INTRODUCTION

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Achromatium oxaliferum is a large sediment-dwelling bacterium found principally in freshwater and brackish environments (1, 12, 17, 32). Notable for its large size, the bacterium also precipitates intracellular calcium carbonate, a property which makes it unique among bacteria. Although the bacterium was first described in 1893 (30), little about its role in the sediments that it inhabits has been elucidated. A. oxaliferum belongs to the -subdivision of the class Proteobacteria and is related to a number of photosynthetic and nonphotosynthetic sulfur-oxidizing bacteria (12). Recent studies have shown that natural communities of A. oxaliferum previously thought to be homogeneous in fact comprised a number of phylogenetically, morphologically, and ecologically distinct subpopulations (6, 7, 9, 13). The degree of homology observed for Achromatium-derived 16S rRNA sequences (<97.5%) was consistent with the occurrence of different Achromatium species both within a single sediment community and at geographically separated locations (6, 7, 9, 13). Thus, it is clear that the majority of Achromatium communities studied in detail comprise several Achromatium species. While this fact was not known at the outset of the current study and was not taken into consideration in the experimental design, the genetic heterogeneity of natural communities of Achromatium has considerable bearing on the interpretation of the data from this study.

In addition to the phylogenetic affiliation of Achromatium spp. with known sulfur-oxidizing chemolithoautotrophs, the presence of sulfur inclusions within Achromatium spp. (17) and the predominance of these organisms in the micro-oxic zone of sediments (2, 8, 12) have suggested that these organisms may also be sulfur-oxidizing chemolithoautotrophs. However, until recently, no direct evidence to support this notion had been obtained. Ecophysiological studies demonstrated that Achromatium cells were capable of oxidizing reduced sulfur to sulfate (8), suggesting that they conserve energy from sulfur oxidation (31), but to date no information on carbon metabolism in Achromatium spp. has been available.

Microautoradiography has the potential to address a number of specific questions relating to substrate uptake by natural populations of uncultured bacteria and has been applied to the study of bacterial activity in a number of distinct environments e.g., references (15), (16), and (21). Bacteria from the genus Achromatium have eluded all attempts at cultivation, and in order to study their metabolic potential, we have applied microautoradiography to the study of carbon and sulfur metabolism in natural communities of these bacteria.

	MATERIALS AND METHODS

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Study sites and sampling. Sediment samples containing Achromatium cells were collected from two locations in the United Kingdom. The sampling site at the margins of Rydal Water (54°27'N, 3°00'W) has been described previously (8, 12). The second site, Hell Kettles (36), is located to the south of Darlington, County Durham, United Kingdom (54°29'N, 1°33'W). Hell Kettles comprise two pools formed by the dissolution and collapse of underlying limestone strata sometime in the year 1179 (36). Samples were taken from Croft Kettle, the more southerly of the two pools. The pH of overlying waters from Rydal Water and Hell Kettles sediments was 6.5.

Radiochemicals. All radiochemicals were purchased from Amersham International (Little Chalfont, Buckinghamshire, United Kingdom). The uptake of radiolabel from sodium [¹⁴C]bicarbonate (2.0 GBq/mmol), [1,2-¹⁴C]acetic acid (2.07 GBq/mmol), D-[U-¹⁴C]glucose (11.2 GBq/mmol), L-U-¹⁴C-protein hydrolysate (>1.85 GBq/milliatom of carbon), and sodium [³⁵S]sulfate (4.162 GBq/mmol) by Achromatium cells was tested.

Uptake of ¹⁴C-labelled compounds. In separate incubations, 0.4 MBq of radiochemical was added. This quantity equated to 0.2 µmol of bicarbonate, acetate, and protein hydrolysate and 0.035 µmol of glucose. The final amount of glucose added was adjusted to 0.2 µmol by the addition of unlabelled glucose to provide similar added specific activities for all substrates. We recognize that the indigenous concentrations of these substrates will affect their specific activities in sediment cores. This fact is principally a consideration if quantitative inferences regarding the level of radiolabelled substrate assimilation by individual cells are made. Consequently, we have not interpreted the level of substrate uptake by individual Achromatium cells quantitatively. Control incubations containing a 2% (wt/vol) final concentration of buffered formaldehyde (pH 6.5, 0.1 M phosphate buffer) were also prepared. Incubations were performed in the dark for 3 h to limit the formation of ¹⁴CO₂ from the degradation of organic substrates by heterotrophic bacteria (15, 16). All incubations were carried out at 20°C. Acetate, glucose, and amino acid uptake experiments were conducted with sediments sampled on 10 June 1997 (Hell Kettles) and 4 July 1997 (Rydal Water). Bicarbonate uptake experiments were conducted with sediments sampled on 18 March 1998 (Hell Kettles) and 28 April 1998 (Rydal Water).

Fate of ³⁵S from radiolabelled sulfate in Achromatium communities from Rydal Water. Na₂³⁵SO₄ (0.5 Mbq) was added to cores sampled on 13 August 1997, producing a final total sulfate concentration of approximately 18 µM. Negative controls were prepared by the addition of buffered formaldehyde (2% [wt/vol] final concentration) or sodium molybdate (2 mM final concentration). Incubation was carried out for 18 h at 20°C. After incubation, the cells were extracted from the cores, partially purified, washed, placed on slides, and dipped as described above. Alternatively, after incubation with Na₂³⁵SO₄, partially purified, washed preparations of Achromatium cells were treated with methanol (300 µl) and incubated overnight to remove elemental sulfur. The extraction of elemental sulfur was determined microscopically, and the extracted cells were washed, placed on slides, and dipped as described above. The percentage of cells assimilating radiosulfur was determined by analyzing 100 randomly selected cells. The presence of radiolabelled elemental sulfur in the methanol extract was determined by an adaptation of a previously published method (35). Briefly, a 20-ml-capacity crimp-top vial (SH Scientific) containing a smaller vial into which lead acetate paper was placed was sealed with a butyl rubber septum and flushed with nitrogen. The methanol extract was injected into the larger outer vial, and 1 M chromous chloride (1 ml) was added. The reduction of dissolved elemental sulfur by chromous chloride to produce hydrogen sulfide was allowed to proceed at room temperature for 6 h (35). The apparatus was dismantled, and Pb³⁵S trapped by the lead acetate paper was detected with a Geiger counter (Morgan series 900 mini-monitor; Mini Instruments Ltd., Burnham-on-Crouch, Essex, United Kingdom).

	RESULTS AND DISCUSSION

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The ability of freshwater colorless sulfur bacteria to couple sulfur oxidation to energy generation or biosynthesis has been a subject of debate (7, 10, 11, 23), and colorless sulfur bacteria have been categorized by their specific metabolic use of reduced sulfur species, inorganic carbon, and organic carbon (29). For example, obligate chemolithoautotrophs obtain all their cellular carbon from inorganic sources, deriving energy and reducing power from the oxidation of reduced sulfur species. Facultative chemolithoautotrophs are able to do the same but are also able to grow mixotrophically or heterotrophically; mixotrophy in sulfur bacteria encompasses the simultaneous use of both organic carbon and inorganic carbon for biosynthesis and energy generation from organic carbon and reduced sulfur compounds (29). Chemolithoheterotrophs conserve energy from sulfur oxidation and use organic carbon for biosynthesis. Sulfur-oxidizing chemoorganoheterotrophs are defined as organisms deriving no energy or reducing power from the oxidation of reduced sulfur, relying instead on organic carbon for both. The oxidation of sulfur in these bacteria has been attributed to detoxification processes (19, 29, 31). Given this nutritional diversity in sulfur bacteria, knowledge of the use of reduced sulfur species and of inorganic and organic carbon is essential for an understanding of the physiology and hence the ecological role of Achromatium.

Assimilation of organic and inorganic carbon in Achromatium communities. Achromatium cells from Rydal Water and Hell Kettles incorporated ¹⁴C from both organic and inorganic carbon sources (Fig. 1 to 3). A proportion of the cells present in both Rydal Water and Hell Kettles proved capable of assimilating carbon from acetate and protein hydrolysate (Fig. 2). The uptake of radiolabelled glucose was observed for only a very small number of cells (Fig. 2). In subsequent experiments, the uptake of radiolabelled bicarbonate was investigated. Although bicarbonate was assimilated by Achromatium cells from Rydal Water and Hell Kettles sediments (Fig. 1A and D and Fig. 3), following decalcification with dilute HCl, only cells from Rydal Water sediments retained the ¹⁴C label (Fig. 1B and E and Fig. 3). The incorporation of ¹⁴C from inorganic carbon into cellular organic material (and thus the potential to fix inorganic carbon for biosynthesis) apparently is a feature only of Achromatium cells present in Rydal Water sediments.

View larger version (164K): [in this window] [in a new window]   FIG. 1.   Microautoradiographs of Achromatium cells purified from sediment cores amended with 14C-labelled bicarbonate. (A to C) Cells from Rydal Water sediments. (D to F) Cells from Hell Kettles sediments. Cells from Rydal Water and Hell Kettles sediments both incorporated 14C from labelled bicarbonate (A and D). However, decalcification (see Materials and Methods) removed 14C from cells present in Hell Kettles sediments (E) but not from cells present in Rydal Water sediments (B). This result indicated that 14C was incorporated into cellular organic carbon in cells from Rydal Water but not in cells from Hell Kettles. Cells from formaldehyde-treated sediment cores did not incorporate any radiolabel (C and F). Note that in decalcified samples, cells incorporating the radiolabel are not visible within the halo of silver halide particles, presumably because removal of the calcite inclusions results in collapse of the cell and an even coating of photographic emulsion over the surface of the cell is obtained. In cells that retain intracellular calcite, a portion of the cell is raised above the surface of the emulsion. Achromatium cells or groups of cells are indicated by arrows. Cells from Hell Kettles are larger than those from Rydal Water (9). The scale bar represents 20 µm and applies to all micrographs.

View larger version (11K): [in this window] [in a new window]   FIG. 2.   Proportion of cells in Achromatium communities incorporating 14C from radiolabelled organic substrates. Uptake of 14C from radiolabelled acetate, glucose, and protein hydrolysate by Achromatium cells from Rydal Water sediments and Hell Kettles sediments was measured. The data represent the mean ± standard error (n = 3). In sediment cores treated with formaldehyde (2% [wt/vol]), no Achromatium cells that had incorporated 14C were detected.

View larger version (11K): [in this window] [in a new window]   FIG. 3.   Proportion of cells in Achromatium communities incorporating 14C-labelled inorganic carbon. Uptake of 14C from radiolabelled sodium bicarbonate by untreated and decalcified Achromatium cells from Rydal Water sediments and Hell Kettles sediments was measured. No decalcified cells from Hell Kettles that had incorporated 14C were detected. The data represent the mean ± standard error (n = 3). In sediment cores treated with formaldehyde (2% [wt/vol]), no Achromatium cells that had incorporated 14C were detected.

Fate of ³⁵S-labelled sulfate in Achromatium-bearing sediments. Microautoradiographic studies of Achromatium communities in Rydal Water sediment clearly demonstrated that sulfur from ³⁵S-labelled sulfate was incorporated into Achromatium cells (Fig. 4A and 5). However, this process was inhibited in the presence of sodium molybdate (Fig. 4B and 5). Given that sodium molybdate probably inhibits the binding and transport of sulfate across the cell membrane (24) and blocks the formation of adenosine-5'-phosphosulfate (APS), an important intermediate in both assimilatory and dissimilatory sulfate reduction (26), there are two possible explanations for these results. First, following the reduction of sulfate to sulfide by dissimilatory sulfate-reducing bacteria, the sulfide was oxidized by Achromatium cells to elemental sulfur, which was deposited intracellularly. When dissimilatory sulfate reduction was inhibited by the addition of sodium molybdate, the production of sulfide and its subsequent conversion to elemental sulfur by Achromatium cells were prevented. Second, Achromatium cells accumulated ³⁵S from radiolabelled sulfate via an assimilatory sulfate reduction pathway and incorporated it into cellular organic sulfur, a process inhibited in the presence of molybdate (28). The first alternative was confirmed by extraction of the radiolabelled cells with methanol to remove elemental sulfur. This procedure resulted in the removal of radioactivity (Fig. 4C and 5). To preclude the possibility that the methanol treatment removed intracellular ³⁵S-labelled sulfate or organosulfur compounds, the methanol extract was treated with chromous chloride. Unlike elemental sulfur, sulfate and organosulfur compounds are not reduced to sulfide by this treatment (3). Consequently, upon treatment with chromous chloride, these compounds remain in the methanol phase, whereas elemental sulfur is converted to ³⁵S-sulfide and can be trapped by lead acetate paper, with the formation of lead sulfide. The recovery of ³⁵S as lead sulfide by this procedure confirmed that ³⁵S-elemental sulfur was present within Achromatium cells.

View larger version (95K): [in this window] [in a new window]   FIG. 4.   Microautoradiographs of Achromatium cells purified from Rydal Water sediment cores amended with 35S-labelled sodium sulfate. (A) Cells from a sediment core treated with radiolabelled sodium sulfate. (B) Cells from a sediment core treated with radiolabelled sodium sulfate and 2 mM sodium molybdate. (C) Cells from a sediment core treated with radiolabelled sodium sulfate and extracted with methanol to remove intracellular elemental sulfur prior to exposure. (D) Cells from a sediment core treated with radiolabelled sodium sulfate and formaldehyde (2% [wt/vol]). Achromatium cells are indicated by arrows. The scale bar represents 20 µm and applies to all micrographs.

View larger version (13K): [in this window] [in a new window]   FIG. 5.   Proportion of cells incorporating 35S from radiolabelled sodium sulfate in the Achromatium community from Rydal Water sediments. The data represent the mean ± standard error (n = 3). In sediment cores treated with formaldehyde (2% [wt/vol]), no Achromatium cells that had incorporated 35S were detected.

Conclusions. Without access to pure cultures, determining the physiology and ecological role of Achromatium has proved difficult (13). This task has been further complicated by the fact that populations of Achromatium originally thought to contain a single species (A. oxaliferum) have now been shown to comprise several ecologically distinct species (9, 13). A simple interpretation of the data presented in this study without this understanding of Achromatium diversity would be that the organism is mixotrophic, utilizing simple organic compounds as well as conserving energy from the oxidation of reduced sulfur species to sulfate and, when conditions allow, fixing inorganic carbon for biosynthesis. However, this simple interpretation is not valid, and Achromatium spp. appear to be nutritionally heterogeneous.