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Institute for Limnology, Austrian Academy of Sciences, Mondseestrasse 9, 5310 Mondsee, Austria
Correspondence
Martin W. Hahn
martin.hahn{at}oeaw.ac.at
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequences of ‘Candidatus Aquirestis calciphila’ and ‘Candidatus Haliscomenobacter calcifugiens' are AJ786341 and AJ786327, respectively.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). One of these groups is the so-called LD2 group which was described in 2002 based on four environmental 16S rRNA gene sequences obtained from two Dutch lakes (Zwart et al., 2002). Phylogenetic analysis of these environmental sequences indicated that the LD2 bacteria were related to Saprospira grandis and Haliscomenobacter hydrossis of the phylum Bacteroidetes (Zwart et al., 2002). Pernthaler et al. (2004) demonstrated by fluorescent in situ hybridization (FISH) of samples obtained from a single lake that bacteria affiliated to the LD2 group share a characteristic filamentous morphology with H. hydrossis. We attempted to isolate bacteria possessing this H. hydrossis-like morphology by utilizing various cultivation methods and media. This resulted in the enrichment of the targeted filamentous bacteria and the establishment of mixed cultures in which up to 42 % of the total number of bacterial cells were of the targeted bacterial morphotype. However, the establishment of pure cultures of the targeted bacteria failed despite the achievement of enrichments of at least one order of magnitude (Schauer & Hahn, 2005).
In order to obtain insights into the phylogeny of the filamentous bacteria characterized by an H. hydrossis-like morphology, primers for the specific amplification of the 16S rRNA gene sequences of the targeted bacteria were developed by a step-wise approach (Schauer & Hahn, 2005). By using these primers, a large number of sequences potentially representing the targeted filamentous bacteria were obtained from enrichment cultures and from various samples from freshwater habitats. Based on these sequences, a suite of five nested FISH probes was developed (Table 1) and these probes were used for verification that the obtained sequences originated from the targeted bacteria with the H. hydrossis-like morphology. By this approach, it was demonstrated that a broad phylogenetic group, provisionally designated the SOL cluster, exclusively harbours bacteria possessing the typical H. hydrossis-like morphology (Schauer & Hahn, 2005).
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). The HAL subcluster is the only subcluster that contains a recognized species, H. hydrossis (van Veen et al., 1973). The HAL subcluster is characterized by a minimum 16S rRNA gene sequence similarity of 98.7 %, suggesting that all members of this subcluster belong to the species H. hydrossis (Rossello-Mora & Amann, 2001). Members of the other two subclusters are more distantly related to H. hydrossis. Sequences affiliated with the GKS2-217 subcluster share 16S rRNA gene sequence similarities of 93.8–95.5 % with members of the HAL subcluster. 16S rRNA gene sequences affiliated with the LD2 subcluster have sequence similarities of 90.1–92.7 % with those of the HAL subcluster. The minimal 16S rRNA gene sequence similarities within the GKS2-217 and LD2 subclusters are 97.6 % and 99.7 %, respectively. The low between-subcluster similarity values indicate that members of the different subclusters are not representatives of H. hydrossis and thus justify the proposal of two novel Candidatus species. Therefore, we propose to classify one member of the GKS2-217 subcluster (reference clone MS-oKlaff1-G) as a candidate for a novel Haliscomenobacter species and propose the designation ‘Candidatus Haliscomenobacter calcifugiens’. Furthermore, we propose to classify one member of the LD2 subcluster (reference clone MS-Falk1-L) as a candidate for a novel species in a new genus and propose the designation ‘Candidatus Aquirestis calciphila’. The novel taxa ‘Candidatus H. calcifugiens' and ‘Candidatus A. calciphila’ share 91.5 % 16S rRNA gene sequence similarity with each other and 94.6 and 91.8 %, respectively, with the type strain of H. hydrossis. Phylogenetic analysis of 16S rRNA gene sequences (Fig. 1) revealed that all three taxa cluster together within the family ‘Saprospiraceae’ (Garrity et al., 2004).
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). All these bacteria appeared as straight, stick-like filaments of variable length ranging from 5 to >100 µm and of quite stable, but narrow, width of 0.25–0.35 µm. A sheath enclosing the filaments was sometimes visible in 4',6-diamidino-2-phenylindole (DAPI)-stained preparations (epifluorescence microscopy). Branching filaments were never detected with the probes specific for the SOL cluster or its subclusters.
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). It seemed that the loss of this trait resulted in the formation of erratically curved filaments which lacked the morphological characteristics originally described for this strain (van Veen et al., 1973). The filamentous bacteria detected by the specific FISH probes in the investigated freshwater lakes shared several of the morphological characteristics originally described for the H. hydrossis type strain (van Veen et al., 1973; Eikelboom, 1975), but showed smaller filament width than that described for the type strain (van Veen et al., 1973). These differences in the results of filament width measurements could result from the application of different sizing methods. Furthermore, we searched for H. hydrossis filaments in several samples from natural freshwater habitats with a species-specific probe (Schauer & Hahn, 2005) but never detected any probe-positive cells. A morphological comparison of filaments detected by the different subcluster-specific probes did not reveal any discriminatory features. Thus, bacteria affiliated with the SOL cluster (including the originally described H. hydrossis phenotype) can be discriminated from other bacteria by their unique morphology, but members of the three different subclusters cannot be discriminated from each other on the basis of morphological traits.
Physiology
Little is known about the physiology of these bacteria due to the lack of pure cultures. In order to estimate the growth rate of ‘Candidatus A. calciphila’ under natural conditions, 0.2 µm-filtered lake water was inoculated with predator-free (without water fleas and other metazooplankton species) water from Lake Mondsee, Austria, and incubated at 20 °C. The growth of the targeted bacteria was monitored by microscopic observations. ‘Candidatus A. calciphila’ bacteria grew in the filtered lake water with doubling times of 3.0 days (growth rate 0.23 day–1) which indicates that the in situ growth rates of these bacteria are low. Growth under anoxic conditions was not observed, while control treatments incubated under oxic conditions showed a clear increase in cell numbers. These observations may indicate an obligately aerobic physiology for ‘Candidatus A. calciphila’.
Ecology
A large number of freshwater habitats have been investigated for the presence of the three SOL subclusters by specific FISH probes (Schauer et al., 2005). Bacteria affiliated to the SOL subcluster were detected in 73 % of the 115 investigated habitats. The LD2 subcluster, which is represented by ‘Candidatus A. calciphila’, was detected in 62 % of all investigated samples, while the GKS2-217 subcluster, represented by ‘Candidatus H. calcifugiens’, was detected in 12 % of the investigated samples. The HAL subcluster, represented by H. hydrossis, was detected in 22 % of the hybridized samples. Members of the subclusters represented by ‘Candidatus A. calciphila’ and ‘Candidatus H. calcifugiens' were never observed to co-occur in the same habitat, while members of the HAL subcluster, represented by H. hydrossis, usually co-occurred with members of one of the other two subclusters. Multivariate statistical analyses revealed that water chemistry parameters mainly control the occurrence of ‘Candidatus A. calciphila’ and ‘Candidatus H. calcifugiens' in stagnant freshwater habitats. ‘Candidatus A. calciphila’ (i.e., subcluster LD2) is restricted to hard-water habitats characterized by medium to high concentrations of calcium and magnesium carbonate, with conductivity values >60 µS cm–1 and pH values 
7.7. ‘Candidatus A. calciphila’ was also observed in the polysaline Lake Qinghai, China (salinity 20 g l–1; Wu et al., 2006), which is the seventh largest saline lake in the world. Thus, this taxon is not restricted to freshwater habitats (salinity <1 g l–1). In contrast, ‘Candidatus H. calcifugiens' is restricted to soft-water (low concentrations of calcium and magnesium carbonate) habitats and was never detected in saline lakes. The stagnant systems inhabited by ‘Candidatus H. calcifugiens' are characterized by low concentrations of calcium and magnesium carbonate with conductivity values 
60 µS cm–1 and pH values in the range 6.4–7.3. Members of both groups (as well as members of the HAL subcluster represented by H. hydrossis) were never observed in acidic freshwater habitats with pH values <6.
‘Candidatus H. calcifugiens' is characterized by a narrow ecological amplitude. The soft-water habitats preferred by this taxon have been found in the Austrian Alps and in northern Sweden (Schauer et al., 2005). In contrast, ‘Candidatus A. calciphila’ was found over a broad range of habitat types which indicates a wide ecological amplitude. For instance, this taxon was detected in a hypertrophic part of Lake Taihu located in the subtropical part of China, in Lake Victoria and Lake Tanganjika located in tropical Africa and in the oligotrophic Lake Attersee in Austria (Fig. 2
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Both Candidatus organisms seem to be restricted to the pelagic zone of stagnant inland waters and were never reported from soil or marine habitats. Both organisms were detected over the entire water column of Lake Mondsee, which has a maximum depth of 68 m. Furthermore, ‘Candidatus A. calciphila’ was detected year-round in the same lake, but showed pronounced seasonal differences in abundance. In two consecutive years differing strongly in climatic conditions, a very similar seasonal pattern of population dynamics was observed. Each year, the ‘Candidatus A. calciphila’ population formed a strong spring peak and showed minor population peaks in summer and early autumn. By contrast, the HAL group (to which H. hydrossis is affiliated) was detected in the same lake only during a short period of a few weeks in early autumn.
‘Candidatus A. calciphila’ was also detected in low numbers in running water systems fed with water from lakes inhabited by populations of these bacteria. On the other hand, these bacteria were not detected in running waters that lacked lakes or ponds located upstream. This observation seems to indicate that the primary habitat of these bacteria is the pelagic zone of stagnant inland waters.
Both ‘Candidatus A. calciphila’ and ‘Candidatus H. calcifugiens' represent an important part of the freshwater bacterioplankton and appear in the water column of freshwater habitats with relative abundances of <1 % to at least 11 % of total bacterial numbers (Schauer & Hahn, 2005). The observed total cell numbers for these taxa ranged from several hundreds to 2x105 cells ml–1. Due to their filamentous morphologies and typical mean filament lengths of 20–50 µm, these bacteria contribute over proportionally to the total bacterial biomass in freshwater habitats. These bacteria comprise about 40 % of the total bacterial biovolume in some habitats (Schauer & Hahn, 2005).
Recently, water flea (Daphnia spp.) and other metazooplankton species were identified as important predators of a ‘Candidatus A. calciphila’ population in the oligo-mesotrophic Lake Mondsee (Schauer et al., 2006). On the other hand, these bacteria were found to be resistant to predation by bacterivorous protists (flagellates and ciliates) which are usually the major predators of planktonic bacteria in freshwater systems (Hahn & Höfle, 2001).
Biogeography
‘Candidatus A. calciphila’ is a cosmopolitan inhabitant of hard-water lakes. This taxon was detected in habitats located in Oceania (Australia, New Zealand), Central America (Mexico), Africa (Lake Victoria, Lake Tanganyika), Eurasia (Austria, China), as well as in habitats located in the temperate, subtropical and tropical zones. Furthermore, this taxon was also detected in a high mountain lake located at an altitude of 4987 m on the Tibetan Plateau (Schauer et al., 2005; Wu et al., 2006). Altogether, these observations indicate that ‘Candidatus A. calciphila’ may inhabit all appropriate hard-water lakes located on all continents and in all climatic zones.
‘Candidatus H. calcifugiens' has so far been exclusively detected in soft-water lakes located in Austria and Sweden. However, soft-water lakes suitable for this taxon located outside of these two regions have not so far been investigated for the presence of ‘Candidatus H. calcifugiens’. Thus, a wider biogeographic distribution for this taxon cannot be ruled out.
Cultivation
Despite extensive efforts to grow representatives of subclusters LD2 and GKS2-217 in pure culture by using various media and isolation methods (Schauer & Hahn, 2005), pure cultures have not yet been successfully established. However, until cultivation of these important representatives of freshwater bacterioplankton is achieved or additional phenotypic data become available, we assign members of the two species-like subgroups of the SOL cluster to the provisional Candidatus status as proposed by Murray & Stackebrandt (1995).
Description of ‘Candidatus Aquirestis calciphila’
‘Candidatus Aquirestis calciphila’ [A.qui.res'tis. L. n. aqua water; L. fem. n. restis thread, rope; N.L. fem. n. Aquirestis water thread; cal.ci'phi.la. L. fem. n. calx limestone (calcium carbonate); Gr. adj. philos loving, friendly to; N.L. fem. adj. calciphila loving limestone].
[(Bacteroidetes) NC; F; NAS (GenBank number AJ786341), oligonucleotide sequence complementary to unique region of 16S rRNA 5'-AAGCTGTGAAGCGGAGCC-3'; FL (freshwater and saline lakes, planktonic); Aer, long straight filaments with width 0.35 µm; M]. Schauer & Hahn, Appl Environ Microbiol 71:1931–1940, 2005.
Description of ‘Candidatus Haliscomenobacter calcifugiens’
‘Candidatus Haliscomenobacter calcifugiens' [cal.ci.fu'gi.ens. L. fem. n. calx limestone (calcium carbonate); L. part. pres. fugiens fleeing, shy; N.L. part. pres. calcifugiens fleeing from limestone].
[(Bacteroidetes, genus Haliscomenobacter); NC; F; NAS (GenBank AJ786327), oligonucleotide sequence complementary to unique region of 16S rRNA 5'-CGGATTGTGTGTCCAAGCGAA-3'; FL (freshwater lakes with soft-water, planktonic); Aer, long straight filaments with width 0.35 µm; M]. Schauer & Hahn, Appl Environ Microbiol 71:1931–1940, 2005.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Garrity, G. M., Bell, J. A. & Lilburn, T. G. (2004). Taxonomic outline of the Prokaryotes. In Bergey's Manual of Systematic Bacteriology, 2nd edn, release 5.0. New York: Springer. DOI: 10.1007/bergeysoutline200405
Hahn, M. W. & Höfle, M. G. (2001). Grazing of protozoa and its effect on populations of aquatic bacteria. FEMS Microbiol Ecol 35, 113–121.[CrossRef][Medline]
Murray, R. G. E. & Stackebrandt, E. (1995). Taxonomic note: Implementation of the provisional status Candidatus for incompletely described procaryotes. Int J Syst Bacteriol 45, 186–187.
Pernthaler, J., Zöllner, E., Warnecke, F. & Jürgens, K. (2004). Bloom of filamentous bacteria in a mesotrophic lake: identity and potential controlling mechanism. Appl Environ Microbiol 70, 6272–6281.
Rossello-Mora, R. & Amann, R. (2001). The species concept for prokaryotes. FEMS Microbiol Rev 25, 39–67.[Medline]
Schauer, M. & Hahn, M. W. (2005). Diversity and phylogenetic affiliations of morphologically conspicuous large filamentous bacteria occurring in the pelagic zones of a broad spectrum of freshwater habitats. Appl Environ Microbiol 71, 1931–1940.
Schauer, M., Kamenik, C. & Hahn, M. W. (2005). Ecological differentiation within a cosmopolitan group of planktonic freshwater bacteria (SOL cluster, Saprospiraceae, Bacteroidetes). Appl Environ Microbiol 71, 5900–5907.
Schauer, M., Jiang, J. & Hahn, M. W. (2006). Recurrent seasonal variations in abundance and composition of filamentous SOL cluster bacteria (Saprospiraceae, Bacteroidetes) in oligomesotrophic Lake Mondsee (Austria). Appl Environ Microbiol 72, 4704–4712.
van Veen, W. L., van der Kooij, D., Geuze, E. C. W. A. & van der Vlies, A. W. (1973). Investigations on the sheathed bacterium Haliscomenobacter hydrossis gen.n., sp.n., isolated from activated sludge. Antonie van Leeuwenhoek 39, 207–216.[CrossRef][Medline]
Wagner, M., Amann, R., Kämpfer, P., Assmus, B., Hartmann, A., Hutzler, P., Springer, N. & Schleifer, K.-H. (1994). Identification and in situ detection of Gram-negative filamentous bacteria in activated sludge. Syst Appl Microbiol 17, 405–417.
Wu, Q. L., Zwart, G., Schauer, M., Kamst-van Agterveld, M. P. & Hahn, M. W. (2006). Bacterioplankton community composition along a salinity gradient of sixteen high-mountain lakes located on the Tibetan Plateau, China. Appl Environ Microbiol 72, 5478–5485.
Zwart, G., Crump, B. C., Kamst-van Agterveld, M. P., Hagen, F. & Han, S.-K. (2002). Typical freshwater bacteria: an analysis of available 16S rRNA gene sequences from plankton of lakes and rivers. Aquat Microb Ecol 28, 141–155.[CrossRef]
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