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Applied and Environmental Microbiology, January 2003, p. 390-398, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.390-398.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Morphological and Phylogenetic Characterizations of Freshwater Thioploca Species from Lake Biwa, Japan, and Lake Constance, Germany

Hisaya Kojima,¹ Andreas Teske,² and Manabu Fukui^1*

Department of Biological Science, Graduate School of Science, Tokyo Metropolitan University, Tokyo 192-0397, Japan,¹ Biology Department, Redfield Laboratory, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543²

Received 3 June 2002/ Accepted 30 September 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
Filamentous, gliding, sulfide-oxidizing bacteria of the genus Thioploca were found on sediments in profundal areas of Lake Biwa, a Japanese freshwater mesotrophic lake, and were characterized morphologically and phylogenetically. The Lake Biwa Thioploca resembled morphologically Thioploca ingrica, a brackish water species from a Danish fjord. The diameters of individual trichomes were 3 to 5.6 µm; the diameters of complete Thioploca filaments ranged from 18 to 75 µm. The cell lengths ranged from 1.2 to 3.8 µm. In transmission electron microscope specimens stained with uranyl acetate, dense intracellular particles were found, which did not show any positive signals for phosphorus and sulfur in an X-ray analysis. The 16S rRNA gene of the Thioploca from Lake Biwa was amplified by using newly designed Thioploca-specific primers (706-Thioploca, Biwa160F, and Biwa829R) in combination with general bacterial primers in order to avoid nonspecific amplification of contaminating bacterial DNA. Denaturing gradient gel electrophoresis (DGGE) analysis of the three overlapping PCR products resulted in single DGGE bands, indicating that a single 16S rRNA gene had been amplified. With the same method, the Thioploca from Lake Constance was examined. The 16S rRNA sequence was verified by performing fluorescence in situ hybridization targeted at specific motifs of the Lake Biwa Thioploca. Positive signals were obtained with the bacterial probe EUB-338, the -proteobacterial probe GAM42a, and probe Biwa829 targeting the Lake Biwa Thioploca. Based on the nearly complete 16S rRNA sequence and on morphological similarities, the Thioploca from Lake Biwa and the Thioploca from Lake Constance are closely related to T. ingrica and to each other.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Members of the genus Thioploca are gliding, filamentous, sulfur-oxidizing bacteria that are distinguished from the related genus Beggiatoa by a morphological trait, the common sheath that surrounds the trichomes (11, 17). Since the discovery of dense mats of large Thioploca spp. on the continental shelf of South America (7), the extraordinarily dense and widespread communities of these bacteria have been surveyed and described in more detail (5, 10, 20, 27, 29, 31, 32, 34). Most of the current information about this genus, including phylogenetic relationships and ecological and physiological traits, has been gained from studies of marine Thioploca mats in coastal upwelling areas, the largest recognized habitat.

Although pure cultures have not been obtained so far, physiological features of marine Thioploca spp. have been revealed to some extent by experiments performed with freshly collected Thioploca samples (20, 27). Thioploca cells accumulate high concentrations of nitrate in large intracytoplasmatic vacuoles and reduce the nitrate to ammonium, with concurrent oxidization of sulfide to sulfate (27). Thioploca is capable of autotrophic carbon fixation, but it can also grow mixotrophically and assimilates substrates such as acetate and amino acids (20). Considering their large biomass and their physiological capabilities, Thioploca mats play a critical role as a link for sulfur, nitrogen, and carbon cycles in marine coastal ecosystems.

On the other hand, the phylogenic relationships and the physiology of freshwater Thioploca are still unknown, although the genus originally was described from freshwater lakes and rivers (12, 13, 15, 37). Many of the previous Thioploca freshwater communities have declined or disappeared, in part due to human impact, and are no longer available for study (18). However, mats of Thioploca spp. were recently found in Lake Biwa, a Japanese freshwater lake (26). This finding provides an opportunity to investigate freshwater Thioploca in detail. In this study, a Thioploca sp. from Lake Biwa was characterized morphologically and by 16S rRNA sequencing in order to provide the basis for further detailed studies. In addition, a Thioploca sp. from Lake Constance, Germany, was characterized by 16S rRNA sequencing in order to analyze phylogenetic relationships among freshwater Thioploca spp. from different geographical locations.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sampling sites and collection of Thioploca specimens.
Thioploca spp. were collected at station A (35°23.4'N, 136° 7.7'E) at a depth of 90 m in the northern part of Lake Biwa. Sediment samples were obtained with a Ekman-Birge grab sampler (15 by 15 cm). Thioploca filaments (sheaths with trichomes) were also collected with a dredge net originally used for collection of macrozoobenthos (26). After 5 min of dredging, numerous Thioploca filaments accumulated on the front part of the dredge net. Dense clumps of filaments on the frame of the dredge net were collected with forceps and immediately transferred into a container with sediment and water from the collection site.

The sediment sample from Lake Constance that contained Thioploca filaments was collected by a diver at a depth of 22 m near the Island of Mainau (Germany).

Light microscopy.
The morphological traits of Thioploca were observed by light microscopy. Micrographs were taken with a phase-contrast microscope (Axioplan; Zeiss, Göttingen, Germany) equipped with a charge-coupled device digital camera (Axiocam; Zeiss).

TEM and X-ray analysis.
Thioploca bundles from Lake Biwa used for transmission electron microscopy (TEM) observation were fixed, dehydrated, and embedded as described previously (19). Thin sections were cut with a diamond knife, mounted on 100- or 200-mesh copper grids, and poststained with uranyl acetate and lead citrate. TEM micrographs were taken with a JEM 1010 microscope (JEOL, Akishima, Japan) at 80 kV. The X-ray analysis was done with a JEM2000 FX instrument (JEOL).

FISH.
For whole-cell fluorescence in situ hybridization (FISH) of the Thioploca from Lake Biwa, the following probes (5' labeled with fluorescein isothiocyanate [FITC]) were used: EUB338 (for the domain Bacteria); ALF19b, BET42a, and GAM42a (for the , ß, and subclasses of the class Proteobacteria, respectively); SRB385 and SRB385Db (for sulfate-reducing bacteria belonging to the families Desulfovibrionaceae and Desulfobacteriaceae, respectively, in the subclass of the Proteobacteria); and 462-Thioploca and 829-Thioploca (for large, marine Thioploca spp.). rSRB385, the complement sequence of SRB385, was used as a negative control (2, 3, 22, 28, 34). Fixation, hybridization, and washing procedures were carried out as previously described (2). To compare results obtained with all probes under the exactly same conditions, Thioploca samples were set on a Teflon-coated glass slide with 10 windows, and hybridizations with all probes were performed on the slide. Hybridization was carried out at 37°C for 10 h. The concentration of formamide in buffer was adjusted to 20%. The results of FISH with these probes were evaluated by determining fluorescence intensity. Fluorescence images of hybridized Thioploca trichomes were obtained with a laser scanning microscope (MRC-600; Bio-Rad, Richmond, Calif.), and fluorescence intensities were measured for 168 trichomes for each probe.

In addition to the previously designed probes, a new probe, Biwa829 (5'-AGGTATACCCTTCCAACGTC-3'), was designed to match specifically the sequence of the Lake Biwa Thioploca. This probe targets the same Escherichia coli nucleotide positions as the previously designed probe 829-Thioploca for large, marine Thioploca spp. (5'-GGATTAATTTCCCCCAACATC-3'), which has 10 mismatches with Biwa829 (34). First, probe Biwa829 was tested with Beggiatoa alba DSM 1416 as a negative control in a direct comparison with the Lake Biwa Thioploca. Fixed samples of Thioploca and B. alba were placed on the same slide and hybridized with EUB338, GAM42a, rSRB385, and Biwa829 (also 5' labeled with FITC). After approximately 4 h of hybridization at 37°C, the slide was washed with washing buffer containing 20% formamide. Following the experiment to confirm specificity and hybridization conditions for Biwa829, another hybridization experiment was performed to compare fluorescence intensities for EUB338, rSRB385, and Biwa829.

DNA extraction, PCR, and denaturing gradient gel electrophoresis (DGGE) of Thioploca.
To identify the Thioploca from Lake Biwa phylogenetically, DNA was extracted from purified trichomes for 16S ribosomal DNA (rDNA) sequencing. Thioploca filaments were picked from the sediment, and trichomes were withdrawn from the sheaths with forceps. The trichomes were rinsed in phosphate-buffered saline. The washed trichomes were homogenized in a 1.5-ml Eppendorf tube, with gradual addition of a mixture of 567 µl of Tris-EDTA buffer and 30 µl of 10% sodium dodecyl sulfate. Then 3 µl of a proteinase K solution (20 mg/ml) was added, and this was followed by 60 min of incubation at 37°C. After proteinase K-sodium dodecyl sulfate digestion, 100 µl of 5 M NaCl and 80 µl of a 10% cetyltrimethylammonium bromide-0.7 M NaCl solution were added. After mixing, the sample was incubated for 10 min at 65°C. After centrifugation following addition of 780 µl of chloroform-isoamyl alcohol (24:1, vol/vol) and mixing, the supernatant was transferred to a new tube and mixed with the same volume of phenol-chloroform-isoamyl alcohol (25:24:1, vol/vol/vol). From the aqueous supernatant obtained after centrifugation, DNA was precipitated by addition of isopropanol and centrifugation. The resulting pellets were rinsed with 70% ethanol.

Since it was difficult to completely remove all non-Thioploca bacteria before DNA extraction, the 16S rRNA gene of Thioploca was selectively amplified with Thioploca-specific primers and DGGE primers. Two primer sets were used. Primer ig706F (5'-ATTAGGAGGAACACCAGTGG-3') was designed based on the sequence of Thioploca ingrica and was used in combination with the general bacterial 16S rDNA primer GM4r-GC (5'-GC clamp-TACCTTGTTACGACTT-3'; E. coli positions 1492 to 1507) (23). The reverse version of ig706F, ig706R (5'-CCACTGGTGTTCCTCCTAAT-3'), was used in combination with primer GC-109f2 (5'-GC clamp-ACGGGTGAGTAATGYMT-3'), the forward version of primer 109r2 (14). PCR were performed as follows: 5 min of denaturation at 94°C, 1 min of annealing at 65°C, and 3 min of elongation at 72°C. Touchdown cycles were performed by decreasing the temperature in 2-min annealing steps by 1°C every second cycle, from 65 to 55°C. DGGE was performed as previously described (24). The products of PCR obtained with each primer set were analyzed by DGGE to confirm that only a single fragment was obtained; then the sequences were determined. Since these two primer sets amplified two mutually nonoverlapping portions of the 16S rRNA genes, it was necessary to confirm that the partial sequences were derived from the same 16S rRNA gene. The overlap region was amplified with newly designed primers GC-Biwa160F (5'-GC clamp-ATAAGTCTTTTTTAACGAAA-3') and Biwa829R (5'-AGGTATACCCTTCCAACGTC-3'). The resulting PCR product was also analyzed by DGGE and then sequenced.

For phylogenetic identification of the Thioploca from Lake Constance, DNA was extracted from washed filaments (not from sheath-free trichomes), and 16S rRNA gene fragments were amplified with three primer sets as described above. Since it was not known whether these primers matched the sequence of the Thioploca from Lake Constance, the stringency of the PCR conditions was lowered. Amplification was performed for 30 cycles, with each cycle consisting of 2 min of denaturation at 94°C, 1.5 min of annealing at 45°C, and 2 min of elongation at 72°C. The PCR fragments were checked by DGGE and sequenced.

Phylogenetic analysis.
The 16S rRNA sequences of the thioplocas from Lake Biwa and Lake Constance were aligned with other - and -proteobacterial 16S rRNA sequences by using the sequence editor SeqPup (8). Positions 135 to 1487 of the 16S rRNA gene (E. coli numbering) were used for phylogenetic analysis, and an analysis with the partial region from position 358 to position 906 was also performed to include marine Thioploca species and Thiomargarita. PCR primer positions were excluded. Phylogenetic trees were inferred with the program PAUP 4.0^* (33). The minimum evolutionary tree for the Lake Biwa Thioploca and the Lake Constance Thioploca was obtained by using the Kimura two-parameter model and was checked with 1,000 bootstrap replicates in minimum evolution and parsimony analyses. Members of the subclass of the Proteobacteria were used as outgroups; these organisms included two clones obtained from a filamentous bacterial mat at the 17 Southern East Pacific Rise hydrothermal vents and the filamentous episymbiont of the vent invertebrates Rimicaris exoculata and Alvinella pompeiana.

Nucleotide sequence accession numbers.
The 16S rRNA gene sequences of the Lake Biwa Thioploca sp. and the Lake Constance Thioploca sp. have been deposited in the GenBank database under accession no. AF452892 and AY115530, respectively.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Morphological characteristics.
Thioploca filaments were easily picked from the surfaces of sediment samples with a pair of forceps. Individual Thioploca sheaths harbored from 1 to 75 trichomes. Numerous sulfur inclusions were observed in trichomes (Fig. 1). The trichome tips were tapered. The trichomes of the Thioploca from Lake Biwa had a mean diameter of 3.93 µm (standard deviation, 0.555 µm; range, 3.0 to 5.6 µm; n = 102). The sheath diameters ranged from 18 to 75 µm, and the mean diameter was 49.5 µm (standard deviation, 14.26 µm; n = 35). The cell lengths ranged from 1.2 to 3.8 µm, and the mean cell length was 2.91 µm (standard deviation, 0.635 µm; n = 16). Active gliding movement of trichomes was observed. Some trichomes occasionally protruded from the open end of a sheath. Each trichome glided independently; simultaneous gliding movements with different directions and speeds were observed for trichomes sharing the same sheath. The mean gliding rate was 3.0 µm · s^-1 at 10°C. The sheaths showed constricted zones and sometimes were bent at these constricted zones, as described previously for T. ingrica (37). The intervals between constrictions seemed to be random. Based on these characteristics, the Thioploca from Lake Biwa resembled most closely the species T. ingrica. The morphology of the Thioploca from Lake Constance was similar to that of the Thioploca from Lake Biwa. The trichomes had a mean diameter 4.2 µm, as determined with 58 individual trichomes that ranged from 3.0 to 5.2 µm in diameter.

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FIG. 1. Micrographs of the Thioploca from Lake Biwa. (A) Sheath having a relatively small diameter. The arrows indicate constrictions. (B) Phase-contrast image of a wide sheath with trichomes. A constriction is indicated by arrows. (C) Phase-contrast image of a trichome tip. S, sulfur droplets. Bars = 10 µm.

TEM and X-ray analysis.
TEM micrographs of the Thioploca from Lake Biwa are shown in Fig. 2. Numerous Thioploca trichomes (in this example approximately 60 trichomes) formed a single bundle (Fig. 2A). The sheath surrounding the trichomes had a striated, fibrous texture (Fig. 2B), similar to the texture of the sheath material of T. ingrica, as described by Maier and Murray (21). In addition to sulfur globules, the cells contained high numbers of dense round particles that were 100 to 200 nm in diameter, which were stained intensively with uranyl acetate (Fig. 2C and D). The TEM specimens were also examined by X-ray analysis, but the unidentified dense particles did not show any positive signal for phosphorus or sulfur. Similar globules that were 100 to 200 nm in diameter (and not stained with lead tartrate) were also abundant in the cytoplasm of T. ingrica cells (21). These particles may be polyhydroxybutyrate globules or another storage compound that is free of sulfur and phosphorus. In microscopic and ultrastructural studies of Beggiatoa strains, the cytoplasm frequently contained conspicuous polyhydroxybutyrate globules (16).

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FIG. 2. TEM micrographs of the Thioploca from Lake Biwa. (A) Cross section of Thioploca filament containing multiple trichomes. (B) Material between sheath and trichomes. (C) Longitudinal section of trichome. (D) Longitudinal cross section of a Thioploca trichome, with individual cells separated by cell walls. White sulfur droplets (S) were present in the cells. In panels B to D intracellular dense particles stained with uranyl acetate appear as black particles. Bars = 500 nm.

Phylogenetic affiliation.
For the sample of the Thioploca from Lake Biwa, the products of PCR performed with two primer sets (GC-109f2 and ig706R; ig706F and GM4r-GC) were analyzed by DGGE, and they produced only a single band each. After sequencing of both DGGE fragments, which corresponded to two nonoverlapping halves of the 16S rRNA gene, new primers were designed for PCR amplification of an overlapping sequence region (Biwa160F and Biwa829R). Amplification of the overlapping region with the new primer set resulted in a single DGGE band, which matched the sequences of the two partial 16S rRNA gene sequences. Based on the consensus, the three sequences were combined to obtain a nearly complete 16S rDNA sequence for the Lake Biwa Thioploca.

From the Lake Constance sample, fragments of 16S rDNA were amplified with three primer sets (GC-109f2 and ig706R; GC-Biwa160F and Biwa829R; ig706F and GM4r-GC). The products obtained with two of the primer sets gave only one DGGE band each, but the products obtained with primers ig706F and GM4r-GC gave two DGGE bands. These four bands were sequenced, and three of them matched in the overlapping sequence regions and were combined to obtain a nearly complete 16S rDNA sequence. There was one mismatch between Biwa160F and the sequence of the corresponding position determined with GC-109f2 and ig706R, but PCR performed at a lower stringency allowed positive amplification with primers GC-Biwa160F and Biwa829R. The additional DGGE band obtained with primers ig706F and GM4r-GC was closely related to Corynebacterium tuberculostearicum (accession no. X84247), a gram-positive bacterium that had three mismatches with ig706F. This band represented bacterial contaminants, most likely in the sheath material of the complete Thioploca filaments (sheath plus trichomes) that were used to extract DNA from the Lake Constance samples.

The phylogenetic analysis based on nearly complete sequences showed that the thioplocas from Lake Biwa and Lake Constance are closely related to T. ingrica (Fig. 3A). These organisms form a branch separate from the large, marine, vacuolated, nitrate-accumulating Beggiatoa and Thioploca spp., which are represented by the Beggiatoa spp. from the Bay of Concepcion and from Monterey Canyon (1, 35). The same result was obtained by analysis of partial 16S rDNA sequences, including those of the large, vacuolated, nitrate-accumulating sulfide oxidizers Thioploca araucae, Thioploca chileae (34), and Thiomargarita namibiensis (30), which group together with large, marine, nitrate-accumulating Beggiatoa species (Fig. 3B).

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FIG. 3. Phylogenetic relationships of the Thioploca spp. from Lake Biwa and Lake Constance based on nearly complete 16S rDNA sequences (E. coli positions 135 to 1487) (A) and on partial sequences (E. coli positions 358 to 906), including those of marine Thioploca species and T. namibiensis, for which only partial sequences are available (B). Bootstrap values (minimum evolution/parsimony) are indicated at the nodes with more than 50% bootstrap support.

FISH analysis of Thioploca sp.
The 16S rDNA sequence analysis results were supported by the FISH results. The results of FISH with previously designed probes are summarized in Table 1. Hybridizations with probes that do not match the rRNA sequence of the Lake Biwa Thioploca sp., including the nonsense probe rSRB385, did not give hybridization signals that were greater than a common level of autofluorescence (obtained with probes ALF19b, BET42a, SRB385, SRB385Db, and rSRB385). Hybridizations with probe EUB338 and the -proteobacterial probe GAM42a resulted in strongly positive fluorescence hybridization signals. Probe 829-Thioploca, previously designed for large marine Thioploca species, gave a negative result, which is consistent with numerous mismatches with the Thioploca from Lake Biwa (10 mismatches in 21 bases). The relatively high intensity obtained with probe 462-Thioploca was unexpected, since this probe has six mismatches in 23 bases. This finding may be related to the fact that the hybridizations were performed with 20% formamide in order to compare the results with the results obtained with other probes under the same conditions. However, the original tests of probe 462-Thioploca with marine Thioploca spp. showed that there was detectable hybridization with 20% formamide (34), which was therefore chosen as the negative control hybridization stringency for freshwater Thioploca spp. Further testing of probe 462-Thioploca is needed in order to evaluate its reliability for Thioploca hybridization.

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TABLE 1. Fluorescence intensities of FISH results

In the experiment with the newly designed probe, the B. alba reference strain gave positive hybridization results with EUB338 and GAM42a and negative results with probes rSRB385 and Biwa829. Under exactly the same conditions on the same slide, Thioploca specimens gave much higher hybridization intensities with probes EUB338, GAM42a, and Biwa829 than with the negative control probe rSRB385.

Under the verified hybridization conditions, fluorescence intensities were measured for EUB338, Biwa829, and rSRB385 (86 to 106 trichomes for each probe). This experiment was performed with samples that had been stored several months, and the level of autofluorescence seemed to be decreased by storage; the difference between the intensities observed with EUB338 and rSRB385 was larger than the difference in the experiment performed with fresh samples. The mean fluorescence intensity observed with Biwa829 was quite similar to that observed with EUB338, and the intensities were significantly greater than the intensity observed with rSRB385 (Table 1 and Fig. 4). Therefore, newly designed probe Biwa829 that matched the Lake Biwa Thioploca 16S rRNA sequence gave a positive signal and indicated that the sequence obtained by PCR from the Lake Biwa Thioploca was indeed the correct 16S rRNA sequence of this organism.

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FIG. 4. FISH hybridization images for the Thioploca from Lake Biwa, obtained with probes labeled with FITC. (A) Bundle of trichomes labeled with probe EuB338. (B) Bundle of trichomes labeled with Biwa829. (C) Negative control with rSRB385. All photographs were taken under the same conditions. Positive fluorescence signals were obtained with probes EuB338 and Biwa829 (A and B). In the negative control, trichomes showed a small amount of autofluorescence independent of the excitation wavelength and filter (C).

Top Abstract Introduction Materials and Methods Results Discussion References

 
The classification of species in the genus Thioploca has been based on trichome diameter and habitat preference. Three types of Thioploca were found in Lake Constance in the early 20th century, Thioploca schmidlei (trichome diameter, 5 to 9 µm), T. ingrica (trichome diameter, 2 to 4.5 µm), and "Thioploca minima" (trichome diameter, 0.8 to 1.5 µm) (13, 15); in a later study the researchers found only T. ingrica in Lake Constance (18). Based on trichome diameter (3.9 and 4.2 µm), the Thioploca sp. from Lake Biwa and the Thioploca sp. from Lake Constance that were examined in this study are most similar to T. ingrica. Besides the similarity in trichome diameter, the thioplocas examined in this study share another morphological feature with T. ingrica, sheath constrictions, which have never been reported in marine species. These morphological similarities are consistent with 16S rDNA sequence data. The sequenced specimen of T. ingrica (from Randersfjord, Denmark) and the Lake Biwa and Lake Constance Thioploca spp. are closely related, with very few 16S rRNA mismatches. These three thioplocas form a tight phylogenetic cluster (100% bootstrap support) apart from the lineage of the large, marine Thioploca and Beggiatoa species (>70% bootstrap support) (Fig. 3). So far, these two phylogenetic groups seem to be consistent in terms of morphology and habitats. However, a Thioploca sp. with a relatively large trichome diameter has been found in Lake Baikal, a Russian freshwater lake (38), and a marine Thioploca sp. with narrow trichomes has been reported from the coast of Chile (31). The 16S rRNA sequences and phylogenetic positions of these interesting morphotypes are not known yet.

Analyses of 16S rRNA sequences for the Thioploca community in the sediment off the coast of Chile have shown that several distinct morphotypes of Thioploca are also genetically distinct (32, 34). On the other hand, a large Chilean Beggiatoa sp. and T. araucae, which have similar trichome diameters, have almost identical 16S rRNA sequences (35). These data suggest that there is a correlation between morphological similarity, based primarily on trichome diameter and cell dimensions, and 16S rRNA relatedness. The data for the Lake Biwa and Lake Constance Thioploca spp. support this working hypothesis. These two Thioploca spp. from two widely separated locations, Lake Biwa in Japan and the German part of Lake Constance, are hardly distinguishable by mean trichome diameter (3.9 versus 4.2 µm) and morphology and are closely related on the basis of their 16S rRNA sequences (two mismatches). Similar cosmopolitan occurrence patterns for other sulfur bacteria are not unprecedented. Very closely related hydrothermal vent strains of the genus Thiomicrospira were found at the Mid-Atlantic Ridge and at the East Pacific Rise (36).

In Lake Biwa and Lake Constance, all three 16S rDNA-targeted DGGE-PCR primer combinations yielded a single Thioploca DGGE band, indicating that the Thioploca populations are genetically homogeneous at the sampling sites in Lake Biwa and Lake Constance. In contrast, Thioploca spp. of several types coexist sympatrically in the Chilean Thioploca mats (31, 32). Also, sulfur-oxidizing Achromatium populations in a single freshwater lake showed considerable genetic diversity at the level of 16S rDNA (9). In principle, the possibility of different genotypes of Thioploca in Lake Biwa and Lake Constance cannot be eliminated, and the genetic diversity of Thioploca in freshwater habitats should be the subject of further studies. For example, it would be interesting to locate a population of the freshwater type species, T. schmidlei, with a larger trichome diameter and, if the morphotype-genotype correlation holds, a divergent 16S rRNA sequence.

A sufficient supply of hydrogen sulfide is needed for sustaining Beggiatoa or Thioploca mats. In some habitats, such as hydrothermal vents, sulfide of geothermal origin is provided (25). In benthic sediments off the coast of Chile and other localities, sulfide is produced by high rates of sulfate reduction, based on the oxidation of abundant organic matter by sulfate-reducing bacteria in sulfate-rich seawater sediments (4). However, Thioploca spp. can be found in relatively sulfate-poor, freshwater lakes. For continuous existence of these organisms, highly efficient sulfide uptake mechanisms could be involved, similar to those in Chilean marine Thioploca spp. which take up sulfide at very low external sulfide concentrations (10), or the organisms could receive sulfide from sulfate-reducing Thioploca epibionts (6). The freshwater Thioploca of Lake Biwa provides an opportunity to compare marine and freshwater Thioploca species and to understand habitat-related differences in the physiology and ecology of these organisms.

 
We are grateful to Takuo Nakajima, Machiko Nishino, and the crew members of the R/V Hakken-Go for their support in sampling in Lake Biwa. The sample of the Lake Constance Thioploca was donated by Alexander Galushko. We thank Isao Uemura for technical assistance with TEM and X-ray analysis. Susumu Takii is acknowledged for his advice and for discussions.

This work was supported by grant 12440219 from the Ministry of Education, Culture, Sports, Science and Technology, Japan, to Manabu Fukui. Andreas Teske was supported by an Independent Studies Award from the Mellon Foundation.

 
* Corresponding author. Mailing address: Department of Biological Science, Graduate School of Science, Tokyo Metropolitan University, Minami-ohsawa 1-1, Hachioji, Tokyo 192-0397, Japan. Phone: 81-426-77-2581. Fax: 81-426-77-2559. E-mail: fukui-manabu{at}c.metro-u.ac.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, January 2003, p. 390-398, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.390-398.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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