 4, 5). All the primer pairs used for these genes (soxB, dsrA, aprA and sqr)
Sulfuricella denitrificans skB26T (AB506458) ‘Thiobacillus plumbophilus’ DSM 6690 (EF618604) Sulfuritalea hydrogenivorans sk43HT (AB506458) Sulfurisoma sediminicola BSN1T (AB842428) Thiobacillus aquaesulis DSM 4255T (EF618597) Thiobacillus thioparus DSM 505T (AJ294326) Thiobacillus denitrificans ATCC 25259 (CP000116) 97 Thiobacillus denitrificans DSM 12475T (EF618607) 77 Herminiimonas arsenicoxydans ULPAs1T (CU207211) Leptothrix cholodnii SP-6 (CP001013) Methylibium petroleiphilum PM1T (CP000555) ‘Dechloromonas aromatica’ RCB (CP000089) Anaeromyxobacter dehalogenans 2CP-C (CP000251) Ralstonia solanacearum GMI1000 (AL646052) Cupriavidus taiwanensis LMG 19424 (CU633749) Ralstonia metallidurans CH34 (CP000352) 100 Thiovirga sulfuroxydans A7 (EF618610) Polaromonas sp. JS666 (CP000316) ‘Aquifex aeolicus’ VF5 (AE000657)
Fig. 2. Phylogenetic position of BSN1T based on soxB gene analysis. This minimum-evolution tree was reconstructed from amino acid sequences deduced from soxB gene sequences (149 amino acid positions were used). Aquifex aeolicus is included as an outgroup. Numbers at nodes are percentage values for 1000 bootstrap resamplings (values larger than 50 % are shown). Bar, 0.1 substitutions per amino acid position. http://ijs.sgmjournals.org
1589
H. Kojima and M. Fukui Burkholderiales bacterium JOSHI_001 (CM001438)
0.1
60
‘Sideroxydans lithotrophicus’ ES-1 (CP001965)
66
Sulfurisoma sediminicola BSN1T (AB842429) 99
65
Sulfuritalea hydrogenivorans sk43HT (AP012547) Sulfuricella denitrificans skB26T (AP013066) Thiobacillus thioparus DSM 505T (EU155054) 100
Thiobacillus denitrificans ATCC 25259 (CP000116) Chlorobaculum tepidum TLST (AE006470)
Fig. 3. Phylogenetic position of BSN1T based on dsrA gene analysis. This minimum-evolution tree was reconstructed from amino acid sequences deduced from dsrA gene sequences (82 amino acid positions were used). Chlorobaculum tepidum is included as an outgroup. Numbers at nodes are percentage values for 1000 bootstrap resamplings (values larger than 50 % are shown). Bar, 0.1 substitutions per amino acid position.
generated PCR products of the expected size from the genomic DNA of Sulfuritalea hydrogenivorans sk43HT, but not from that of G. toluolica G5G6T. In contrast, the primer pair for the bssA gene generated a PCR product from G. toluolica G5G6T, but not from strains BSN1T or sk43HT. The novel strain BSN1T showed high sequence divergence from its closest relative, enough to be regarded as a representative of novel species. According to the 16S rRNA gene-based phylogeny, only the genus Georgfuchsia can accommodate strain BSN1T without loss of monophyleticity (Fig. 1). However, physiological characteristics of the novel strain are clearly different from those of the sole species in
61
55
71
Description of Sulfurisoma gen. nov. Sulfurisoma (Sul. fu.ri.so9ma. L. neut. n. sulfur sulfur; Gr. neut. n. soma body; N.L. neut. n. Sulfurisoma sulfur-oxidizing body).
100 Thiodictyon bacillosum DSM 234T (EF641915) Thiocapsa roseopersicina DSM 217T (EF641907) 84 Thiorhodococcus minor DSM 11518T (EF641913) Thiocystis violacea DSM 207T (EF641912) Thiocystis violascens DSM 198T (EF641910) Thiocystis gelatinosa DSM 215T (EF641911) Thiocapsa pendens DSM 236T (EF641914) Lamprocystis purpurea DSM 4197T (EF641909) Thiothrix nivea DSM 5205 (EF641919) Thiothrix sp. 12730 (EF641918) Thiobacillus aquaesulis DSM 4255T (EF641916)
55
0.05
this genus. Strain BSN1T could not grow in the medium used for G. toluolica G5G6T, supplemented with succinate or pyruvate. In the respective media, these strains exhibited distinctly different physiological characters (Table 1). Furthermore, the PCR-based analysis also indicated significant functional differences between the two strains. All these results indicate that the novel strain should not be placed into any existing genus, and thus strain BSN1T is proposed to represent a novel species of a novel genus, with the name Sulfurisoma sediminicola gen. nov., sp. nov.
Sulfuricella denitrificans skB26T (AB506457) ‘Thiobacillus plumbophilus’ DSM 6690 (EF641917) Thiobacillus denitrificans DSM 807 (EF641922) Thiobacillus thioparus DSM 505T (EF641920) 63 Thiobacillus denitrificans DSM 739 (EF641923) 73 74 Thiobacillus denitrificans DSM 12475T (EF641924)
96
Thiodictyon sp.f4 (EF641921) Sulfuritalea hydrogenivorans sk43HT (AB552843) Sulfurisoma sediminicola BSN1T (AB842430) Chlorobium limicola DSM 257 (EF641903)
Fig. 4. Phylogenetic position of BSN1T based on aprA gene analysis. This minimum-evolution tree was reconstructed from amino acid sequences deduced from aprA gene sequences (119 amino acid positions were used). Chlorobium limicola is included as an outgroup. Numbers at nodes are percentage values for 1000 bootstrap resamplings (values larger than 50 % are shown). Bar, 0.05 substitutions per amino acid position. 1590
International Journal of Systematic and Evolutionary Microbiology 64
Sulfurisoma sediminicola gen. nov., sp. nov.
67 66 72
0.1
62 98
Thiobacilus denitrificans ATCC 25259 (CP000116) Thiobacillus thioparus DSM 505T (EF641920) Sulfurisoma sediminicola BSN1T (AB842431) Sulfuritalea hydrogenivorans sk43HT (AP012547) ‘Sideroxydans lithotrophicus’ ES-1 (CP001965)
64
Sulfuricella denitrificans skB26T (AP013066) Thiomonas intermedia K12 (CP002021) Burkholderiales bacterium JOSHI_001 (CM001438) Chlorobium tepidum TLST (AE006470)
Fig. 5. Phylogenetic position of BSN1T based on sqr gene analysis. This minimum-evolution tree was reconstructed from amino acid sequences deduced from sqr gene sequences (146 amino acid positions were used). Chlorobaculum tepidum is included as an outgroup. Numbers at nodes are percentage values for 1000 bootstrap resamplings. Bar, 0.1 substitutions per amino acid position.
Grows chemolithoautotrophically by the oxidation of thiosulfate, elemental sulfur and hydrogen. Heterotrophic growth occurs on organic acids. Based on 16S rRNA gene sequence analysis, phylogenetically affiliated to the class Betaproteobacteria. The type species is Sulfurisoma sediminicola.
Table 1. Differential physiological properties of BSN1T and the most closely related strain Strains: 1, BSN1T; 2, Georgfuchsia toluolica G5G6T. Data for G. toluolica were obtained from Weelink et al. (2009). For both strains, electron donors were tested with nitrate as the electron acceptor. Electron acceptors were tested with acetate and toluene, respectively. +, Positive; 2, negative. Characteristic Electron donors Acetate Lactate Succinate Fumarate Malate Propionate Pyruvate Phenol Toluene m-Cresol Hydrogen Electron acceptors Oxygen Fe(III) MnO2
http://ijs.sgmjournals.org
1
2
+ + + + + + + 2 2 2 +
2 2 2 2 2 2 2 + + + 2
+ 2 2
2 + +
Description of Sulfurisoma sediminicola sp. nov. Sulfurisoma sediminicola (se.di.mi.ni9co.la. L. n. sedimen inis sediment; L. suff. -cola inhabitant, dweller; N.L. n. sediminicola sediment-dweller, referring to the source of the type strain). Cells are Gram-stain-negative, rod-shaped, 1.0–2.6 mm in length and 0.4–0.6 mm in width. Facultatively anaerobic and reduce nitrate. Autotrophic growth occurs with oxidation of thiosulfate, elemental sulfur and hydrogen. Catalase-positive and oxidase-positive. Heterotrophic growth occurs on acetate, fumarate, lactate, malate, propionate, pyruvate and succinate. Growth occurs at temperatures lower than 34 uC, with optimum growth at 30–32 uC. The pH range for growth is pH 6.8–8.8, and optimum growth occurs at pH 7.8–8.1. The type strain BSN1T (5DSM 26916T5NBRC 109412T) was isolated from sediment of a freshwater lake in Japan. The G+C content of genomic DNA is 67 mol%.
Acknowledgements We thank K. Ishikawa and the crew members of the R/V Hakken-Go for their support in sampling of lake sediment. We also thank R. Tokizawa and M. Watanabe for technical assistance. This work was supported by a grant from the Institute for Fermentation, Osaka, to H. K.
References Botton, S., van Harmelen, M., Braster, M., Parsons, J. R. & Ro¨ling, W. F. M. (2007). Dominance of Geobacteraceae in BTX-degrading
enrichments from an iron-reducing aquifer. FEMS Microbiol Ecol 62, 118–130. Katayama-Fujimura, Y., Komatsu, Y., Kuraishi, H. & Kaneko, T. (1984). Estimation of DNA base composition by high-performance
liquid chromatography of its nuclease PI hydrolysate. Agric Biol Chem 48, 3169–3172. 1591
H. Kojima and M. Fukui Kojima, H. & Fukui, M. (2011). Sulfuritalea hydrogenivorans gen. nov., sp. nov., a facultative autotroph isolated from a freshwater lake. Int J Syst Evol Microbiol 61, 1651–1655. Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid
Meyer, B. & Kuever, J. (2007). Molecular analysis of the diversity of sulfate-reducing and sulfur-oxidizing prokaryotes in the environment, using aprA as functional marker gene. Appl Environ Microbiol 73, 7664–7679.
Techniques in Bacterial Systematics, pp. 115–175. Edited by E. Stackebrandt & M. Goodfellow. Chichester: Wiley.
Weelink, S. A., van Doesburg, W., Saia, F. T., Rijpstra, W. I., Ro¨ling, W. F., Smidt, H. & Stams, A. J. (2009). A strictly anaerobic
Lovley, D. R. (2013). Dissimilatory Fe(III)- and Mn(IV)-reducing
betaproteobacterium Georgfuchsia toluolica gen. nov., sp. nov. degrades aromatic compounds with Fe(III), Mn(IV) or nitrate as an electron acceptor. FEMS Microbiol Ecol 70, 575–585.
prokaryotes. In The Prokaryotes, 4th edn, pp. 287–308. Edited by E. Rosenberg, E. F. DeLong, S. Lory, E. Stackebrandt & F. Thompson. Berlin: Springer. of sulfur-oxidizing bacteria community in sulfide removing bioreactor. Appl Microbiol Biotechnol 90, 769–778.
Widdel, F. & Bak, F. (1992). Gram-negative mesotrophic sulfatereducing bacteria. In The Prokaryotes, 2nd edn, vol. 4, pp. 3352–3378. Edited by A. Balows, H. G. Tru¨per, M. Dworkin, W. Harder & K.-H. Schleifer. New York: Springer-Verlag.
1592
International Journal of Systematic and Evolutionary Microbiology 64
Luo, J. F., Lin, W. T. & Guo, Y. (2011). Functional genes based analysis
DOI 10.1099/ijs.0.057281-0
Sulfurisoma sediminicola gen. nov., sp. nov., a facultative autotroph isolated from a freshwater lake Hisaya Kojima and Manabu Fukui Correspondence
The Institute of Low Temperature Science, Hokkaido University. Kita-19, Nishi-8, Kita-ku, Sapporo 060-0819, Japan
Hisaya Kojima [email protected]. jp
A novel facultatively autotrophic bacterium, strain BSN1T was isolated from sediment of a freshwater lake in Japan. The cells were rod-shaped, motile and Gram-stain-negative. As sole energy sources for autotrophic growth, the strain oxidized thiosulfate, elemental sulfur and hydrogen. Strain BSN1T was a facultative anaerobe utilizing nitrate as an electron acceptor. Growth was observed at temperatures lower than 34 6C, and the optimum growth was observed at 30–32 6C. The range of pH for growth was pH 6.8–8.8, and the optimum pH was pH 7.8–8.1. The optimum growth of the isolate occurred at concentrations of NaCl less than 50 mM. The G+C content of genomic DNA was 67 mol%. The major component in the fatty acid profile of strain BSN1T grown on fumarate was summed feature 3 (C16 : 1v7c and/or iso-C15 : 0 2-OH). Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain was a member of the class Betaproteobacteria, and it showed the highest sequence similarity with Georgfuchsia toluolica G5G6T (96.2 %). Phylogenetic analyses were also performed on genes involved in sulfur oxidation. On the basis of its phylogenetic and phenotypic properties, strain BSN1T (5DSM 26916T5NBRC 109412T) is proposed as the type strain of a novel species of a novel genus, Sulfurisoma sediminicola gen. nov., sp. nov.
Members of the family Rhodocyclaceae within the class Betaproteobacteria are diverse in physiological properties. Recently, a facultatively autotrophic member of this family was isolated and characterized, as Sulfuritalea hydrogenivorans sk43HT (Kojima & Fukui, 2011). This strain is capable of autotrophic growth on reduced sulfur species and hydrogen under anoxic conditions and utilizes organic acids as growth substrates. Such physiological properties are largely different from those of its closest relative, Georgfuchsia toluolica G5G6T, a strict anaerobe that degrades aromatic compounds by reducing Fe(III), Mn(IV) or nitrate (Weelink et al. 2009). In the present study, a novel sulfur oxidizer related to these organisms was isolated and characterized. Strain BSN1T was isolated from sediment of a freshwater lake in Japan, Lake Biwa. The sediment sample was obtained from a site with 90 m water depth. The basal medium used for enrichment and isolation was bicarbonate-buffered low-salt defined medium (Kojima & Fukui, 2011). The composition of the medium was as follows (l21): 0.2 g MgCl2 . 6H2O, 0.1 g CaCl2 . 2H2O, 0.1 g NH4Cl, 0.1 g KH2PO4, 0.1 g KCl, 1 ml trace element solution, 1 ml selenite–tungstate solution, 1 ml vitamin mixture solution, The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain is AB842427. The accession numbers for the soxB, dsrA, aprA and sqr genes are AB842428, AB842429, AB842430 and AB842431, respectively.
057281 G 2014 IUMS
Printed in Great Britain
1 ml vitamin B12 solution, 1 ml thiamine solution, 30 ml NaHCO3 solution and 1.5 ml Na2S2O3 solution. All stock solutions were prepared as described previously (Widdel & Bak, 1992). To establish the first enrichment, 0.5 ml sediment was inoculated into 70 ml medium. Into the culture, anoxic stock slurry of poorly crystalline Fe(III) oxide was added to obtain a final concentrations of 10 mM, as a scavenger of sulfide generated by thiosulfate disproportionation. The slurry was prepared by neutralizing FeCl3 solution with NaOH and repeated washing with pure water (Lovley, 2013). The head space of the bottle was filled with N2/CO2 (80 : 20 v/v), and incubation was performed at 15– 18 uC. After progression of disproportionation was confirmed by the change in colour of the precipitate in the culture (from reddish brown to black), a small portion of culture (1–2 % volume of fresh medium) was transferred to the medium of the same composition to obtain subsequent cultures. From the fifth culture of disproportionation, sulfur oxidizers were enriched by changing culture conditions. To enhance growth sulfur-oxidizing bacteria, elemental sulfur (approximately 0.5 g l21) and nitrate (20 mM) were added in the basal medium instead of Fe(III) oxide. After several transfers of the culture growing on elemental sulfur, the electron donor was further changed to 20 mM thiosulfate. Finally, the isolate was obtained by agar shake dilution (Widdel & Bak, 1992). Purity of the isolate was checked by phase-contrast light microscopy and sequencing of the 16S rRNA gene 1587
H. Kojima and M. Fukui
fragments amplified with several universal PCR primer pairs. The tests of Gram-staining, catalase activity and oxidase activity were performed as described previously (Kojima & Fukui, 2011). The G+C content of the genomic DNA was determined using HPLC methods as descried previously (Katayama-Fujimura et al., 1984). For the characterization of the strain, medium modified for aerobic culture was used unless otherwise specified. The composition of the modified medium was almost the same as that of the basal medium used for isolation, but it was buffered with 20 mM MOPS–NaOH and contained no NaHCO3. The aerobic cultures were incubated at 25 uC without shaking, and each experiment was performed in duplicate. Effects of salt concentration (0, 20, 50, 100, 150, 180 and 200 mM NaCl) and temperature (5, 8, 10, 15, 18, 22, 25, 28, 30, 32, 34, 36 and 37 uC) on growth of the strain were tested under aerobic conditions by using the MOPSbuffered medium (pH 7.5) amended with 2.5 mM sodium fumarate. Sensitivity to kanamycin (100 mg ml21) was tested in the same medium at 32 uC. The effect of pH on growth was tested with modified media containing 5 mM sodium fumarate as substrate, buffered with 20 mM MES (pH 5.7, 6.1, 6.2, 6.4, 6.6, 6.8 and 7.0), PIPES (pH 6.4, 6.8 and 7.1), MOPS (pH 6.6, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0 and 8.2) or Tricine (pH 7.7, 8.1, 8.2, 8.4, 8.7, 8.8 and 9.0). Utilization of organic substrates was tested in MOPS-buffered media, each containing one of the substances listed later. For comparison with the closest relative, growth on organic substrates was also tested under nitrate-reducing conditions, using bicarbonate-buffered basal medium. The capability of autotrophic growth on inorganic compounds was assessed with the same medium under a gas mixture (N2/CO2, 80 : 20). The utilization of electron acceptors was tested in the basal medium amended with 10 mM sodium acetate. Stock slurry of MnO2 was prepared by mixing solutions of KMnO4 and MnCl2 (Lovley, 2013). Growth in ordinary complex liquid media was tested for R2A, NB, LB and TSB at 32 uC without shaking. Growth in defined medium for the closest relative (Weelink et al., 2009) was tested with succinate and pyruvate as substrates.
by PCR and then sequenced directly. The fragments of soxB, dsrA and sqr genes were amplified with the primer pairs soxB 704F/1199R, dsrA 625F/877R and sqr 473F/ 982R, respectively (Luo et al., 2011). The fragments of aprA genes were amplified with Apr-1-FW/Apr-5-RV (Meyer & Kuever, 2007). The PCR conditions for these primer sets were as follows: initial denaturation for 2 min at 94 uC; 34 cycles of denaturation (30 s at 94 uC), annealing (30 s at 55 uC), and extension (45 s at 72 uC)); and the final extension for 10 min at 72 uC. All these primer sets were also tested with genomic DNA of the most closely related species, Sulfuritalea hydrogenivorans sk43HT and G. toluolica G5G6T. The genomic DNA of strain G5G6T was purchased from DSMZ and that of strain sk43HT was obtained from a culture maintained in our laboratory. With the genomic DNA samples of three strains, a primer set targeting bssA, bssA_f/bssA_r was also tested under the PCR conditions previously described (Botton et al., 2007). The gene encodes benzylsuccinate synthase, a key enzyme for anaerobic degradation of toluene and xylenes. Phylogenetic analyses of 16S rRNA gene and functional genes were performed as described previously (Kojima & Fukui, 2011). The cells of the obtained isolate BSN1T were motile Gramstain-negative rods (1.0–2.6 mm long and 0.4–0.6 mm wide). Spore formation was not observed. The tests for catalase and oxidase both gave positive results. Growth of the isolate was inhibited by kanamycin. The G+C content of the genomic DNA of the isolate was 67 mol%. The fatty acid profile of strain BSN1T grown on fumarate was characterized by outstanding abundance of summed feature 3 (C16 : 1v7c and/or iso-C15 : 0 2-OH; 70.5 %). The other fatty acids detected were C16 : 0 (14.3 %), C10 : 0 3-OH (6.9 %), C18 : 1v7c (4.5 %), C18 : 3v6c (1.7 %), C16 : 1v5c (1.1 %), C10 : 0 (0.7 %) and C14 : 0 (0.3 %).
A fragment of the 16S rRNA gene was amplified with the primer pair 27F and 1492R (Lane, 1991), and the resulting PCR product was directly sequenced. The PCR amplification was initiated with 2 min of denaturation at 94 uC, followed by 25 cycle of 30 s at 94 uC, 30 s at 55 uC, and 90 s at 72 uC. The final extension was carried out for 10 min at 72 uC. Partial fragments of four genes for sulfur oxidation (soxB encoding sulfate thioesterase/sulfate thiohydrolase, dsrA encoding dissimilatory sulfite reductase, aprA encoding adenosine-59-phosphosulfate reductase and sqr encoding sulfide–quinone oxidoreductase) were also amplified
The isolate could grow heterotrophically on the following substrates in the presence of oxygen or nitrate: pyruvate (5 mM), lactate (5 mM), acetate (5 mM), propionate (2.5 mM), succinate (2.5 mM), fumarate (2.5 mM) and malate (2.5 mM). The following substrates could not support aerobic or anaerobic growth of the novel strain: benzoate (2.5 mM), butyrate (2.5 mM), isobutyrate (2.5 mM), methanol (5 mM), ethanol (2.5 mM), formate (5 mM), citrate (5 mM), lactose (2.5 mM), glucose (2.5 mM), xylose (2.5 mM), phenol (2 mM), o-cresol (1 mM), m-cresol (1 mM) and toluene (250 mM). Under nitrate-reducing conditions, the isolate could grow chemolithotrophically on H2 (H2/N2/CO2 50 : 40 : 10, v/v; 200 kPa total pressure), thiosulfate (20 mM) and S0 (0.5 g l21). The end product of sulfur oxidation was sulfate. Sulfide (2 mM), sulfite (5 mM), or FeSO4 (20 mM) did not support autotrophic growth of the novel strain. The strain could utilize oxygen and nitrate (20 mM) as electron acceptors. During growth with nitrate reduction, gas production was observed and nitrite did not accumulate. Sulfite (5 mM), poorly crystalline Fe(III) oxide (10 mM), Fe(III)
1588
International Journal of Systematic and Evolutionary Microbiology 64
The fatty acid profile of the strain was analysed with cells aerobically grown on fumarate, harvested at stationary phase. The fatty acid analysis was performed at Techno Suruga (Shizuoka, Japan), by using the Sherlock Microbial Identification System (Version 6.0; database, TSBA40; MIDI).
Sulfurisoma sediminicola gen. nov., sp. nov.
87 53 53
0.02
Sulfurisoma sediminicola BSN1T (AB842427) Georgfuchsia toluolica G5G6T (EF219370) Sulfuritalea hydrogenivorans sk43HT (AB552842)
51
Denitratisoma oestradiolicum AcBE2-1T (AY879297)
94
Sterolibacterium denitrificans Chol-1ST (AJ306683) Methyloversatilis universalis FAM5T (DQ442273)
78
Rhodocyclus purpureus 6770T (M34132) Azospira oryzae N1 (DQ863512)
92
‘Dechloromonas aromatica’ RCB (AY032610) Thauera aromatica K172T (X77118) Escherichia coli ATCC 11775T (X80725)
Fig. 1. Phylogenetic position of BSN1T within the family Rhodocyclaceae, based on 16S rRNA gene sequence analysis. Escherichia coli is included as an outgroup. The tree was reconstructed with the minimum-evolution method using 1405 sites, and an identical tree was obtained with the neighbour-joining method. Numbers at nodes represent percentage values for 1000 bootstrap resamplings (values larger than 50 % are shown). Bar, 0.02 substitutions per nucleotide position.
nitrilotriacetic acid (10 mM) or MnO2 (20 mM) could not support anaerobic growth of the strain as electron acceptors. The strain exhibited no growth on R2A, NB, LB and TSB. The growth of strain BSN1T was observed over a temperature range between 34 uC and 8 uC, with an optimum at 30– 32 uC. The range of pH for growth was pH 6.8–8.8, and the optimum pH was pH 7.8–8.1. Growth of the isolate was observed in medium containing 100 mM or lower NaCl, and the optimum growth was observed with 0–50 mM NaCl. The 16S rRNA gene analysis revealed that strain BSN1T represented a member of the family Rhodocyclaceae within
57
0.1
94
the class Betaproteobacteria (Fig. 1). The characterized strain which showed highest sequence similarity (96.2 %) to strain BSN1T was G. toluolica G5G6T (Weelink et al., 2009). It has been reported that G. toluolica could not utilize carboxylic acids or hydrogen as electron donor. The next closest cultivated relative was Sulfuritalea hydrogenivorans sk43HT, a facultative autotroph (Kojima & Fukui, 2011). Similarity to Sulfuritalea hydrogenivorans was consistently observed in phylogenetic analyses of genes involved in sulfur oxidation, but novelty of the isolate was also shown by these analyses (Figs 2, 3, 4, 5). All the primer pairs used for these genes (soxB, dsrA, aprA and sqr)
Sulfuricella denitrificans skB26T (AB506458) ‘Thiobacillus plumbophilus’ DSM 6690 (EF618604) Sulfuritalea hydrogenivorans sk43HT (AB506458) Sulfurisoma sediminicola BSN1T (AB842428) Thiobacillus aquaesulis DSM 4255T (EF618597) Thiobacillus thioparus DSM 505T (AJ294326) Thiobacillus denitrificans ATCC 25259 (CP000116) 97 Thiobacillus denitrificans DSM 12475T (EF618607) 77 Herminiimonas arsenicoxydans ULPAs1T (CU207211) Leptothrix cholodnii SP-6 (CP001013) Methylibium petroleiphilum PM1T (CP000555) ‘Dechloromonas aromatica’ RCB (CP000089) Anaeromyxobacter dehalogenans 2CP-C (CP000251) Ralstonia solanacearum GMI1000 (AL646052) Cupriavidus taiwanensis LMG 19424 (CU633749) Ralstonia metallidurans CH34 (CP000352) 100 Thiovirga sulfuroxydans A7 (EF618610) Polaromonas sp. JS666 (CP000316) ‘Aquifex aeolicus’ VF5 (AE000657)
Fig. 2. Phylogenetic position of BSN1T based on soxB gene analysis. This minimum-evolution tree was reconstructed from amino acid sequences deduced from soxB gene sequences (149 amino acid positions were used). Aquifex aeolicus is included as an outgroup. Numbers at nodes are percentage values for 1000 bootstrap resamplings (values larger than 50 % are shown). Bar, 0.1 substitutions per amino acid position. http://ijs.sgmjournals.org
1589
H. Kojima and M. Fukui Burkholderiales bacterium JOSHI_001 (CM001438)
0.1
60
‘Sideroxydans lithotrophicus’ ES-1 (CP001965)
66
Sulfurisoma sediminicola BSN1T (AB842429) 99
65
Sulfuritalea hydrogenivorans sk43HT (AP012547) Sulfuricella denitrificans skB26T (AP013066) Thiobacillus thioparus DSM 505T (EU155054) 100
Thiobacillus denitrificans ATCC 25259 (CP000116) Chlorobaculum tepidum TLST (AE006470)
Fig. 3. Phylogenetic position of BSN1T based on dsrA gene analysis. This minimum-evolution tree was reconstructed from amino acid sequences deduced from dsrA gene sequences (82 amino acid positions were used). Chlorobaculum tepidum is included as an outgroup. Numbers at nodes are percentage values for 1000 bootstrap resamplings (values larger than 50 % are shown). Bar, 0.1 substitutions per amino acid position.
generated PCR products of the expected size from the genomic DNA of Sulfuritalea hydrogenivorans sk43HT, but not from that of G. toluolica G5G6T. In contrast, the primer pair for the bssA gene generated a PCR product from G. toluolica G5G6T, but not from strains BSN1T or sk43HT. The novel strain BSN1T showed high sequence divergence from its closest relative, enough to be regarded as a representative of novel species. According to the 16S rRNA gene-based phylogeny, only the genus Georgfuchsia can accommodate strain BSN1T without loss of monophyleticity (Fig. 1). However, physiological characteristics of the novel strain are clearly different from those of the sole species in
61
55
71
Description of Sulfurisoma gen. nov. Sulfurisoma (Sul. fu.ri.so9ma. L. neut. n. sulfur sulfur; Gr. neut. n. soma body; N.L. neut. n. Sulfurisoma sulfur-oxidizing body).
100 Thiodictyon bacillosum DSM 234T (EF641915) Thiocapsa roseopersicina DSM 217T (EF641907) 84 Thiorhodococcus minor DSM 11518T (EF641913) Thiocystis violacea DSM 207T (EF641912) Thiocystis violascens DSM 198T (EF641910) Thiocystis gelatinosa DSM 215T (EF641911) Thiocapsa pendens DSM 236T (EF641914) Lamprocystis purpurea DSM 4197T (EF641909) Thiothrix nivea DSM 5205 (EF641919) Thiothrix sp. 12730 (EF641918) Thiobacillus aquaesulis DSM 4255T (EF641916)
55
0.05
this genus. Strain BSN1T could not grow in the medium used for G. toluolica G5G6T, supplemented with succinate or pyruvate. In the respective media, these strains exhibited distinctly different physiological characters (Table 1). Furthermore, the PCR-based analysis also indicated significant functional differences between the two strains. All these results indicate that the novel strain should not be placed into any existing genus, and thus strain BSN1T is proposed to represent a novel species of a novel genus, with the name Sulfurisoma sediminicola gen. nov., sp. nov.
Sulfuricella denitrificans skB26T (AB506457) ‘Thiobacillus plumbophilus’ DSM 6690 (EF641917) Thiobacillus denitrificans DSM 807 (EF641922) Thiobacillus thioparus DSM 505T (EF641920) 63 Thiobacillus denitrificans DSM 739 (EF641923) 73 74 Thiobacillus denitrificans DSM 12475T (EF641924)
96
Thiodictyon sp.f4 (EF641921) Sulfuritalea hydrogenivorans sk43HT (AB552843) Sulfurisoma sediminicola BSN1T (AB842430) Chlorobium limicola DSM 257 (EF641903)
Fig. 4. Phylogenetic position of BSN1T based on aprA gene analysis. This minimum-evolution tree was reconstructed from amino acid sequences deduced from aprA gene sequences (119 amino acid positions were used). Chlorobium limicola is included as an outgroup. Numbers at nodes are percentage values for 1000 bootstrap resamplings (values larger than 50 % are shown). Bar, 0.05 substitutions per amino acid position. 1590
International Journal of Systematic and Evolutionary Microbiology 64
Sulfurisoma sediminicola gen. nov., sp. nov.
67 66 72
0.1
62 98
Thiobacilus denitrificans ATCC 25259 (CP000116) Thiobacillus thioparus DSM 505T (EF641920) Sulfurisoma sediminicola BSN1T (AB842431) Sulfuritalea hydrogenivorans sk43HT (AP012547) ‘Sideroxydans lithotrophicus’ ES-1 (CP001965)
64
Sulfuricella denitrificans skB26T (AP013066) Thiomonas intermedia K12 (CP002021) Burkholderiales bacterium JOSHI_001 (CM001438) Chlorobium tepidum TLST (AE006470)
Fig. 5. Phylogenetic position of BSN1T based on sqr gene analysis. This minimum-evolution tree was reconstructed from amino acid sequences deduced from sqr gene sequences (146 amino acid positions were used). Chlorobaculum tepidum is included as an outgroup. Numbers at nodes are percentage values for 1000 bootstrap resamplings. Bar, 0.1 substitutions per amino acid position.
Grows chemolithoautotrophically by the oxidation of thiosulfate, elemental sulfur and hydrogen. Heterotrophic growth occurs on organic acids. Based on 16S rRNA gene sequence analysis, phylogenetically affiliated to the class Betaproteobacteria. The type species is Sulfurisoma sediminicola.
Table 1. Differential physiological properties of BSN1T and the most closely related strain Strains: 1, BSN1T; 2, Georgfuchsia toluolica G5G6T. Data for G. toluolica were obtained from Weelink et al. (2009). For both strains, electron donors were tested with nitrate as the electron acceptor. Electron acceptors were tested with acetate and toluene, respectively. +, Positive; 2, negative. Characteristic Electron donors Acetate Lactate Succinate Fumarate Malate Propionate Pyruvate Phenol Toluene m-Cresol Hydrogen Electron acceptors Oxygen Fe(III) MnO2
http://ijs.sgmjournals.org
1
2
+ + + + + + + 2 2 2 +
2 2 2 2 2 2 2 + + + 2
+ 2 2
2 + +
Description of Sulfurisoma sediminicola sp. nov. Sulfurisoma sediminicola (se.di.mi.ni9co.la. L. n. sedimen inis sediment; L. suff. -cola inhabitant, dweller; N.L. n. sediminicola sediment-dweller, referring to the source of the type strain). Cells are Gram-stain-negative, rod-shaped, 1.0–2.6 mm in length and 0.4–0.6 mm in width. Facultatively anaerobic and reduce nitrate. Autotrophic growth occurs with oxidation of thiosulfate, elemental sulfur and hydrogen. Catalase-positive and oxidase-positive. Heterotrophic growth occurs on acetate, fumarate, lactate, malate, propionate, pyruvate and succinate. Growth occurs at temperatures lower than 34 uC, with optimum growth at 30–32 uC. The pH range for growth is pH 6.8–8.8, and optimum growth occurs at pH 7.8–8.1. The type strain BSN1T (5DSM 26916T5NBRC 109412T) was isolated from sediment of a freshwater lake in Japan. The G+C content of genomic DNA is 67 mol%.
Acknowledgements We thank K. Ishikawa and the crew members of the R/V Hakken-Go for their support in sampling of lake sediment. We also thank R. Tokizawa and M. Watanabe for technical assistance. This work was supported by a grant from the Institute for Fermentation, Osaka, to H. K.
References Botton, S., van Harmelen, M., Braster, M., Parsons, J. R. & Ro¨ling, W. F. M. (2007). Dominance of Geobacteraceae in BTX-degrading
enrichments from an iron-reducing aquifer. FEMS Microbiol Ecol 62, 118–130. Katayama-Fujimura, Y., Komatsu, Y., Kuraishi, H. & Kaneko, T. (1984). Estimation of DNA base composition by high-performance
liquid chromatography of its nuclease PI hydrolysate. Agric Biol Chem 48, 3169–3172. 1591
H. Kojima and M. Fukui Kojima, H. & Fukui, M. (2011). Sulfuritalea hydrogenivorans gen. nov., sp. nov., a facultative autotroph isolated from a freshwater lake. Int J Syst Evol Microbiol 61, 1651–1655. Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid
Meyer, B. & Kuever, J. (2007). Molecular analysis of the diversity of sulfate-reducing and sulfur-oxidizing prokaryotes in the environment, using aprA as functional marker gene. Appl Environ Microbiol 73, 7664–7679.
Techniques in Bacterial Systematics, pp. 115–175. Edited by E. Stackebrandt & M. Goodfellow. Chichester: Wiley.
Weelink, S. A., van Doesburg, W., Saia, F. T., Rijpstra, W. I., Ro¨ling, W. F., Smidt, H. & Stams, A. J. (2009). A strictly anaerobic
Lovley, D. R. (2013). Dissimilatory Fe(III)- and Mn(IV)-reducing
betaproteobacterium Georgfuchsia toluolica gen. nov., sp. nov. degrades aromatic compounds with Fe(III), Mn(IV) or nitrate as an electron acceptor. FEMS Microbiol Ecol 70, 575–585.
prokaryotes. In The Prokaryotes, 4th edn, pp. 287–308. Edited by E. Rosenberg, E. F. DeLong, S. Lory, E. Stackebrandt & F. Thompson. Berlin: Springer. of sulfur-oxidizing bacteria community in sulfide removing bioreactor. Appl Microbiol Biotechnol 90, 769–778.
Widdel, F. & Bak, F. (1992). Gram-negative mesotrophic sulfatereducing bacteria. In The Prokaryotes, 2nd edn, vol. 4, pp. 3352–3378. Edited by A. Balows, H. G. Tru¨per, M. Dworkin, W. Harder & K.-H. Schleifer. New York: Springer-Verlag.
1592
International Journal of Systematic and Evolutionary Microbiology 64
Luo, J. F., Lin, W. T. & Guo, Y. (2011). Functional genes based analysis
