1 School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK
2 Centre for Ecology, Evolution and Conservation, University of East Anglia, Norwich NR4 7TJ, UK
Correspondence
Philip Bond
phil.bond{at}uea.ac.uk
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ABSTRACT |
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Supplementary tables with details of the proteins induced under various conditions are available with the online version of this paper.
Present address: Molecular Biology, Umeå University, S-90187 Umeå, Sweden.
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INTRODUCTION |
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Ferroplasma spp. are of particular interest as they have been isolated from the most acidic and metal-rich natural environment reported to date (Edwards et al., 2000; Nordstrom & Alpers, 1999
), as well as having been detected from a number of other AMD sites and commercial bioleaching plants (Dopson et al., 2004b
; Golyshina et al., 2000
; Gonzalez-Toril et al., 2003
; Kinnunen & Puhakka, 2004
; Okibe et al., 2003
). The genus includes Ferroplasma acidarmanus Fer1 isolated from Iron Mountain, CA, USA, where typically the drainage water pH is between 0·5 and 1·0 and metal ion concentrations are in the range of tens of grams per litre (Edwards et al., 2000
). The Fer1 genome has been sequenced (97 % complete) and draft results are available at http://genome.ornl.gov/microbial/faci/. Other Ferroplasma strains include Ferroplasma acidiphilum YT, Y-2, MT17 and DR1, all isolated from bioreactors for metal removal (Dopson et al., 2004b
; Golyshina et al., 2000
; Okibe et al., 2003
; Pivovarova et al., 2002
). A further Ferroplasma lineage is detected from an Iron Mountain biofilm via DNA sequencing directly from the biofilm, termed Ferroplasma Type II (Tyson et al., 2004
). Finally, a Ferroplasma-like isolate, which grows optimally at 55 °C, has recently been identified from a chalcocite heap leach operation in Perth, Western Australia (Hawkes et al., 2005
).
Ferroplasma spp. are capable of chemo-organotrophic growth on yeast extract (Baumler et al., 2005; Dopson et al., 2004b
). They also gain energy via oxidation of Fe2+ (Dopson et al., 2004b
; Golyshina et al., 2000
). However, with the exception of Acidithiobacillus ferrooxidans (Brasseur et al., 2004
; Elbehti et al., 1999
; Yarzabal et al., 2003
, 2004
), little is known regarding the biochemistry of oxidation of Fe2+ in acidophiles. But, from a biotechnological viewpoint and in conditions of very low pH AMD, Fe2+ oxidation is probably more important in Ferroplasma spp., as they are the predominant iron oxidizers present at low pH and they have been found in many industrial bioleaching operations (Edwards et al., 2000
). Furthermore, preliminary investigations of acidophile Fe2+ oxidation indicate that mechanisms of other acidophiles differ from that of A. ferrooxidans (Barr et al., 1990
; Blake et al., 1993
; Hart et al., 1991
). A putative electron transport chain for Ferroplasma Type II is suggested to contain components involved in oxidation of both organic carbon and Fe2+ (Tyson et al., 2004
).
Availability of organic carbon is important to the microbial biochemistry at AMD-producing sites. Organic carbon inhibits the growth of many extreme acidophiles, a result of their reversed membrane potential and accumulation of organic acids that results in acidification of the cytoplasm (Alexander et al., 1987). However, Ferroplasma spp. grow chemo-organotrophically by aerobic and anaerobic respiration at pH 1·0 (Baumler et al., 2005
; Dopson et al., 2004b
). It is likely that acid-producing sites such as Iron Mountain receive little fixed carbon from external sources. Presently there is some incongruity concerning the autotrophic capabilities of Ferroplasma spp. From environmental genomic information some acidophiles detected at Iron Mountain are considered to be autotrophic. Genes for CO2 fixation attributed to Leptospirillum strains have been detected, and a mechanism for CO2 fixation is suggested for Ferroplasma Type II (Tyson et al., 2004
). However, both Ferroplasma strains MT17 and Fer1 are described as organotrophic iron oxidizers (Okibe & Johnson, 2004
). In contrast, Ferroplasma YT and Y-2 are described as able to fix CO2 for organic carbon and only require yeast extract as an essential growth factor, and these strains are classified as autotrophs (Golyshina et al., 2000
; Pivovarova et al., 2002
). Our recent characterization failed to conclusively prove CO2 fixation by either strain YT or strain Fer1 (Dopson et al., 2004b
). Ferroplasma spp. in general and the issue of CO2 fixation in particular has been recently reviewed (Golyshina & Timmis, 2005
). It is of critical importance to elucidate the exact biochemical mechanisms of CO2 fixation/carbon assimilation in these organisms because of their importance as intermediates in key geochemical cycling, such as the mobilization of heavy metals (Dopson et al., 2003
), and their role in catalysing AMD and bioleaching.
The Ferroplasmaceae represent a particularly important group of organisms from both an environmental (AMD genesis) and biotechnological perspective (bioleaching capability and a possible source of acid-tolerant proteins for industrial applications). In this study we combine proteomic analyses of different growth states with the characterization of electron transport. This is believed to constitute the first detailed proteomic examination of growth in an acidophilic archaeon, and insight has been gained into metabolic and physiological details of Ferroplasma strains.
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METHODS |
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Growth in continuous culture.
For comparison of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) protein expression profiles, cells were grown to steady state in glass air-lift continuous-culture vessels (Dopson et al., 2004b). All vessels were sparged with
800 ml air min1; the dissolved oxygen concentration was measured for chemomixotrophic growth and found to be 96·1 % of the oxygen concentration in air at 8·6 mg protein l1. Continuous flow of the medium was as stated in Table 1
and the vessels were regularly sampled and protein concentration measured (as described above). Flow rates for the chemomixotrophic and chemo-organotrophic continuous cultures were based on maximum rates obtained for batch growth under the respective conditions [4·2 and 9·6 h for chemomixotrophic and chemo-organotrophic conditions, respectively (Dopson et al., 2004b
)].
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Protein excision and mass spectrometry.
A portion of the protein spots of interest were punched out of EZBlue or mass spectrometry compatible silver-stained gels with a sterile micropipette tip. Samples were treated by trypsin digestion and analysed using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (Bruker Reflex III; Hesketh et al., 2002). MALDI-TOF peptide mass fingerprint data were matched directly against the F. acidarmanus Fer1 genome sequence data (http://genome.ornl.gov/microbial/faci/) using MASCOT (http://www.matrixscience.com). Results were only included when peptide matching was statistically significant (MOWSE score greater than 75). Putative protein functions were inferred from the analyses contained in the annotated genome site (BLAST, KEGG hit, COGS Comparison, PFam model Comparison, and InterPro). Inferences regarding gene operons were drawn from STRING (http://www.bork.embl-heidelberg.de/STRING/).
Extraction and analysis of membrane proteins.
F. acidarmanus Fer1 cells were harvested by centrifugation at 10 000 g for 20 min and washed twice in MSM pH 1·2. Pellets were resuspended in 15 ml 50 mM Tris pH 8+250 mM sucrose+1 mM EDTA+Complete protease inhibitor cocktail (Roche). The cells were disrupted by passing twice through a French press at 1100 p.s.i. (7·6 MPa) and centrifuged at 5000 g for 10 min to remove whole cells and large debris. Membranes were pelleted by centrifugation at 120 000 g for 30 min. The resulting membrane pellet was resuspended in 500 µl 10 mM Tris+1 mM EDTA (TE)+2 % dodecyl maltoside and proteins were dissolved by rocking gently overnight at 4 °C. Undissolved proteins were removed by centrifugation at 120 000 g for 30 min and the remaining solubilized protein concentration was quantified using Bio-Rad protein assay reagent (as above). Protein was separated via one-dimensional polyacrylamide gel electrophoresis (1D-PAGE; Sambrook et al., 1989) and stained with either Coomassie or haem stain (Thomas et al., 1976
). Differentially expressed protein bands in the 1D-PAGE gel were detected and quantified using Quantity One (Bio-Rad) and chosen bands excised and identified by mass spectrometry (as described above). UV-visible spectra of membrane proteins were obtained using a Hitachi U4001 UV-visible spectrophotometer and membrane fractions were analysed as prepared (oxidized) and after addition of sodium dithionite (reduced). Fe(CN)6 (2 mM) was added to ensure that the membrane redox proteins were fully oxidized, whilst 2 and 1·5 mM dithionite for the chemomixotrophic and chemo-organotrophic membranes, respectively, was added to reduce the proteins.
Measurement of Fe2+ oxidation in the presence and absence of inhibitors.
F. acidarmanus Fer1 cells were grown in continuous culture vessels (as described above) and harvested by centrifugation at 10 000 g for 10 min. The cells were washed in MSM and then Fe2+ oxidation was detected in the presence and absence of inhibitors in a thermostatted, stirred reaction chamber at 37 °C. The reaction mixture (1 ml total volume) contained MSM (without trace elements), inhibitors (as indicated), and cell mass equivalent to 10 µg protein. The cells were allowed to equilibrate at 37 °C for 3 min in the thermostatted chamber before the reaction was initiated by the addition of 50 µM Fe2+. Fer1 resting cells were stored 2 h prior to use. Samples (100 µl) were removed at the indicated time points, diluted in a Tris buffer (100 mM Tris, pH 7), and the Fe2+ concentration analysed utilizing ferrozine (Dawson & Lyle, 1990
).
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RESULTS |
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Growth of Ferroplasma strains in continuous culture
For the purpose of proteomic analyses, all four Ferroplasma strains were grown in continuous culture to steady state under substrate-limiting conditions (Table 1). Increases in yeast extract resulted in increased steady-state protein concentrations, e.g. with 0·02 and 0·04 % (w/v) yeast extract the F. acidarmanus Fer1 steady-state protein concentration was 7·5±2·4 and 16·5±2·4 mg protein l1, respectively (n=7). The Fe2+ concentration inside a subsequent chemomixotrophic continuous-culture vessel incubated under the same conditions was found to be 0·5±0·2 mM Fe2+ (n=11 over a 17 day period). F. acidarmanus Fer1 steady-state protein concentration under chemo-organotrophic conditions was lower than that observed for chemomixotrophic growth on Fe2+ and yeast extract, probably reflecting the extra energy the organism obtains while oxidizing Fe2+ as opposed to growing on organic carbon alone (Table 1
). For protein expression comparisons, cells were harvested from the continuous-culture vessel during steady-state growth (except for incubations with no organic carbon addition), thus avoiding pitfalls in comparison of the vastly uneven growth conditions and states that can occur in batch culture.
Comparative protein expression of F. acidarmanus Fer1 grown under chemomixotrophic and chemo-organotrophic conditions
When F. acidarmanus Fer1 was grown under chemomixotrophic (Fig. 1a) and chemo-organotrophic (Fig. 1b
) conditions, many of the proteins were common between the sets of 2D-PAGE separations. However, 52 % or the proteins were more than twofold differentially expressed when comparing the two conditions.
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Under chemo-organotrophic conditions, F. acidarmanus Fer1 up-regulated 15 proteins with high homology to proteins having a suggested function in central metabolic pathways (see Supplementary Table S1). These included proteins similar to a transketolase that is a link between glycolysis and the pentose phosphate pathway; glyceraldehyde-3-phosphate dehydrogenase, which plays an important role in glycolysis and gluconeogenesis; a flavoprotein involved in central fermentation pathways; a further flavoprotein that has been implicated in the transfer of electrons from a dehydrogenase to a ubiquinone oxidoreductase; and glucose dehydrogenase. Four enzymes with similarity to those involved in the tricarboxylic acid (TCA) cycle were up-regulated; these include two subunits (expressed in a single operon in Thermoplasma acidophilum and Thermoplasma volcanium) of the pyruvate/2-oxoglutarate dehydrogenase complex that produces acetyl-CoA feeding into the TCA cycle (Patel & Roche, 1990), aconitate hydratase, succinyl-CoA synthetase
subunit, and succinate dehydrogenase. Proteins with homology to 3-hydroxyacyl-CoA dehydrogenase, glutamate/leucine dehydrogenase and acyl-CoA dehydrogenase involved in fatty acid metabolism (another central metabolic process) were also up-regulated. Other proteins up-regulated had homology to the thermosome
subunit and the 20S proteasome. In archaea these stress proteins protect intracellular proteins from aggregation as well as aiding protein translocation across the membrane (Klumpp & Baumeister, 1998
). A protein similar to a member of the major facilitator permease family was also up-regulated. This probable sugar permease may be up-regulated to provide substrate for central metabolic pathways, in contrast to chemomixotrophic growth, where energy would be obtained from Fe2+ oxidation.
F. acidiphilum YT proteins expressed in the presence and absence of yeast extract
Differences of protein expression from F. acidiphilum YT were detected during continuous culture on Fe2+ in the presence and absence of organic carbon (Fig. 1eg). While steady-state growth was not achieved in the absence of yeast extract addition, cells were harvested for protein extraction following continuous culture incubation for three reactor volumes (28 days). The proteome of these cells would be indicative of extremely oligotrophic conditions. During incubation of F. acidiphilum YT on Fe2+ in the absence of organic carbon, four proteins with homology to anti-oxidants and related proteins were up-regulated (see Supplementary Table S2). These were superoxide dismutase, which converts toxic superoxides to the less toxic hydrogen peroxide (Cannio et al., 2000
); two proteins similar to a peroxiredoxin subunit (an alkyl hydroperoxide reductase), which is the primary scavenger for hydrogen peroxide in E. coli (Seaver & Imlay, 2001
); and finally, a protein with homology to superoxide-inducible cysteine synthase. Other proteins up-regulated during incubation of F. acidiphilum YT in the absence of organic carbon were similar to thermosome
and
subunits (chaperone HSP60 family group II) and a component of the H+ ATP synthase. Furthermore, two proteins with significant DNA sequence homology to methionine synthase II were expressed. This appears to be a gene duplication event, and although the sequences had different percentage identities, the same gene arrangement is apparent in the genomes of Thermoplasma spp. and Pyrococcus spp.
A F. acidiphilum YT protein with significant gene sequence homology to an anti-oxidant hydroperoxide reductase (Supplementary Table S2) was up-regulated during chemomixotrophic growth compared to incubation without added organic carbon. Three proteins with homology to transcription and translation components were up-regulated; these were a ribosomal protein L7A, an eIF-2B initiation factor, and a DNA primase, possibly reflecting the increased growth rate. In addition, proteins similar to a number of electron transport and metabolic proteins possibly involved in growth on organic substrates were up-regulated. Among others, these included pyruvate/ferredoxin oxidoreductase, which forms acetyl-CoA, and an uncharacterized NAD(FAD)-dependent dehydrogenase, which possibly feeds electrons into the electron transport chain.
Comparative protein expression of chemomixotrophic growth between F. acidiphilum YT and F. acidarmanus Fer1
To investigate differences between Ferroplasma spp., protein expression profiles during chemomixotrophic growth were compared (Fig. 1a, e, h). Of the proteins detected, 21 and 31 % were uniquely expressed by F. acidarmanus Fer1 and F. acidiphilum YT, respectively (data not shown). Many of the proteins up-regulated in F. acidiphilum YT had high homology to proteins involved in central energy production from organic carbon (see Supplementary Table S3). These included malate dehydrogenase in the TCA cycle, fructose 1,6-bisphosphatase and phosphoglycerate mutase involved in gluconeogenesis and both subunits of an aerobic carbon monoxide dehydrogenase involved in acetyl-CoA metabolism. Finally, proteins with high similarity to electron transfer proteins that may be involved in energy conservation were also up-regulated (contig 166, gene 1240; contig 166, gene 1220; and contig 168, gene 1522; see Supplementary Table S3).
Identification of respiratory complexes
Spectrophotometric scans of chemomixotrophically and chemo-organotrophically grown F. acidarmanus Fer1 membranes were successfully obtained with high signal-to-noise ratio (Fig. 2). Importantly, the membranes from the two growth conditions have quite distinct spectral characteristics, indicating differential expression of redox proteins. Each of the spectra has a number of different features that suggest contributions from a range of different protein-bound cofactors, including haems and ironsulfur clusters. From the chemomixotrophically grown cells the broad oxidized peak from approximately 415 to 455 nm is due to the Soret band and may be made up of a combination of cytochromes b and c from a terminal oxidase (Brasseur et al., 2004
; Komorowski et al., 2002
). The shift of the 423 nm peak to 437 nm is indicative of the reduction of a partially oxidized haem protein (423 nm) to the reduced state (437 nm). Characteristics of the observed spectra reflect differential protein expression. For example, the broad absorbance at 460510 nm is typical of FeS centres and this correlates with proteins identified as up-regulated in chemomixotrophic growth (contig 168, gene 1454 and contig 157, gene 545). The peak at 583 nm suggests the presence of cytochrome a583 found in Sulfolobus solfataricus strain 7 that has been tentatively suggested to have a cytochrome c-like function in terminal oxidases (Schafer et al., 1996
). The peak at 596 nm is possibly a blue copper-haem sulfocyanin, although it has been shown that the absorbance associated with a purified, truncated sulfocyanin is lost upon reduction (Komorowski & Schafer, 2001
). Such a protein may have an important role mediating Fe2+ oxidation similar to rusticyanin (as in A. ferrooxidans) or SoxE (as in Sulfolobus acidocaldarius), replacing the function of cytochrome c (Komorowski & Schafer, 2001
). An alternative explanation is that the peak is due to a cytochrome aa3 oxidase (peak at 597 nm) that has been identified in A. ferrooxidans when grown on Fe2+ (Brasseur et al., 2004
), although no genes encoding a cytochrome aa3 oxidase have been identified on the 97 % complete Fer1 genome. In addition, a protein containing c-type haem was detected in F. acidarmanus Fer1 grown under chemomixotrophic, but not chemo-organotrophic conditions (by haem-stained 1D-PAGE gel; Fig. 3
).
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F. acidarmanus Fer1 growth and ferrous iron oxidation in the presence of electron transport inhibitors
F. acidarmanus Fer1 was grown in the presence of electron transport inhibitors (Table 2) and their effect on Fe2+ oxidation in F. acidarmanus Fer1 resting cells was measured (Fig. 4
). Rotenone (a competitive inhibitor of NADH-ubiquinone reductase) did not have a statistically significant effect on chemomixotrophically grown F. acidarmanus Fer1, whereas it did inhibit chemo-organotrophic growth (Table 2
). Quinacrine (a competitive inhibitor of succinate dehydrogenase) inhibited 51 % of chemomixotrophic growth, but had no statistically significant effect on chemo-organotrophic growth (Table 2
). Antimycin A non-competitively reduces electron flow through the cytochrome b and ironsulfur protein in a bc1 complex or its analogue, whilst 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) inhibits most quinol oxidases and reductases. The point of cytochrome b inhibition by HQNO is at the site of ubiquinone reduction. Both antimycin A and HQNO strongly reduced chemomixotrophic and chemo-organotrophic growth (Table 2
), despite no cytochrome bc1 complex genes having been identified on the genome (Tyson et al., 2004
). It is possible that a cytochrome b and a FeS protein, detected in the membrane spectra, may act as a bc1 complex [similar to S. acidocaldarius (Komorowski et al., 2002
)] and inhibition of electron flow in this portion of the complex may result in the lowered growth. Although both antimycin A and HQNO inhibited growth, they do not significantly affect Fe2+ oxidation by F. acidarmanus Fer1 resting cells, even at high concentrations (Fig. 4a, b
). This suggests that electrons from the oxidation of Fe2+ bypass a bc1 complex or its analogue. In contrast, azide (a terminal oxidase inhibitor) completely inhibited Fe2+ oxidation at 100 µM, and only allowed 40 % of the available Fe2+ to be oxidized after 20 min at 1 µM (Fig. 4c
). The degree of inhibition by azide on chemomixotrophic and chemo-organotrophic growth was not statistically different.
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DISCUSSION |
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To facilitate discussion of our proteomic, genomic, membrane spectra, and inhibition studies, a very speculative model for electron transport in Ferroplasma spp. is provided (Fig. 5), with the suggested points of inhibition for each of the electron transport inhibitors given. It is possible that Fe2+ oxidation is mediated by a blue copper-haem sulfocyanin that is suggested to have a function similar to S. acidocaldarius SoxE and A. ferrooxidans rusticyanin (Tyson et al., 2004
). Following that, we suggest that transfer of the electron from Fe2+ oxidation to the cbb3 terminal oxidase occurs, without passing higher up the electron transport chain (Fig. 5
). This hypothesis is supported by the membrane scans, which indicated that a blue copper-haem sulfocyanin at 596 nm was present during chemomixotrophic growth but greatly reduced or absent during chemo-organotrophic growth. Our suggested model is also supported by the biochemical data, whereby HQNO and antimycin A did not inhibit Fe2+ oxidation, whereas the terminal electron acceptor inhibitor azide inhibited virtually 100 % of this activity at 10 µM. Only a few redox proteins were identified from 2D-PAGE to support this hypothesis. It is possible that Fe2+-oxidizing proteins are constitutively expressed, although detection of redox proteins from membrane scans suggests that some regulation is occurring. More likely, the lack of proteomic data is due to the accepted difficulty of resolving membrane proteins on 2D-PAGE gels (Smith, 2000
).
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A key issue remaining for characterization of the genus Ferroplasma is the question of chemolithoautotrophic growth. Previously we maintained a viable culture of F. acidiphilum in a continuous-culture reactor for more than three culture volumes in the absence of organic carbon, but the protein level was very close to the concentration calculated from the theoretical washout rate (Dopson et al., 2004b). However, F. acidarmanus Fer1 and Ferroplasma Type II contain candidate genes for CO2 fixation via the reductive acetyl-CoA pathway (Tyson et al., 2004
). The pathway works by reduction of CO2 to form acetyl-CoA and then pyruvate and is found in anaerobic prokaryotes. A key enzyme in this pathway is a Ni-Fe carbon monoxide dehydrogenase, which is unlikely to be functional in the aerobic iron-oxidizing conditions of Ferroplasma spp. In contrast, we have demonstrated the expression of proteins with similarity to two subunits (CutM and CutL) of an aerobic Mo-Fe-flavin carbon monoxide dehydrogenase. One possibility is that this dehydrogenase may be utilized to fix CO2 via the reductive pentose phosphate pathway (Ferry, 1995
). However, this also seems unlikely, as key genes for this process have not been identified in Ferroplasma spp. and we found these proteins to be up-regulated in chemomixotrophic growth compared to incubation in the absence of added organic carbon (Supplementary Table S2). Tyson et al. (2004)
suggested that due to the large numbers of sugar and amino acid transporters identified, Ferroplasma Type II may predominantly grow chemo-organotrophically, and F. acidiphilum MT17 has been described as an obligate chemo-organotroph (Okibe & Johnson, 2004
). Nothing in our results contradicts these hypotheses that Ferroplasma spp. are chemo-organotrophs. We also find no evidence indicating that these strains can fix CO2, although lack of evidence does not preclude that it occurs.
During incubation in the absence of organic carbon, a complete pathway of anti-oxidant proteins was expressed in F. acidiphilum YT, possibly due to a general lack of intracellular reducing power and to oxidative damage caused by interaction of Fe2+ with superoxide and hydrogen peroxide to form highly toxic hydroxyl radicals (Andrews et al., 2003). During chemo-organotrophic growth, enzymes associated with central metabolic pathways were up-regulated. These include components of gluconeogenesis, glycolysis, the TCA cycle, and fatty acid metabolism. None of these proteins were up-regulated during any of the other comparisons between growth states. Along with the probable sugar transporter identified during chemo-organotrophic growth, this was almost certainly due to the increased energy requirement from organic carbon during growth in the absence of Fe2+. The identification of flavoproteins in the chemo-organotrophic membrane spectra could be one of the up-regulated proteins identified from the proteomic analysis, for example succinate dehydrogenase and an electron transfer flavoprotein (Supplementary Table S1).
Differences in the protein profile between chemomixotrophically grown F. acidiphilum YT and F. acidarmanus Fer1 revealed that a number of central metabolic proteins were up-regulated in F. acidiphilum YT. These proteins were similar to those up-regulated during chemo-organotrophic growth of F. acidarmanus Fer1 compared to chemomixotrophic growth. For instance, components of the TCA cycle, gluconeogenesis, and electron transfer proteins, which feed electrons into the electron transport chain at a more negative redox potential than Fe2+ oxidation, were up-regulated under both conditions. These results may indicate a more versatile physiology of F. acidiphilum YT compared to F. acidarmanus Fer1 in response to changes in organic carbon in its environment.
This study is believed to constitute the first in-depth proteomic analysis of growth states in acidophilic archaea. This study also addresses key biochemical and bioenergetic issues from the characterization of the genus. The results do not support the utilization of any known chemolithoautotrophic CO2 fixation pathway in Ferroplasma spp., suggesting that these organisms are chemo-organotrophs. Also, membrane scans and inhibitor experiments suggest that Fe2+ oxidation and organization of the redox components are different from the SoxM terminal oxidase supercomplex from S. acidocaldarius (Fig. 5). Protein expression during chemo-organotrophic growth suggests that many of the central metabolic pathways are up-regulated to compensate for the lack of energy derived from Fe2+ oxidation. Finally, key intermediates in central metabolic pathways are also up-regulated in chemomixotrophically grown F. acidiphilum YT compared to F. acidarmanus Fer1.
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ACKNOWLEDGEMENTS |
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Received 21 July 2005;
revised 7 September 2005;
accepted 9 September 2005.
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