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1 School of Pharmacy and Biomedical Sciences, University of Portsmouth, St Michael's Building, White Swan Rd, Portsmouth PO1 2DT, UK
2 Max-Planck-Institut für marine Mikrobiologie, Celsiusstr. 1, D-28359 Bremen, Germany
3 IWW, Rheinisch-Westfälisches Institut für Wasserchemie und Wassertechnologie, Moritzstr. 26, 45476 Mülheim/Ruhr, Germany
4 IFREMER, Centre de Brest, DRV/VP/BMH, BP 70, 29280 Plouzané, France
Correspondence
Iwona B. Beech
Iwona.Beech{at}port.ac.uk
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ABSTRACT |
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7c were the predominant species. Fatty acid profile and complete 16S rRNA gene sequencing demonstrated the similarity between strain Al1T and members of the genus Desulfovibrio. The position of strain Al1T within the phylogenetic tree indicated that it clustered closely with Desulfovibrio vietnamensis DSM 10520T (98·9 % sequence similarity), a strain recovered from a similar habitat. However, whole-cell protein profiles, Fourier-transform infrared studies and DNA–DNA hybridization demonstrated that, in spite of the high level of 16S rRNA gene sequence similarity, there is sufficient dissimilarity at the DNA sequence level between D. vietnamensis DSM 10520T and strain Al1T (10·2 % similarity) to propose that strain Al1T belongs to a separate species within the genus Desulfovibrio. Based on the results obtained, the name Desulfovibrio alaskensis sp. nov. is therefore proposed, with Al1T (=NCIMB 13491T=DSM 16109T) as the type strain.
Published online ahead of print on 26 March 2004 as DOI 10.1099/ijs.0.63118-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Al1T is Y11984.
The fatty acid profile and an AFM image of strain Al1T, FT-IR spectra of various SRB and a dendrogram based on these spectra are available as supplementary material in IJSEM Online.
Present address: IBVF – Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de la Cartuja, Av. Américo Vespucio 49, 41092 Sevilla, Spain. 
Present address: IIQ – Instituto de Investigaciones Químicas, Centro de Investigaciones Científicas Isla de la Cartuja, Av. Américo Vespucio 49, 41092 Sevilla, Spain.
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; Beech et al., 1994; Zinkevich et al., 1996), was isolated from material collected by E. van der Vende from a soured oil reservoir in Alaska (March 1991), a habitat with direct links to the marine environment, as the sea water from Purdu Bay was used in a secondary oil recovery system.
SRB enrichment was carried out using lactate as carbon source in marine Postgate medium B (Postgate, 1984) and purification was completed on semi-solid marine Postgate medium E (Postgate, 1984) as described elsewhere (Zinkevich et al., 1996). Cultures were maintained anaerobically at 37 °C as stationary batch cultures in marine Postgate medium C (Postgate, 1984).
Light microscopy and atomic force microscopy (AFM) were used to study cell morphology. A Leitz light microscope (Laborlux S) was used to determine the Gram reaction (Gregersen, 1978), as well as cell shape and motility. AFM imaging was conducted in a Discoverer TMX2000 SPM (Veeco Metrology Group) as described by Feio et al. (1998) and a micrograph is available as Supplementary Fig. A in IJSEM Online. The physiological characterization of isolate Al1T included determination of the temperature, pH and salinity ranges that allowed bacterial growth. These parameters were evaluated by growing cells in marine Postgate medium B for a period of 28 days under a range of conditions. The ability of cells to use different carbon and energy sources and electron donors was also tested. Results of the morphological and physiological characterization are given in Table 1 and summarized in the species description.
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). Lipids from lyophilized cells (50–100 mg) were extracted following a modified Bligh–Dyer method (White et al., 1979). The extracted lipids were fractionated into neutral lipids, glycolipids and polar lipids by silicic acid column chromatography using appropriate volumes of chloroform, acetone and methanol, respectively. Phospholipids were subjected to mild alkaline methanolysis and the resulting fatty acid methyl esters were purified by TLC, GC and GC-MS (Guezennec, 1991). The position and geometry of the double bond of each monounsaturated fatty acid were determined using dimethyl disulphide derivatives according to a procedure described previously (Nichols et al., 1986; Guezennec, 1991). The fatty acid profile of strain Al1T (Supplementary Table A) revealed considerable amounts of monounsaturated fatty acids (21·2 % total fatty acids), of which 10·4 % were isoC17 : 17c, a specific biomarker for the genus Desulfovibrio (Vainshtein et al., 1992).
SRB chromosomal DNA was obtained using the guanidine isothiocyanate method (Zinkevich & Beech, 2000) from cultures grown for 7 days at 37 °C in 10 ml marine Postgate medium B. 16S rRNA genes of purified genomic DNA were amplified by PCR using eubacterial universal primers (Lane, 1991). Appropriate PCR products were purified using the QIAquick PCR purification kit (Qiagen) and cloned according to standard methods (Sambrook et al., 1989) in pGEM-T Easy vector (Promega). Restriction enzymes used were obtained from New England Biolabs. Escherichia coli JM 109 (Promega; Messing et al., 1981) was used as a host strain for molecular cloning. E. coli JM 109 was grown in LB medium (Sambrook et al., 1989) and SOC medium (Promega) at 37 °C. The solid LB medium was supplemented with 100 µg ampicillin ml–1, 100 µg X-Gal ml–1 and 0·5 mM IPTG. Recombinant plasmid DNA was purified using a Qiagen plasmid mini kit. Both strands of the purified plasmid DNA (after restriction analysis) were sequenced by Cambridge BioSciences, Cambridge, UK.
The 16S rRNA gene sequence from Al1T was added to an alignment of about 15 000 homologous bacterial 16S rRNA gene sequences using the alignment tool of the ARB program package (Strunk et al., 1999). Phylogenetic trees were constructed using subsets of data that included representative sequences of members of the 
-Proteobacteria. Only sequences with at least 1300 nt were used. Distance matrix and maximum-likelihood methods, as implemented in the programs PHYLIP (Felsenstein, 1993), ARB and FASTDNAML (Maidak et al., 2000), were used.
The comparison between the 16S rRNA gene sequences of Al1T and some SRB strains of the genus Desulfovibrio revealed sequence similarities above 86 % with the majority of the species used. However, the closest relatives to strain Al1T were Desulfovibrio acrylicus DSM 10141T (89·0 %), Desulfovibrio vulgaris subsp. vulgaris Hildenborough ATCC 29579T (89·4 %) and Desulfovibrio vietnamensis DSM 10520T (98·9 %). This latter strain was recovered from the water phase of a crude oil storage tank of an offshore oil platform in Vietnam (Nga et al., 1996). The constructed phylogenetic trees were in good agreement with previously published ones (Devereux et al., 1990; Feio et al., 1998, 2000). Al1T and Desulfovibrio vietnamensis DSM 10520T formed a group in a lineage with an origin very close to the base of the family ‘Desulfovibrionaceae’ (Fig. 1). A 16S rRNA gene sequence similarity of 97 % is commonly considered as the upper limit for the definition of separate species (Stackebrandt & Goebel, 1994). Although more than 97 % similarity indicates that strains may belong to the same species, it is now generally acknowledged that this rule does not always apply. DNA–DNA analysis was therefore performed to determine whether or not Desulfovibrio vietnamensis DSM 10520T and Al1T were sufficiently dissimilar for the latter to be considered to belong to a novel species.
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. DNA–DNA hybridization was carried out as described by De Ley et al. (1970), with modifications reported by Huß et al. (1983) and Escara & Hutton (1980), using a model 2600 spectrophotometer equipped with a model 2527-R thermoprogrammer and plotter (Gilford Instrument Laboratories). Renaturation rates were computed with the program TRANSFER.BAS (Jahnke, 1992). In spite of the high similarity between strain Al1T and Desulfovibrio vietnamensis DSM 10520T observed at the 16S rRNA level, DNA–DNA hybridization revealed only 10·2 % relatedness. This result confirmed that the two strains are not related at the species level when the threshold value of 70 % for the definition of species is considered (Wayne et al., 1987). Furthermore, the observed difference in melting temperatures between the DNA of the two strains (11 °C) indicates considerable difference in their DNA base composition, further confirming that strain Al1T belongs to a novel species.
To further distinguish Desulfovibrio vietnamensis DSM 10520T and Al1T at the phenotypic level, whole-cell protein profiles and Fourier-transform infrared (FT-IR) spectroscopy analysis were performed. SDS-PAGE of whole-cell proteins is a rapid method for distinguishing bacterial species and has a similar level of discrimination to DNA–DNA hybridization (Jackman, 1987). Although there is not a genus-specific pattern (Jackman, 1987), differences in the protein patterns of whole cells reflect differences in the genomic content of the organism. Therefore, bacterial cells that are grown and recovered in an identical manner generate reproducible protein patterns, which can be used as fingerprints for their identification.
Whole-cell protein profile comparisons between Desulfovibrio vietnamensis DSM 10520T and strain Al1T were carried out with cultures grown under identical conditions in marine Postgate medium C for 5 days. Cells were harvested from 10 l batch cultures by centrifugation at 3000 g for 30 min. The pelleted cells were washed in 30 ml cold 50 mM MOPS buffer (pH 7·4) with 0·15 M NaCl and the pellet, after subsequent centrifugation at 3000 g for 30 min, was resuspended in 30 ml MOPS buffer. The cell preparation was then sonicated in a Soniprep 150 sonicator for 10x 1 min bursts at 16 µm amplitude with 30 s intervals. Any unbroken cells and remaining culture debris were then removed by centrifugation at 3500 g for 30 min and the supernatant was stored at –20 °C for whole-cell protein profile analysis. Protein profile analysis was performed by SDS-PAGE in 12·5 % T acrylamide gels according to the method of Laemmli (1970). Gels were stained with Coomassie brilliant blue R-250 (Sigma). The protein profiles obtained for the whole cells of Al1T and Desulfovibrio vietnamensis DSM 10520T (Fig. 2) clearly demonstrated dissimilarities, thus supporting the evidence that strain Al1T belongs to a novel species.
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; Schmitt & Flemming, 1998). It can also be used to discern different bacterial species or even strains, providing cultures are grown under identical conditions. Hence, three independent replicate cultures of the SRB species investigated were grown anaerobically in 10 ml vials in Postgate medium C at 37 °C. Cells were harvested after 2 days incubation by centrifugation at 5000 g. Pelleted cells were freeze-dried after washing with 0·9 % (w/v) NaCl solution. Control replicates of sterile media were also lyophilized and analysed. Spectra were collected using a Mattson RS/2 research series spectrometer (ThermoUnicam) and data were manipulated using WINFIRST software. All spectra were acquired in transmission mode, by the KBr disc method. In each case, cells (2 mg) were diluted in 200 mg KBr powder to achieve a 1 % (w/w) concentration before pressing the disc. After a spectral quality check, data treatment consisted of vector-normalization of the spectra derivatives for statistical evaluation and construction of dendrograms. The FT-IR spectra of Al1T, Desulfovibrio vietnamensis DSM 10520T, Desulfovibrio indonesiensis NCIMB 13468T, Desulfovibrio gabonensis DSM 10636T, Desulfovibrio gigas ATCC 19364T, Desulfovibrio desulfuricans ATCC 27774, Desulfovibrio vulgaris subs. vulgaris strain Hildenborough ATCC 29579T and Desulfovibrio vulgaris strain Woolwich NCIMB 8457 revealed considerable differences, mainly in the region between 1200 and 900 cm–1 (Supplementary Fig. B). This region is characterized by the presence of strain-specific bands that derive predominantly from the -C-O, -C-OH, -C-O-C and -C-O-P stretching vibrations. Statistical cluster analysis of the obtained FT-IR spectra, based on the bands at 1311 cm–1, the phosphate groups with a maximum at 1234 cm–1 and the -C-O, -C-O-C and -C-O-H stretching region with bands at 1160 cm–1, 1083 cm–1 and 969 cm–1, led to the construction of a dendrogram (Supplementary Fig. C). Despite the fact that the dendrogram reflects whole cell composition, it shows a remarkable agreement with phylogenetic trees constructed based on full 16S rRNA gene sequences. This analysis confirmed the high degree of similarity between strain Al1T and Desulfovibrio vietnamensis DSM 10520T but clearly established that they are separate species.
Desulfovibrio gigas ATCC 19364T, Desulfovibrio gabonensis DSM 10636T and Desulfovibrio indonesiensis NCIMB 13468T formed a separate group in FT-IR analysis. This grouping was based on the similarity of these strains in the region between 1200 and 900 cm–1, with a multiple band with peaks at 1128 cm–1, 1083 cm–1 and 1046 cm–1. Previous studies that did not include FT-IR analysis (Feio et al., 1998) placed these three Desulfovibrio strains in the same group, thus verifying the FT-IR data and validating the use of FT-IR spectroscopy of whole cells as a rapid and highly sensitive technique for identification and characterization of SRB.
Despite the high level of similarity found between the 16S rRNA gene sequences of strain Al1T and Desulfovibrio vietnamensis DSM 10520T and the similarities in the environment from which the two isolates were recovered, the remaining evidence, i.e. DNA–DNA hybridization, FT-IR analysis and whole-cell protein profiles, clearly demonstrates the difference between these two strains. Our data strongly indicate that strain Al1T represents a novel species belonging to the genus Desulfovibrio and classification of this isolate as a representative of a novel species, Desulfovibrio alaskensis sp. nov., is therefore proposed.
Description of Desulfovibrio alaskensis sp. nov.
Desulfovibrio alaskensis (al.ask.en'sis. N.L. masc. adj. alaskensis from Alaska, referring to the place of isolation).
Gram-negative, non-spore-forming, vibrio-shaped cells, 1·0–5·0x0·5–1·2 µm. Cells occur singly and are motile by means of a single polar flagellum. Grows at pH 6·5–8·5, 10–45 °C and in 0–10 % (w/v) NaCl. Maximum growth rate under optimal growth conditions in marine Postgate medium C [37 °C, pH 7·0 and 2·5 % (w/v) NaCl] using lactate as carbon source is 0·133 h–1. Vitamins are not required for growth. Strictly anaerobic, reduces sulphate, sulphite and thiosulphate, producing sulphide. Nitrate is not used as an electron acceptor. Substrates that are oxidized by sulphate reduction are lactate, pyruvate and succinate. Ethanol and butanol can be utilized fermentatively (for a limited number of generations). Desulfoviridin-type sulphite reductase is present. DNA G+C content is 64·1 mol%. Major cellular fatty acids are C18 : 0, isoC15 : 0 and isoC17 : 17c.
The type strain is Al1T (=NCIMB 13491T=DSM 16109T), isolated from the production fluids of offshore oilfields in Alaska.
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ACKNOWLEDGEMENTS |
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