| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Institute of Microbiology, Russian Academy of Sciences, Prospect 60-let Octyabrya 7/2, 117811 Moscow, Russia
2 Kluyver Laboratory of Biotechnology, Delft Technical University, Julianalaan 67, 2628 BC Delft, The Netherlands
3 Laboratory for Electron Microscopy, Biological Centre, University of Groningen, 9750 AA Haren, The Netherlands
Correspondence
Dimitry Yu. Sorokin
soroc{at}inmi.da.ru
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
Published online ahead of print on 21 March 2003 as DOI 10.1099/ijs.0.02615-0.
The GenBank accession number for the 16S rDNA sequence of Thialkalivibrio nitratireducens ALEN 2T is AY079010.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
; McHatton et al., 1996; Otte et al., 1999). Such SOB play an important role in mineral cycling and waste removal by linking the sulfur and nitrogen cycles (Kuenen & Robertson, 1987; Robertson & Kuenen, 1992). Of the haloalkaliphilic SOB isolated from soda lakes, currently represented by nearly 100 isolates (Sorokin et al., 2000, 2001a, 2002a, 2002b), only one, described as Thialkalivibrio denitrificans ALJDT, has the potential for anaerobic growth. This strain differs from its neutrophilic analogues Thiobacillus denitrificans and Thiomicrospira denitrificans in that it does not possess a dissimilatory nitrate reductase. It grows with nitrite or N2O as electron acceptor and thiosulfate or polysulfide as electron donor (Sorokin et al., 2001b). Recently, an enrichment culture has been obtained from a sediment sample from an alkaline hypersaline lake in Wadi Natrun (Egypt) that completely reduced nitrate to nitrogen gas, with thiosulfate as electron donor, at pH 10. In this report, the nitrate-reducing member of this haloalkaliphilic autotrophic denitrifying association is described.
Sediment from Lake Fazda, a hypersaline soda lake (250 g total salts l-1; pH 10) in the Wadi Natrun (Egypt), was used as inoculum to enrich for denitrifying SOB. The basic hydrochemical and microbiological characteristics of the Wadi Natrun lakes have been described by Taher (1999) and Imhoff et al. (1979), respectively. The enrichment was performed in 100 ml serum bottles with butyl-rubber stoppers filled with 50 ml alkaline base containing 0·6 M total Na+, pH 10 (Sorokin et al., 2001a), with 20 mM thiosulfate and 30 mM nitrate. Anoxic conditions were achieved by five cycles of evacuation–flushing with argon with active degassing of the liquid. Solid medium was prepared by 1 : 1 mixing of the above-mentioned alkaline base containing a double concentration of substrates and 4 % (w/v) agar at 50 °C. Anaerobic plate incubation was performed using closed jars filled with pure argon in the presence of anaerobic catalyst (Oxoid). Growth with H2 as electron donor was tested in 100 ml bottles closed with butyl-rubber stoppers containing 10 ml medium under an atmosphere of 98 % (v/v) H2 and 2 % (v/v) O2. Methylotrophy was tested with methanol and formate (5 mM) under aerobic and denitrifying conditions. The pH range for growth was tested on media prepared using 0·1 M HEPES/Na2CO3 (pH 7–8) supplemented with 0·6 M NaCl or sodium carbonate/sodium bicarbonate (pH 8–11) with 0·6 M total Na+. Na+ tolerance was tested on sodium carbonate/bicarbonate-based mineral medium containing 0·1–2·0 M total Na+ at pH 10. The same pH and salinity buffers were used in experiments with washed cells. Respiration activity, reduction of nitrogen oxides by washed cells, sulfur-metabolizing activity, denitrifying enzymes and cytochrome spectra were measured as described previously (Sorokin et al., 2002c). Inorganic sulfur compounds, nitrate, nitrite, N2O and protein concentrations were determined by spectrophotometric and GC methods as described previously (Sorokin et al., 2001a, b). DNA extraction, DNA G+C content determination and DNA–DNA hybridization were performed according to standard protocols (Marmur, 1961; De Ley et al., 1970). For amplification and sequencing of the 16S rRNA gene, DNA was obtained by standard phenol/chloroform extraction. The 16S rRNA gene was selectively amplified using primers 5'-AGAGTTTGATCCTGGCTCAG-3' (forward) and 5'-TACGGTTACCTTGTTACGACTT-3' (reverse). PCR products were purified from low-melting-point agarose using the Wizard PCR-Prep kit (Promega) according to the manufacturer's instructions. Almost-complete sequencing (1420 nt) was performed using the Promega Silver Sequencing kit according to the manufacturer's instructions, but with minor modifications. Primary comparative analysis of the 16S rRNA gene sequence of strain ALEN 2T with database sequences was done using BLAST. On the basis of results of the BLAST search, the 16S rRNA gene sequences of strain ALEN 2T and its closest relatives were aligned using CLUSTAL_X (Thompson et al., 1997). Regions that were not sequenced in one or more reference organisms were omitted from subsequent analyses. An unrooted phylogenetic tree based on 16S rRNA gene sequences of the studied bacteria was constructed by the neighbour-joining method available in the TREECON package (Van de Peer & De Wachter, 1994). Bootstrap analysis (100 replications) was used to validate reproducibility of the branching pattern of the tree.
The anaerobic enrichment resulted in a stable binary culture consisting of large irregular coccoid cells with intracellular sulfur globules and thin straight rods that were occasionally motile. The latter were responsible for nitrite reduction. On the basis of their physiology and genetic properties, the thin straight rods resembled the previously described haloalkaliphilic SOB Thialkalivibrio denitrificans (Sorokin et al., 2001b). Further work focused on isolation and characterization of the unusual coccoid morphotype that was responsible for the reduction of nitrate to nitrite.
The coccoid organism was present in relatively low numbers in the mixed denitrifying culture, which made it difficult to isolate. When plated onto nitrate/thiosulfate alkaline agar under an argon atmosphere, a surprisingly low proportion of the total cells present in liquid culture produced colonies. It appeared that the numerically dominant small rods depended on the coccoid phenotype for growth, resulting in formation of mixed colonies. By picking up the colony material containing mostly the coccoid morphotype and replating it, the colony number of the desired organism gradually increased. Eventually, pure colonies of the coccoid phenotype were obtained, and the isolated bacterium was designated strain ALEN 2T. It formed colonies of variable size and shape, 1–3 mm, some flat, some dome-like. The young colonies were shining white and full of sulfur, turning reddish and transparent with time. Cells in the colonies were extremely pleomorphic, mostly coccoid, with multiple intracellular sulfur globules. Cells grown aerobically on liquid medium with thiosulfate at pH 10 were coccoid, 0·8–2·0 µm in diameter and aggregated in chains of different lengths (Fig. 1a, c). The cells grown anaerobically in liquid culture were barrel-shaped and less aggregated (Fig. 1b, d).
|
), which accounts for a heavy sulfur accumulation in cultures.
|
ratio of 2·0–2·5 : 1. The latter implies a one-electron reduction of nitrite to NO. Although NO was not measured, at this stage some N2O in the headspace was registered. However, no anaerobic growth was observed with sulfide and nitrite or N2O as electron acceptors. From these experiments, it was concluded that strain ALEN 2T is a high-capacity nitrate-to-nitrite reducer and, therefore, may serve as a nitrite provider in a denitrifying association with a nitrite reducer. Further experiments with washed cells of strain ALEN 2T confirmed its inability to reduce nitrite and N2O with thiosulfate as electron donor. However, low nitrite-reducing activity was observed with polysulfide and H2 as electron donors (Table 2). In both cases, N2O was produced in the gas phase. Moreover, H2 also stimulated the reduction of elemental sulfur by washed cells of strain ALEN 2T. Despite these facts, the bacterium was incapable of anaerobic growth with H2 as electron donor and nitrite or elemental sulfur as electron acceptors, which implies that some important links between the dissimilatory enzymes and the energy-generating system are missing in this unusual alkaliphilic SOB species.
|
(mg protein)-1 min-1]. This implies that strain ALEN 2T possesses both nitrate and nitrite reductase activities despite the fact that it is unable to use nitrite as an electron acceptor in vivo. Antipov et al. (2003) have demonstrated the presence of a nitrate/nitrite reductase in strain ALEN 2T with unusual properties. Activities of the sulfur-metabolizing enzymes thiosulfate reductase, sulfite dehydrogenase and sulfide dehydrogenase were found in the soluble fraction of cell-free extract of strain ALEN 2T at pH 10 [690, 260 and 60 nmol (mg protein)-1 min-1, respectively]. The cell membranes of strain ALEN 2T contained high amounts of cytochrome types c and b (absorption maxima in the alpha region at 554 and 558 nm, respectively). CO difference spectra of the membranes indicated the presence of a cytochrome oxidase of type bb (troughs in alpha region at 558 and 563 nm).
The G+C content of the DNA of strain ALEN 2T was 64·8±0·5 mol%. Phylogenetic analysis placed this bacterium in the genus Thialkalivibrio (Fig. 2), which accommodates the high-G+C-containing species of haloalkaliphilic SOB (Sorokin et al., 2001a). The isolate had highest similarity (98·2 %) to the thiocyanate-utilizing species Thialkalivibrio paradoxus (Sorokin et al., 2002b). Indeed, strain ALEN 2T resembled this species in its specific cell morphology and accumulation of intracellular sulfur due to a low elemental sulfur oxidizing capacity. These two strains also had the highest level of DNA–DNA relatedness (54 %) compared to values obtained with other species of Thialkalivibrio (<30 %). However, strain ALEN 2T was substantially different physiologically from Thialkalivibrio paradoxus: strain ALEN 2T was incapable of thiocyanate and carbon disulfide oxidation, whereas Thialkalivibrio paradoxus cannot utilize nitrate either as electron acceptor nor as nitrogen source. Overall, these differences suggest that strain ALEN 2T should be regarded as a novel species of the genus Thialkalivibrio, for which the name Thialkalivibrio nitratireducens is proposed.
|
Cells are mostly coccoid or barrel-shaped, often in chains and aggregates, 0·8–2·0 µm in diameter, often with large sulfur globules inside. Non-motile. Colonies are up to 3 mm in size, full of sulfur, turning reddish with age. Obligately chemolithoautotrophic. Oxidizes thiosulfate, sulfide, polysulfide and, much less actively, elemental sulfur and tetrathionate to sulfate. Facultatively anaerobic. Grows anaerobically with nitrate as electron acceptor and thiosulfate, sulfide or polysulfide as electron donor. The sole product of nitrate reduction is nitrite. Obligately alkaliphilic and moderately halophilic. Genetically most closely related to a thiocyanate-oxidizing species, Thialkalivibrio paradoxus.
The type strain is ALEN 2T (=DSM 14787T=UNIQEM 213T), isolated from sediments of Lake Fazda (Wadi Natrun, Egypt), a hypersaline soda lake. Its DNA G+C content is 64·8±0·5 mol% (Tm method). Other properties are as for the genus.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]
Imhoff, J. F., Sahl, H. G., Soliman, G. S. H. & Trüper, H. G. (1979). The Wadi Natrun: chemical composition and microbial mass developments in alkaline brines of eutrophic desert lakes. Geomicrobiol J 1, 219–234.
Kuenen, J. G. & Robertson, L. A. (1987). Ecology of nitrification and denitrification. In The Nitrogen and Sulfur Cycles, pp. 162–218. Edited by J. A. Cole & S. J. Ferguson. Cambridge: Cambridge University Press.
Kuenen, J. G., Robertson, L. A. & Tuovinen, O. H. (1992). The genera Thiobacillus, Thiomicrospira and Thiosphaera. In The Prokaryotes, 2nd edn, pp. 2638–2657. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K.-H. Schleifer. New York: Springer.
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
McHatton, S. C., Barry, J. P., Jannasch, H. W. & Nelson, D. C. (1996). High nitrate concentrations in vacuolate, autotrophic marine Beggiatoa spp. Appl Environ Microbiol 62, 954–958.[Abstract]
Otte, S., Kuenen, J. G., Nielsen, L. P. & 7 other authors (1999). Nitrogen, carbon, and sulfur metabolism in natural Thioploca samples. Appl Environ Microbiol 65, 3148–3157.
Robertson, L. A. & Kuenen, J. G. (1992). The use of natural bacterial populations for the treatment of sulfur-containing wastewater. Biodegradation 3, 239–254.[CrossRef]
Sorokin, D. Y., Robertson, L. A. & Kuenen, J. G. (2000). Isolation and characterisation of obligately chemolithoautotrophic alkaliphilic sulfur-oxidizing bacteria. Antonie van Leeuwenhoek 77, 251–260.
Sorokin, D. Yu., Lysenko, A. M., Mityushina, L. L., Tourova, T. P., Jones, B. E., Rainey, F. A., Robertson, L. A. & Kuenen, J. G. (2001a). Thioalkalimicrobium aerophilum gen. nov., sp. nov. and Thioalkalimicrobium sibericum sp. nov., and Thioalkalivibrio versutus gen. nov., sp. nov., Thioalkalivibrio nitratis sp. nov. and Thioalkalivibrio denitrificans sp. nov., novel obligately alkaliphilic and obligately chemolithoautotrophic sulfur-oxidizing bacteria from soda lakes. Int J Syst Evol Microbiol 51, 565–580.[Abstract]
Sorokin, D. Yu., Kuenen, J. G. & Jetten, M. S. (2001b). Denitrification at extremely high pH values by the alkaliphilic, obligately chemolithoautotrophic, sulfur-oxidizing bacterium Thioalkalivibrio denitrificans strain ALJD. Arch Microbiol 175, 94–101.[CrossRef][Medline]
Sorokin, D. Yu., Gorlenko, V. M., Tourova, T. P., Tsapin, A. I., Nealson, K. H. & Kuenen, G. J. (2002a). Thioalkalimicrobium cyclicum sp. nov. and Thioalkalivibrio jannaschii sp. nov., novel species of haloalkaliphilic, obligately chemolithoautotrophic sulfur-oxidizing bacteria from hypersaline alkaline Mono Lake (California). Int J Syst Evol Microbiol 52, 913–920.[Abstract]
Sorokin, D. Yu., Tourova, T. P., Lysenko, A. M., Mityushina, L. L. & Kuenen, J. G. (2002b). Thioalkalivibrio thiocyanoxidans sp. nov. and Thioalkalivibrio paradoxus sp. nov., novel alkaliphilic, obligately autotrophic, sulfur-oxidizing bacteria capable of growth on thiocyanate, from soda lakes. Int J Syst Evol Microbiol 52, 657–664.[Abstract]
Sorokin, D. Yu., Tourova, T. P., Kolganova, T. V., Sjollema, K. A. & Kuenen, J. G. (2002c). Thioalkalispira microaerophila gen. nov., sp. nov., a novel lithoautotrophic, sulfur-oxidizing bacterium from a soda lake. Int J Syst Evol Microbiol 52, 2175–2182.[Abstract]
Taher, A. G. (1999). Inland saline lakes of Wadi El Natrun depression, Egypt. Int J Salt Lake Res 8, 149–170.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Van de Peer, Y. & De Wachter, R. (1994). TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci 10, 569–570.
This article has been cited by other articles:
|
|
 |
|
  D. Yu. Sorokin, T. P. Tourova, E. M. Spiridonova, F. A. Rainey, and G. Muyzer Thioclava pacifica gen. nov., sp. nov., a novel facultatively autotrophic, marine, sulfur-oxidizing bacterium from a near-shore sulfidic hydrothermal area Int J Syst Evol Microbiol, May 1, 2005; 55(3): 1069 - 1075. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
-Proteobacteria. Numbers on the branches refer to bootstrap values; only values above 90 % are included. Bar, 5 nt substitutions per 100 nt. Accession numbers are shown in parentheses.