Characterization and Localization of Iron-Oxidizing Proteins in Acid Mine Drainage Biofilms
Abstract
As molecular geomicrobiologists, we are interested in the microbially-produced molecules that effect geochemical transformations, particularly proteins involved in lithotrophic energy generation. We have identified two such proteins produced by Leptospirillum group II microbes, which dominate biofilms floating on acidic waters in the Richmond Mine at Iron Mountain, CA. Leptospirillum generates energy by iron oxidation, producing the ferric iron catalyst responsible for pyrite oxidation, subsequent acid generation and toxic metal release. We have shown that a small (~16 kDa) soluble protein, cytochrome-579, extracted from environmental biofilm samples is capable of iron oxidation in vitro, consistent with prior studies on similar cytochromes from L. ferriphilum and ferrooxidans (Blake et al., 1993; Hart et al., 1991). The abundance of cyt579 and its ability to oxidize iron makes it a key link between microbial metabolism and acid mine drainage. Given the importance of cyt579 in biofilm sustenance as well as acid generation, we want to understand more about its distribution and also the architecture of the biofilm environment in which it functions. Using transmission electron microscopy (TEM) on ultrathin sections, we observe biofilms as thin as 15 microns with densely-packed cells in a matrix of polymers. To localize cyt579 in the biofilm, we purified the protein and developed antibodies for immunolabeling. The antibodies were shown to be highly specific for cyt579 using Western blots of whole biofilm lysate. Fluorescence- and gold-labeled secondary antibodies were used to visualize immunolabeled biofilms by confocal laser scanning microscopy and TEM, respectively. Preliminary results suggest that the cytochrome is on the bacterial cell surface or in the periplasm but not throughout the biofilm, as we had postulated due to the abundance of cytochrome in extracellular fractions of biofilm samples. These localization studies will be helpful in determining the mechanism of cyt579 in various biofilms and growth stages. Cytochrome 579 is unique in that its heme spectral signature is not typical of any a, b, or c-type cytochromes (Blake et al., 1993; Ram et al., 2005). Thus, it is interesting to note that we have extracted a second, abundant, membrane-bound cytochrome with a very similar spectrum, differing in that it has a characteristic absorbance peak at 575 nm, instead of 579 nm. N-terminal sequencing indicates that cyt575 is also produced by Leptospirillum group II. Cyt575 and cyt579 may belong to a new class of acid-stable cytochromes. Its abundance suggests that cyt575 is also involved in energy generation from iron oxidation, though we are currently investigating its role in the electron transport chain.
- Publication:
-
AGU Fall Meeting Abstracts
- Pub Date:
- December 2005
- Bibcode:
- 2005AGUFM.B11B..03C
- Keywords:
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- 0330 Geochemical cycles (1030);
- 0414 Biogeochemical cycles;
- processes;
- and modeling (0412;
- 0793;
- 1615;
- 4805;
- 4912);
- 0448 Geomicrobiology;
- 0456 Life in extreme environments;
- 0465 Microbiology: ecology;
- physiology and genomics (4840)