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1 NASA/NSSTC, SD-50, 320 Sparkman Dr., Huntsville, AL 35805, USA
2 Department of Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
3 Laboratory for Structural Biology, The University of Alabama in Huntsville, MSB, Huntsville, AL 35899, USA
4 Department of Microbiology, University of Georgia, Athens, GA 30602-2605, USA
5 American Type Culture Collection, 10801 University Blvd, Manassas, VA 20110, USA
Correspondence
Richard B. Hoover
Richard.B.Hoover{at}NASA.GOV
Elena V. Pikuta
Elena.Pikuta{at}msfc.nasa.gov
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ABSTRACT |
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The GenBank accession number for the 16S rDNA sequence of Spirochaeta americana ASpG1T is AF373921.
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, 1984). Analysis of 16S rRNA gene sequences has shown that the spirochaetes represent a monophyletic phylum within the Bacteria (Paster et al., 1991). At the time of writing, the genus Spirochaeta contained 13 species of bacteria. These organisms occur in a wide variety of freshwater and marine environments (Canale-Parola, 1992), such as aquatic habitats (Spirochaeta aurantia) (Breznak & Canale-Parola, 1975), freshwater mud (Spirochaeta zuelzerae, Spirochaeta stenostrepta) (Canale-Parola, 1980), marine mud (Spirochaeta isovalerica, Spirochaeta litoralis) (Hespell & Canale-Parola, 1970; Harwood & Canale-Parola, 1983), oilfields (Spirochaeta smaragdinae) (Magot et al., 1997) and microbial mats (Teal et al., 1996). Some members of the genus Spirochaeta are microbial extremophiles that inhabit environments with extremes of salinity, alkalinity, pressure and/or temperature. The spirochaete extremophiles may harbour important enzymes and proteins and, consequently, are of particular interest to biotechnologists (Bermudes et al., 1987; Barth et al., 1991; Munson et al., 1993).
Prior studies have shown that some species of the genus Spirochaeta are naturally resistant to sulfide concentrations at levels that inhibit the growth of many other micro-organisms. The mesophilic halophile Spirochaeta halophila was isolated from black mud (smelling strongly of H2S) of Solar Lake, a high-salinity pond on the Sinai shore of the Gulf of Aqaba (Greenberg & Canale-Parola, 1976). The obligate anaerobe strain GS-2 is a mesophilic barophile that was isolated from sulfide-rich mud samples collected at a depth of 2550 m near deep-sea hydrothermal vents at the Galapagos Rift ocean-floor spreading centre (Harwood et al., 1982). ‘Spirochaeta bajacaliforniensis’ (Fracek & Stolz, 1985) is another anaerobic marine spirochaete that was isolated from a microbial mat community in sulfide-rich mud at Laguna Figueroa, Baja California. It was suggested that this might be a strain of S. litoralis, since it had almost identical physiological characteristics and a similar DNA G+C content to S. litoralis (Canale-Parola, 1992). However, 16S rRNA gene sequence data indicate that ‘S. bajacaliforniensis’ and S. litoralis do not represent the same species, as a pairwise comparison shows only 83·8 % similarity between the two species (data from this study).
Other spirochaete extremophiles include anaerobic, thermophilic spirochaetes from hot springs in New Zealand (Patel et al., 1985), moderately thermophilic Spirochaeta caldaria from cyanobacterial mats of freshwater hot springs in Oregon and Utah (Pohlschroeder et al., 1994), and extremly thermophilic Spirochaeta thermophila from a marine littoral hydrotherm of Shiaskotan Island, Russia (Aksenova et al., 1992).
The first alkaliphilic spirochaetes (Spirochaeta alkalica, Spirochaeta africana, Spirochaeta asiatica) were isolated from brine sediments under crusts of Trona [Na3(CO3)(HCO3).2(H2O)] in the alkaline equatorial Lake Magadi in Kenya (Africa) and from sulfide-saturated mud sediments of alkaline Lake Khatyn in Tuva, Siberia (Central Asia) (Zhilina et al., 1996). In this report, we describe a novel, haloalkaliphilic, obligately anaerobic, mesophilic spirochaete isolated from a soda lake in California, USA.
Strain ASpG1T was isolated from a black mud sample (with a strong odour of H2S) collected on 15 August 2000 from soda Mono Lake of northern California, USA. The mud sample was collected anaerobically under shallow water (temperature, 21·6±0·1 °C; salinity, 7 %; pH 9·9±0·02) near the south shore of Mono Lake. To obtain the enrichment culture, 0·5 g of wet sediment material was injected into standard Hungate tubes containing a glucose medium and incubated at 35 °C for 3–4 days. Phase-contrast microscopy of the enrichment cultures indicated good growth of motile, spirochaete-shaped cells. Pure cultures were obtained by serially diluting the enrichment cultures in a fresh medium and filtering 1 ml of these cultures (from 7 dilutions) through a 0·4 µm sterile membrane syringe filter (Gelman Laboratory) into medium containing D-mannitol, as described previously (Canale-Parola, 1992). Three strains of spirochaetes that exhibited indistinguishable morphological, metabolic and physiological characteristics were isolated; strain ASpG1T was selected for further characterization. The culture of strain ASpG1T was judged to be pure because only a single type of colony morphology was observed on the agar surface after growth of the organism, and only one cell type was observed upon microscopic examination during growth of the strain on a variety of substrates.
Strain ASpG1T was grown in a medium modified from Zhilina et al. (1996). The medium contained (l-1) 30 g NaCl, 2·76 g Na2CO3, 24·0 g NaHCO3, 0·2 g KCl, 0·2 g K2HPO4, 0·1 g MgCl2.6H2O, 1·0 g NH4Cl, 0·4 g Na2S.9H2O, 0·001 g resazurin, 0·5 g yeast extract, 5·0 g D-glucose, 2 ml vitamin solution (Wolin et al., 1963) and 1 ml trace mineral solution (Whitman et al., 1982). The final pH was adjusted to 9·5. High-purity nitrogen was used for the gas phase. Growth was observed by phase-contrast microscopy and by measurements of optical density at 595 nm using a spectrophotometer (Genesis 5, Spectronic Instruments).
The morphology and size range of cells of strain ASpG1T were determined by phase-contrast microscopy, fluorescent microscopy and field emission scanning electron microscopy. Cells of strain ASpG1T were 0·20–0·22 µm in diameter, 8–18 µm in length and had regular-shaped spirals (Fig. 1a). They also stained Gram-negative. In the final stage of exponential growth, living cells, spherical bodies (sphaeroplasts) and conglomerates of dead cells of strain ASpG1T were imaged using a Leitz Diaplan Epifluorescent microscope and the BacLight (Molecular Probes) live versus dead fluorescent stain (Fig. 1b). Cells of strain ASpG1T were highly motile and exhibited a rotating undulatory movement typical of spirochaetes. Hitachi S-4000 field emission scanning electron microscope images of strain ASpG1T cells that had been destroyed by osmotic shock showed a flagellum released from the periplasmic space (Fig. 1c).
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). Confluent growth was observed, and the mass was white with veil-like, irregular formations, which were a result of migration of the bacteria through the agar gel. Individual colonies were observed when strain ASpG1T was grown on an agar medium containing 10 µg rifampicin ml-1. These colonies were white and round with smooth, regular edges (0·5–2·0 mm in diameter). Individual colonies were densely granulated at their centres, with the granulation diminishing towards the perimeter of the colonies (1–5 dilutions). In deep agar (6–8 dilutions), colonies of strain ASpG1T were ‘cotton-ball-like’, with an irregular spherical shape and a higher density toward their centres. Strain ASpG1T grew between 10 and 44 °C, with optimum growth at 37 °C; no growth was observed at 5 or 48 °C. Growth of the strain at 37 °C occurred between pH 7·8 and 10·5; no growth was detected at pH 7·0 or 11·0. Strain ASpG1T required NaCl for growth, but no growth was observed in 1 % (w/v) or 15 % (w/v) NaCl; the optimum concentration of NaCl for growth was 3 % (w/v), and the range over which growth was observed was 2–12 % (w/v) NaCl. The novel isolate required sodium ions for growth and did not grow on a medium with K2CO3 instead of Na2CO3. Similarly, when the carbonate buffer was replaced with serine, cells of strain ASpG1T did not grow. Thus, carbonate ions were also required for growth. Growth of strain ASpG1T was not dependent on chloride ions. Growth of the strain was observed after three sequential transfers in a medium in which the chloride salts were replaced with sulfate salts, but the cells became straight and lost their motility. The doubling time of the organism at optimal conditions [37 °C, 3 % (w/v) NaCl, pH 9·5] was 5 h.
The novel isolate was catalase-negative (method of Gerhardt et al., 1981) and only grew under strictly anaerobic conditions. It had a fermentative type of metabolism and was an obligate chemoheterotroph. Growth of strain ASpG1T on organic substrates was determined in the presence of 0·2 g yeast extract l-1, as no growth of the organism was observed in the absence of yeast extract. Strain ASpG1T grew on D-glucose, fructose, maltose, sucrose and D-mannitol. No growth was observed with acetate, lactate, pyruvate, propionate, butyrate, citrate, formate, methanol, ethanol, glycerin, peptone, yeast extract, Casamino acids, betaine or trimethylamine. Although yeast extract was required for growth of strain ASpG1T, it could not support growth when used as the sole substrate. The end products of fermentation in liquid phase were determined by HPLC. Separation was done on an Aminex HPX-87H (Bio-Rad) column with 5 mM H2SO4 as the mobile phase. Gases were measured using a gas chromatograph 3700 (Varian) equipped with a Porapak Q column and a TCD detector (nitrogen was the carrier gas). During growth of strain ASpG1T on glucose, H2 was the main end product of fermentation, and formate, acetate and ethanol were minor end products. CO2 production could not be measured due to the high carbonate level in the medium. The susceptibility of strain ASpG1T to kanamycin, gentamicin, tetracycline, rifampicin (all at 250 µg ml-1) and chloramphenicol (125 µg ml-1) was tested. Strain ASpG1T was resistant to kanamycin and rifampicin, but sensitive to gentamicin, tetracycline and chloramphenicol.
Fatty acid methyl esters (FAMEs) were extracted from fresh biomass and identified following the procedure recommended for the Microbial Identification System (MIDI, Sherlock Microbial Identification System Version 4.0, MIS Operating Manual March 2001). Cells of strain ASpG1T were harvested from a broth culture grown in the modified medium described above and incubated at 37 °C for 48 h. Extract preparation included sonication, methylation, organic extraction (transferring FAMEs from an aqueous to an organic phase) and a base wash. Then, the cell extracts were subjected to GC analysis (MIDI). The major FAMEs of strain ASpG1T were C14 : 0 (11·94 %), C16 : 0 (14·42 %), dimethyl acetate C16 : 0 (12·55 %), C18 : 1 (9·75 %) and a mixture of C18 unsaturated fatty acids in both the cis and trans configurations (31·58 %). Aldehydes were present in lower amounts: C16 : 0 (4·69 %), C16 : 1 (5·79 %), C18 : 0 (5·21 %) and C20 : 1 (2·63 %).
The G+C content of the genomic DNA of strain ASpG1T was determined by the HPLC method (Mesbah et al., 1989), except that an Alltima C18 column [250x4·6 mm, 5 µm particle size (Alltech)] and 8 % (v/v) methanol were used. The result reported is the mean of two determinations for each of two degradations of the DNA. The G+C content of the genomic DNA of strain ASpG1T was 58·5±0·2 mol% (mean±SD, n=4).
Genomic DNA was isolated through phenol/chloroform extraction followed by ethanol precipitation (Ausubel et al., 1987). The 16S rRNA gene of strain ASpG1T was selectively amplified by PCR using primers designed by us: 5'-AGAGTTTGATCCTGGCTCAG-3' (forward, Escherichia coli positions 8–27); 5'-TACGGCTACCTTGTTACGAC-3' (reverse, E. coli positions 1512–1493). PCR was performed with 30 pmol of each primer in a total volume of 50 µl, using 1·2 U Pfu polymerase (Promega) in the provided buffer, 10 nmol of each dNTP and 10 % DMSO. The thermal cycling profile used was 5 min at 95 °C (initial denaturation), followed by 10 cycles of 2 min at 95 °C (denaturation), 45 s at 56 °C (annealing) and 3 min at 73 °C (extension), then 20 additional cycles with annealing at 50 °C (other parameters were the same as for the first 10 cycles). The final extension step was for 15 min at 73 °C. The amplified fragment was extracted from a 1·5 % agarose gel using the Qiaquick extraction kit (Qiagen), then subcloned using the Zero Blunt TOPO PCR Cloning kit (Invitrogen). Three clones were sequenced in both directions using the dye terminator AmpliTaq FS cycle sequencing kit (Applied Biosystems), with both vector-specific primers (T3 and T7) and the following primers (designed by us), which were specific to internal regions of the 16S rRNA gene: 5'-CGGAATTACTGGGCGTAAAG-3' (E. coli positions 555–574); 5'-GTGCTGCATGGCTGTCGTC-3' (E. coli positions 1047–1065); 5'-GGGTTGCGCTCGTTGCGGG-3' (E. coli positions 1113–1095); 5'-GCGCTCGCTTTACGCCCAAT-3' (E. coli positions 581–562). The 16S rRNA gene sequence of strain ASpG1T was aligned with those of 12 closely related sequences found in GenBank after a BLAST search (Altschul et al., 1990). The alignment was done using the PILEUP program from the Genetic Computer Group (GCG) package. Pairwise distances were computed with MEGA version 2.0 (Kumar et al., 2001) using the Jukes–Cantor model (Jukes & Cantor, 1969). An unrooted phylogenetic tree was constructed with the same MEGA program using the neighbour-joining method (Saitou & Nei, 1987).
A sequence covering 1467 nt of the 16S rRNA gene of strain ASpG1T was obtained, corresponding to positions 28–1551 of the E. coli 16S rDNA sequence. The G+C content of this sequence was 55·96 mol%. The sequence was compared with all sequences presently available in the GenBank database and appeared to be highly similar to sequences from the spirochaete group of organisms. A dendrogram showing the phylogenetic relationship of strain ASpG1T to other spirochaetes, based on 1223 common nucleotide positions, was constructed (Fig. 2). Strain ASpG1T appears to belong to the alkaliphilic–halophilic cluster of spirochaetes, with similarity values ranging between 88·4 and 99·3 % (distances based on 1223 nt positions). The highest similarity was observed with S. alkalica Z-7491T. A pairwise alignment of the 16S rDNA sequence of strain ASpG1T with the complete available sequence of the S. alkalica 16S rRNA gene showed 99·2 % identity (1446 nt) with 1457 nt positions compared.
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and Gillis et al. (1970). The Tm value for the genomic DNA of strain ASpG1T was 68±2 °C (mean±SD, n=3), whereas it was 74±2 °C (mean±SD, n=3) for S. alkalica Z-7491T. The genome size of strain ASpG1T was 2·98x109 Da, as calculated using the equation described by Gillis et al. (1970) and the reported genome size of S. alkalica Z-7491T (Zhilina et al., 1996). A comparison of the genome size of strain ASpG1T with those of previously reported haloalkaliphilic spirochaetes is provided in Table 1.
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and Johnson (1985). Genomic DNA from both of these micro-organisms was sonicated using a Sonic Dismembrator 50 (Fisher Scientific) to generate DNA fragments of between 400 and 800 bp in size. Residual RNA or single-stranded DNA was removed by using RNase I (Sigma) or S1 nuclease (US Biochemical) (Ausubel et al., 1987), respectively. The concentration and purity of the DNA were determined at 260 nm and a ratio of 260/280 nm, respectively, by using a Shimadzu UV-160 spectrophotometer. Initial reassociation kinetics were determined by linear regression analysis. DNA–DNA hybridization established 48·7 % homology between the genomes of strain ASpG1T and S. alkalica Z-7491T based on the reassociation kinetics and the equation described by Gillis et al. (1970). All statistical analyses were performed using Microsoft Excel.
Strain ASpG1T was isolated from Mono Lake, an ancient (
750 000 years old), continental, athalassic, sulfurous, hypersaline (80 g NaCl l-1), alkaline (pH 9·4–10·5) soda lake in California. Mono Lake is a closed basin with high evaporation rates that is in close proximity to active volcanoes (Tierney, 1997). Dominant ions of Mono Lake water (pH 9·7) are sodium (1400 mM), carbonate (400 mM) and sulfate (130 mM) (Oremland & Miller, 1993). The salinity of Mono Lake varies with the water level and has ranged from 50 to 100 g salt l-1 (when Mono Lake was at its lowest level, in 1982).
The volcanic island Paoha, with a hot (90 °C) alkaline spring, emerged only 350 years ago in the middle of Mono Lake. In the winter months, along the south shoreline, metastable minerals such as gaylussite [CaNa2(CO3)2.5(H2O)] and ikaite (CaCO3.6H2O) precipitate abundantly (Bischoff et al., 1991, 1993; Council & Bennett, 1993). In warmer months, these minerals transform into tufa (anhydrous CaCO3), and tufa columns grow where calcium-rich spring water mixes with alkaline lake water. The microbial community of Mono Lake exhibits a complete cycle of trophic chains: the primary producers are phototrophs, such as cyanobacteria, diatoms and other algae, and the primary micro-organisms responsible for the destruction of organic matter under anaerobic conditions include species of Spirochaeta (strain ASpG1T) and Tindallia (novel strain APO), some cellulolytic bacteria and secondary anaerobes, such as Desulfonatronum sp. (novel sulfate-reducer strain MLF1), using the end products of primary anaerobes.
On the basis of its phenotypic characteristics (e.g. cell morphology, Gram-negative cell wall and fermentation of sugars), strain ASpG1T was identified as a member of the genus Spirochaeta. Among the alkaliphilic spirochaete species, strain ASpG1T is the only haloalkaliphile capable of growth in a medium containing 12 % NaCl and with a pH of 9·5. Strain ASpG1T was also shown to be a member of the genus Spirochaeta by a molecular phylogenetic analysis. The significant differences between the morphological and physiological characteristics of strain ASpG1T and its closest phylogenetic relatives of the genus Spirochaeta are shown in Table 1
. Strain ASpG1T is notably different from its nearest phylogenetic neighbour, S. alkalica Z-7491T, in both its cell morphology and size; both strains also exhibit differences in important physiological and metabolic characteristics, such as the ability to use D-mannitol, production of lactate from glucose fermentation and their relationship to oxygen (strain ASpG1T is an obligate anaerobe, whereas S. alkalica Z-7491T is a facultative anaerobe). In addition, strain ASpG1T does not exhibit visible pigmentation (its biomass is white), but the biomass of S. alkalica cells is orange. Furthermore, cells of strain ASpG1T are regular-shaped spirals, but S. alkalica Z-7491T cells are irregular-shaped coils. Fatty acid analysis demonstrates the absence of branched-chain fatty acids in strain ASpG1T, as was also observed for S. alkalica, S. africana, S. asiatica, S. halophila and S. litoralis. DNA–DNA hybridization of genomic DNA between strain ASpG1T and S. alkalica Z-7491T exhibited 48·7 % homology. The Tm value for the genomic DNA of S. alkalica Z-7491T was more than 5 °C higher than the Tm value for the genomic DNA of strain ASpG1T. Finally, the genome size of strain ASpG1T has been determined to be higher than that of S. alkalica Z-7491T, and the G+C content of strain ASpG1T is 1·4 mol% higher than that of S. alkalica Z-7491T.
A comparison of the phenotypic and genotypic data for strain ASpG1T with those of S. alkalica Z-7491T indicates that there are sufficient differences between the two strains for strain ASpG1T to be considered a separate species of the genus Spirochaeta. Based upon the taxonomic criteria set forth by Wayne et al. (1987), the DNA reassociation value of 48·7 % between strain ASpG1T and S. alkalica Z-7491T supports this assertion. Two of the alkaliphilic species of the genus Spirochaeta were named after the continent from which they were collected (S. africana Z-7692T from soda Lake Magadi in Central Africa and S. asiatica Z-7591T from Lake Khatyn of Central Asia; Zhilina et al., 1996). In accordance with this convention, the name Spirochaeta americana is proposed for strain ASpG1T, which represents the first haloalkaliphilic spirochaete isolated from the American continents.
Description of Spirochaeta americana sp. nov.
Spirochaeta americana (a.mer.i.can'a. N.L. fem. adj. americana of American continent, isolated from soda Mono Lake, California, USA).
Cells are motile and helix-shaped. Flagellum present in periplasmic space. Gram-negative. Cells have regular, unstable primary coils. Sphaeroplasts are formed at the end of the growth phase. Haloalkaliphile that cannot grow at pH 7·0. Growth is dependent upon the presence of carbonate and sodium ions in the medium. No growth occurs below 2 % (w/v) NaCl or above 12 % (w/v) NaCl. Mesophilic. Cells can be stored frozen in a liquid medium. Strictly anaerobic, catalase-negative chemoheterotroph with fermentative type of metabolism. Preferred substrates are D-glucose, fructose, maltose, sucrose, starch and D-mannitol. Requires vitamins and yeast extract for growth. Primary end product of glucose fermentation is H2; the minor end products in the stationary phase of growth are acetate, formate, ethanol and CO2. Resistant to kanamycin and rifampicin, but sensitive to gentamicin, tetracycline and chloramphenicol. Other characteristics of the species can be found in Table 1.
The type strain of Spirochaeta americana is ASpG1T (=ATCC BAA-392T=DSM 14872T). The Tm of its genomic DNA is 68±2 °C. Isolated from mud sediments of the alkaline, hypersaline, meromictic, soda Mono Lake in northern California, USA.
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ACKNOWLEDGEMENTS |
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