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1 The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, 7 MBL Street, Woods Hole, Massachusetts
2 The BioCurrents Center, Marine Biological Laboratory, 7 MBL Street, Woods Hole, Massachusetts
* To whom correspondence should be addressed. E-mail: sogin{at}evol5.mbl.edu
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Abstract |
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Earth harbors many extreme environments. Previous investigations of the microbial diversity in these environments have been constrained by preconceived notions about the range of habitability for both eukaryotic and prokaryotic microorganisms. We have been exploring the genetic and physiological diversity of organisms living at pH extremes, both acidic (pH < 3) and alkaline (pH > 10). Our research ranges from environments like the warm (45 °C), acidic (pH 2.7) Nymph Creek in Yellowstone National Park to temperate alkaline lakes in the Sandhills region of western Nebraska. The focus of this report is the acidic, heavy-metal-rich Rio Tinto in southwestern Spain.
The Rio Tinto flows 100 km through the world’s largest pyritic (FeS2) belt. The river gets its red color from the high levels of iron dissolved in its acidic waters (pH 
2.0). Ferric hydroxide (Fe3+/Fe(OH)3, pKa 2.5) and SO42-/HSO4- (pKa 2.0) act as buffers to maintain the pH of the river at about 2. The concentration of iron can be as high as 20 g/l, and the river also contains other heavy metals at concentrations orders of magnitude higher than those in typical freshwater environments.
Much of the past research on the Rio Tinto has focused on the prokaryotes that play an important role in shaping the acidic environment of the Rio Tinto through their metabolism of iron-rich pyrite and chalcopyrite. Recent paleontological research shows that iron-oxidizing bacteria existed in the Rio Tinto river basin 300,000 years ago, long before its 5000-year mining history (Leblanc et al., 2000). Other chemolithotrophs such as sulfur-oxidizing bacteria and archaea also contribute to the river’s probably ancient ecosystem structure (Gónzalez-Toril et al., 2001).
Some of these prokaryotes, along with fungi, contribute to the formation of biofilms on the surface of rocks. These biofilms, in turn, are the site of metal and mineral precipitation that ultimately forms stromatolites. Biofilms provide a substrate for communities to develop within the river. However, in many parts of this river basin, eukaryotic microbes (Fig. 1) are the major contributors of biomass (López-Archilla et al., 2001). Eukaryotes not only form the foundations of some of these biofilm communities, but they are also conspicuous inhabitants of them (López-Archilla et al., 1993, 1994, 1995; López-Archilla and Amils, 1999). This makes the Rio Tinto system unique among acidic environments described to date.
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Our studies also revealed a diversity that we were not able to readily characterize using molecular techniques. Clones such as RT5iin16 and RT5iin14 are most likely examples of novel eukaryotic lineages that at best branch at the base of the animal-fungal-nucleariid radiation. Other clones (RT5iin21 and RT5iin44) branched with the recently sequenced filose amoeba Filamoeba nolandi, whose own taxonomic placement is equivocal. Because our study was not exhaustive, we surmise that there are still more undiscovered novel lineages in the river.
Despite our growing knowledge of the Tinto’s eukaryotic diversity, we know little about the role eukaryotes play in shaping the varied ecosystems that occur along the river. For example, we do not know if these biofilm communities have microenvironments that enhance survival of their members. Could fungal metal sequestration protect nontolerant species? Furthermore, we know little about how these organisms have evolved adaptations to extreme concentrations of acid and metals.
To explore these questions, we have been isolating organisms from the river for ex situ physiological experiments. We have established monocultures of Chlamydomonas sp., Euglena cf. mutabilis, Chlorella sp., and Vannella sp. isolated from enrichments of river water and are currently exploring the physiology of these protists from extreme environments.
We have initiated our physiological studies on an acidophilic species of a chlamydomonad alga isolated from the river—Chlamydomonas sp. Our first question about the physiology of the Tinto acidophiles was the nature of the cytosolic pH (pHi). There are published reports of acidophiles from all domains of life with internal pH values that deviate from neutral—these include the archaebacterium Picrophilus oshimae, pHi = 4.6 (van de Vossenberg et al., 1998); the eubacterium Bacillus acidocaldarius, pHi = 5.6–5.8 (Thomas et al., 1976); and the eukaryotic alga Euglena mutabilis, pHi = 5.0–6.4 (Lane and Burris, 1981). Using the fluorescent H+ indicator BCECF, we determined that our acidophilic chlamydomonad isolate maintains an average internal pH of 6.6 at an external pH of 2 (M. A. Messerli, L. A. Amaral Zettler, S.-K. Jung, P. J. S. Smith, and M. L. Sogin, unpubl.). Our other isolates await similar measurements.
Given that there is a 40,000-fold difference in hydrogen ion activity between the inside and the outside of these cells, we propose the existence of active transport mechanisms that help these organisms regulate their internal pH. We hypothesize that novel diversity in H+-ATPases may explain the ability of different protist species to thrive in the low pH, high-metal Rio Tinto environment. There are two major families of H+-ATPases: the V/F/A-ATPases and the P-type-ATPases. The V-type ATPases can occur in the plasma membrane of eukaryotes (but are more commonly associated with vacuolar membranes) and consist of at least 11 subunits and a molecular mass approaching 106 Da. In contrast, eukaryotic P-type ATPases consist of either monosubunits (as with H+-ATPases) or a hetero-subunit (alpha and beta, as found in the Na+/K+-ATPases and H+/K+-ATPases); have a molecular weight of about 100 kDa; and form a phosphorylated intermediate during the course of ATP hydrolysis. Indirect evidence of novel ATPases comes from studies of the protozoan parasite Leishmania donovani, which has the ability to switch between living in a neutral environment, pH 7.5, as a promastigote (flagellated stage) and in an acidic environment, pH 5.0, as an amastigote (nonflagellated stage) (Meade et al., 1989). The plasma membrane of this organism contains a P-type ATPase that has two isoforms with slightly different sequences. Isoform 1a is expressed in both promastigotes and amastigotes, whereas isoform 1b is expressed more abundantly in the amastigotes (Meade et al., 1989). This difference suggests the use of a sequence change to accommodate the acidic condition. Modifications to ion regulatory machinery might be reflected by convergent amino acid substitution patterns or by accelerated rates of change in acidophilic protist lineages, as revealed in phylogenetic analyses. For example, portions of membrane-bound V- and P-type ATPases that are exposed to the acidic external environment may display different amino acid substitution patterns than do domains that face the cytoplasm.
We are currently using degenerate primers designed against two conserved regions, the phosphorylation site and the ATP-binding site, to amplify members of the P-type superfamily of ion transporters. Thus far, all of our clones fall into the heavy-metal P-type class but may represent different metal transporters. We have found more diverse sequences in the acidophilic Chlamydomonas than in the neutrophilic C. reinhardtii. We are screening additional clones for H+-transporting ATPases.
Once we obtain ion-transporter sequence information from these acidophiles, we will focus on correlating the expression of these transporters in space and time to biogeochemical characteristics in the river. This will bring us beyond the study of biodiversity in the river to questions at the heart of potential genomic interactions between members of the microbial consortia. With this kind of approach, we may also be able to determine whether symbiotic interactions are occurring in this environment.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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This article has been cited by other articles:
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  B. J. Baker, M. A. Lutz, S. C. Dawson, P. L. Bond, and J. F. Banfield Metabolically Active Eukaryotic Communities in Extremely Acidic Mine Drainage Appl. Envir. Microbiol., October 1, 2004; 70(10): 6264 - 6271. [Abstract] [Full Text] [PDF]
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