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ABSTRACT Recently, there has been intense interest in the role of electron transfer by microbial communities in biogeochemical systems. We examined the process of iron oxidation by microbial

biofilms in one of the most extreme environments on earth, where the inhabited water is pH 0.5–1.2 and laden with toxic metals. To approach the mechanism of Fe(II) oxidation as a means of

cellular energy acquisition, we isolated proteins from natural samples and found a conspicuous and novel cytochrome, Cyt572, which is unlike any known cytochrome. Both the character of its

covalently bound prosthetic heme group and protein sequence are unusual. Extraction of proteins directly from environmental biofilm samples followed by membrane fractionation, detergent

solubilization and gel filtration chromatography resulted in the purification of an abundant yellow-red protein. The purified protein has a cytochrome _c_-type heme binding motif, CxxCH, but

a unique spectral signature at 572 nm, and thus is called Cyt572. It readily oxidizes Fe2+ in the physiologically relevant acidic regime, from pH 0.95–3.4. Other physical characteristics

are indicative of a membrane-bound multimeric protein. Circular dichroism spectroscopy indicates that the protein is largely beta-stranded, and 2D Blue-Native polyacrylamide gel

electrophoresis and chemical crosslinking independently point to a multi-subunit structure for Cyt572. By analyzing environmental genomic information from biofilms in several distinctly

different mine locations, we found multiple genetic variants of Cyt572. MS proteomics of extracts from these biofilms substantiated the prevalence of these variants in the ecosystem. Due to

its abundance, cellular location and Fe2+ oxidation activity at very low pH, we propose that Cyt572 provides a critical function for fitness within the ecological niche of these acidophilic

microbial communities. SIMILAR CONTENT BEING VIEWED BY OTHERS MULTI-HEME CYTOCHROME-MEDIATED EXTRACELLULAR ELECTRON TRANSFER BY THE ANAEROBIC METHANOTROPH ‘_CANDIDATUS_ METHANOPEREDENS

NITROREDUCENS’ Article Open access 30 September 2023 INTRACYTOPLASMIC MEMBRANES DEVELOP IN _GEOBACTER SULFURREDUCENS_ UNDER THERMODYNAMICALLY LIMITING CONDITIONS Article Open access 07 April

2023 SPONTANEOUS ASSEMBLY OF REDOX-ACTIVE IRON-SULFUR CLUSTERS AT LOW CONCENTRATIONS OF CYSTEINE Article Open access 11 October 2021 INTRODUCTION Acid mine drainage (AMD) is a global

environmental problem that occurs when metal sulfide ore deposits, dominated by FeS2 (pyrite) and containing a range of other metal sulfide phases, are exposed to air and water (Baker and

Banfield, 2003). Pyrite undergoes oxidative dissolution through several reactions, depicted in Equation (1), forming solutions of low pH that generate high levels of toxic metals.

Experiments have shown that microbial ferrous iron [Fe(II)] oxidation can accelerate Equation (1) over the inorganic rate by up to 106 (Singer and Stumm, 1970). Based on such experiments, it

has been inferred that the microbial catalyzed reaction is the rate-limiting step for AMD generation. Consequently, the study of enzymatic mechanisms of microbial iron oxidation is central

to understanding the biology and environmental impact of AMD (Druschel et al., 2004). Previous approaches to understanding biological iron oxidation under acidophilic conditions have

concentrated on isolating organisms from acidic, metal rich environments and culturing them in the laboratory to obtain proteins that oxidize Fe(II) (Blake et al., 1993). An alternative

strategy is to isolate proteins directly from natural microbial consortia and interrogate their functions by biochemical techniques (Kruger et al., 2003). This enables the identification of

important proteins in their natural environment produced by organisms that are difficult to culture under laboratory conditions. For this reason, we have chosen to study low-diversity

microbial communities that establish floating biofilms in extremely acidic water (pH 0.5–1.0) at high temperatures (30–50 °C) in the Richmond Mine at Iron Mountain, California (Bond et al.,

2000). The mine water in this environment contains high levels of metals, including 0.2–0.4 M Fe(II) and millimolar levels of As, Zn and Cu. Genomic analyses of a biofilm isolated from the

Richmond Mine at two different sites (Supplementary Figure S1, for map) reveals that _Leptospirillum_ group II (_Lepto_II) dominates these communities, with _Leptospirillum_ group III

(_Lepto_III) and several archaeal members present in lower abundance (Tyson et al., 2004; Lo et al., 2007). Proteomic mass spectrometry (MS) analyses of a related biofilm collected at the AB

end site identified 2033 proteins, of which >500 were expressed hypothetical proteins (Ram et al., 2005); a subset of these were among the most abundant proteins. We anticipate that many

of the proteins of unknown function are central to processes that are essential for survival in the AMD habitat, including the oxidation of Fe(II) to drive cellular metabolism. Outer

membrane-bound _c_-type cytochromes have been frequently implicated as electron transfer proteins that interact directly with metals in the environment (Newman and Banfield, 2002; Marshall

et al., 2006; Weber et al., 2006). For acidophilic Fe(II) oxidation by _Acidithiobacillus ferrooxidans_, a bacterium often observed in less acidic (pH 2–3) AMD environments, biochemical and

gene expression analyses have implicated an outer-membrane _c_-type cytochrome (Cyc2) as the iron oxidase (Yarzabal et al., 2002, 2004). Here, we report the identification and purification

of a conspicuous cytochrome belonging to _Lepto_II from biofilms obtained in the Richmond Mine. Biochemical studies of this protein indicate that it is membrane bound, contains a unique heme

group, is not homologous to known _c_-type cytochromes and oxidizes iron at low pH, a possible link between the microbial community and the generation of AMD. This work illustrates that a

classical biochemical approach combined with new proteomics and genomics methods can be used to identify proteins of interest from a natural, heterogeneous microbial community. MATERIALS AND

METHODS SAMPLE COLLECTION Biofilm samples were collected from the C-drift region (AMD dam, March 2005, and 15 m beyond the AMD dam into the C-drift, November 2005; Supplementary Figure S1)

of Richmond Mine near Redding CA, USA (Ram et al., 2005). Purified Cyt572 from both samples had identical spectral and redox properties over a range of pH from 0.95 to 5.0. Samples were

frozen in 50 ml aliquots in dry ice at the site, and later moved to −80 °C for storage. PROTEIN PURIFICATION A sample of frozen biofilm (50 ml) was slowly thawed and mine water removed by

centrifugation at 5000 _g_ at 4 °C for 10 min. Biofilm was resuspended in 4 volumes of H2SO4 (pH 1.1) using a glass Dounce homogenizer. Cells were collected at 12 000 _g_ at 4 °C for 10 min,

and similarly resuspended in 50 mM MES-NaOH, pH 5 (MES (2-(_N_-morpholino) ethanesulfonic acid) buffer), to a volume of 50 ml. Cells were kept on ice and broken by sonication (Misonix,

Farmingdale, NY, USA; 50% intensity, 20 cycles of 30 s on, 1 min off). After centrifugation at 12 000 _g_ at 4 °C for 10 min, membranes in the opaque yellow supernatant were sedimented by

centrifugation at 100 000 _g_ at 4 °C for 1 h, resulting in a translucent reddish pellet. This was resuspended again in MES buffer to 50 ml by passing through a narrow bore needle several

times to obtain a homogeneous suspension, then pelleted again and resuspended in 20 mM Tris-HCl pH 7, 10 mM EDTA (TE buffer) to a volume of 3 ml. This membrane suspension was loaded onto a

discontinuous sucrose gradient and centrifuged in a Beckman SW41 Ti swinging bucket rotor at 39 000 r.p.m. at 4 °C for 18 h. Sucrose concentrations (w/w in TE buffer) and volumes per tube

were: 60% (0.4 ml); 55% (0.9 ml); 50% (2 ml); 45% (2 ml); 40% (2 ml); 35% (2 ml) and 30% (2 ml). Yellow colored bands of membrane were removed from the gradient and diluted into 50 ml TE

buffer. The membranes were then pelleted at 100 000 _g_ for 1 h and washed three times in TE buffer to remove sucrose. The membranes were resuspended to a final concentration of 1 mg ml−1 in

TE buffer. Proteins were extracted from membranes with n-dodecyl-β-D-maltoside (DM, ULTROL grade Calbiochem, Gibbstown, NJ, USA). DM was chosen as the solubilizing agent as it is a mild

detergent that has been shown to retain activity in a number of isolated integral membrane complexes (Seddon et al., 2004), and because in preliminary experiments it was better at extracting

proteins than _n_-octyl-β-d-glucopyranoside, amidosulfobetaine-14 or Triton X-100. DM was added to the membrane sample to a final concentration of 1%, and the suspension was incubated with

gentle mixing at 4 °C for 3 h. Insoluble material was pelleted by centrifugation at 8000 _g_ for 20 min, the supernatant was recovered and concentrated in Centricon spin concentrators

(Millipore, Billerica, MA, USA) with a 3 kDa molecular weight cutoff. Samples were typically concentrated to 5 mg ml−1, as determined by a detergent compatible protein assay (DC protein

assay, BioRad, Hercules, CA, USA). Approximately 1.3 mg protein in the detergent extract was run on a 1 × 30 cm Superdex 200HR column in TE buffer containing 0.05% DM. Each peak fraction was

pooled, and where necessary, concentrated in Centricon spin concentrators to 5–10 mg ml−1 (for example, for spectroscopic assays requiring dilution into acidic buffers). PROTEIN

ELECTROPHORESIS AND STAINING For routine analysis of membrane proteins, samples were treated to remove excess detergent and lipid (PAGEprep Advance, Pierce, Rockford, IL, USA) prior to

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. Staining for heme binding proteins following SDS-PAGE was carried out using _o_-dianisidine (Francis and Becker, 1984).

Prior to MS analysis, individual protein bands were excised from colloidal-Coomassie stained polyacrylamide gels, reduced, alkylated and digested with trypsin. SPECTRAL ANALYSIS OF CYT572

Spectrophotometric analysis was carried out on a Pharmacia Ultraspec 3000Pro. Protein samples were diluted to approximately 0.1 mg ml−1 in buffer at the required pH. Four buffers were used:

H2SO4 (pH 0.95–1.1), 50 mM KCl-HCl (pH 1 to 2), 50 mM glycine-HCl (pH 2.2–3.4) and 50 mM MES-NaOH (pH 5). For pH values from 0.95 to 3.4, 1 ml of Cyt572 solution was oxidized by addition of

10 μl of 0.1 g per 1 ml Fe(III)SO4·H2O (23% Fe) and reduced by addition of 10 μl of 0.7 M Fe(II)SO4·7H2O (7 mM final concentration). Because Fe(III) is only soluble in acidic buffers,

reaction of iron with Cyt572 was not carried out above pH 3.5. Instead, at pH 5, Cyt572 was oxidized by addition of 10 μl of 0.2 M ammonium cerium (IV) nitrate (2 mM final concentration) and

reduced with 10 μl of 0.5 M sodium dithionite (5 mM final concentration). Under these conditions, redox activity and spectra are similar at both pH 3.4 and 5. 2D BLUE-NATIVE ELECTROPHORESIS

For analysis of membrane protein complexes, 2D Blue-Native PAGE was used (Stenberg et al., 2005). Briefly, 10–20 μg protein in TE buffer with 1% DM (w/w) was mixed with Coomassie blue G250

to a final dye concentration of 0.25% (w/w), loaded onto a 5–20% acrylamide gradient gel, and separated by electrophoresis in a Mini-Protean II system at 15 mA constant current. After

electrophoresis in the first dimension, gel strips were excised, laid on a glass plate and soaked in 2% SDS in 250 mM Tris-HCl pH 6.8 for 30 min. The second glass plate was placed on top,

and a 15% acrylamide gel containing 0.1% SDS was poured. A 5% acrylamide stacking gel was poured around the first dimension gel slice, and the denaturing gel was run according to standard

protocols and then stained with silver. PROTEIN IDENTIFICATION BY MASS SPECTROMETRY All protein samples were denatured, reduced and digested with sequencing grade trypsin (Promega, Madison,

WI, USA) or pepsin (Sigma, St Louis, MO, USA), and analyzed by MS as described previously (Lo et al., 2007). For detailed LC-MS methods, see Supplementary Methods. RESULTS IDENTIFICATION AND

PURIFICATION OF A NOVEL MEMBRANE-BOUND CYTOCHROME While examining extracts of microbial biofilms taken from the toxic drainage water of a pyrite mine, we searched for heme containing

proteins potentially involved in Fe(II) oxidation. One of the membrane proteins most highly detected by MS proteomic analyses was identified as a hypothetical gene product from our biofilm

community genomics data set, corresponding to scaffold 630-gene 6 (630-6) from _Lepto_II (Ram et al., 2005). This protein has been detected with relatively high spectral counts in membrane

fractions from five distinct biofilms (Supplementary Table S1A). The encoded 570 amino-acid (61.3 kDa) sequence has no significant homology to any known proteins, but does contain a single

heme binding motif, CxxCH, found in _c_-type cytochromes. To purify this putative cytochrome, biofilm samples obtained at the C-drift site (Supplementary Figure S1) were disrupted by

sonication and membranes were isolated by sucrose density gradient centrifugation to reduce protein heterogeneity. The major density band was analyzed by MS and found to contain several

prominent proteins, including flagellar proteins and porins indicative of outer membranes (Figure 1B). This fraction also contained proteins localized to the cytoplasmic membrane, most

likely due to mixing by the sonication process; however, the majority of the proteins could be assigned to the outer membrane. Similar results have been obtained from protein purified from

membranes without using a sucrose gradient (data not shown). The protein detected with the highest number of mass spectral counts was the 630-6 gene product, the dominant protein stained by

both Coomassie blue and silver staining following separation by SDS-PAGE. DM was used to release the cytochrome from membranes for purification. DM consistently extracted approximately 75%

of the protein from isolated and washed membranes. Proteins in the DM extract were separated by size exclusion chromatography into four major fractions (Figure 1A). The second peak (Fraction

2), eluting at an apparent molecular weight of 400 kDa, had a distinct yellow color. Analysis of fractions following SDS-PAGE indicated that Fraction 2 contained a 57 kDa heme binding

protein of 97% relative purity (Figure 1B). This represents a 30% yield of protein from the crude membrane preparation, which, along with spectral counts from MS analysis, indicates the

abundance of this cytochrome. Moreover, absorption spectra of the column fractions confirm a heme containing cytochrome in Fraction 2 (Figure 1C). The absorbance of the α-band at 572 nm is

unique among known cytochromes and is the basis for the name Cytochrome 572 (Cyt572). To further characterize the heme group of purified Cyt572, an alkaline pyridine hemochrome spectrum of

purified Cyt572 reduced with sodium dithionite was taken (Figure 2). The α-band of Cyt572 in the pyridine hemochrome spectrum was observed at 568 nm, instead of 550 nm for known _c_-type

cytochromes (Berry and Trumpower, 1987), thus indicating novel properties of the Cyt572 heme. Edman degradation of purified Cyt572 determined the N-terminal 10-amino-acid sequence,

YPGFARKYNF, which matches exactly to the translated 630-6 gene. This sequence is preceded by a signal peptide, as predicted by the SignalP program (Bendtsen et al., 2004), with a signal

peptidase I cleavage site between Ala and Tyr in the sequence AANA/YPGF. The identity of the purified protein was confirmed using LC-MS/MS analysis after treatment with either trypsin or

pepsin (Figure 3a; see Supplementary Tables S2A and S2B for sequence coverage and detailed mass spectral data, respectively). In the Cyt572 sequence there are five long stretches of amino

acids that lack either R or K residues, so trypsin digestion resulted in large peptides that are more difficult to detect by standard LC-MS/MS methods; nevertheless, tryptic peptides were

detected over 30% of the sequence, giving a positive identification of the 630-6 gene. A higher number of peptides were detected following the pepsin digest, with 72% of the sequence covered

(Figure 3a). Using a deeper 2D LC-MS/MS analysis of the pepsin digest, 92% of the sequence was detected; moreover, Cyt572 was the only protein significantly identified. The 22 amino-acid

stretch from the observed N-terminus containing the single heme binding site and flanking residues was not detected by MS, most likely due to the interference by the covalently bound heme.

Analysis of the detailed reconstruction of the _Lepto_II genome from environmental sequence data (Tyson et al., 2004) revealed five strain variants from the 5-way site, each at ⩾99%

amino-acid sequence identity with 630-6; subsequent analyses indicated that gene 630-6 is a composite of these sequences. A second environmental genomic data set was obtained from a biofilm

isolated at a different site in the UBA location within the Richmond mine (Supplementary Figure S1), in which three additional _Lepto_II Cyt572 homologs were identified (Lo et al., 2007). To

determine if Cyt572 is also present in the less abundant bacterium, _Lepto_III, we examined these sequences and found nine different homologs. Alignment of the composite 630-6 sequence with

one representative from each of these three sets of sequence variants indicates greater divergence of UBA _Lepto_II and 5-way _Lepto_III from the 5-way _Lepto_II strain variants (Figure

3b). The protein purified is most likely one or a mixture of 5-way _Lepto_II variants, with nearly all residues in peptides detected by MS in these particular sequences. The relationship

between the 17 Cyt572 variant sequences from both bacterial types is especially strong in the region surrounding the CxxCH heme binding site, implicating an N-terminal cytochrome _c_-like

structural domain of approximately 80 amino acids (Supplementary Figure S2). Alignment of Cyt572 with the Cyc2, the putative iron oxidase from _A. ferrooxidans_, illustrates that despite

significant sequence divergence (15% identity), the position of the heme binding site and some surrounding residues are conserved (Yarzabal et al., 2002) (Figure 3c). SPECTROSCOPIC AND REDOX

PROPERTIES OF CYT572 The absorption spectrum of the purified cytochrome contains a Soret band at 434 nm and α-band at 572 nm (Figure 1C), both red-shifted compared with the spectra of known

_c_-type cytochromes. Reduced Cyt572 was stable to oxidation for prolonged periods under ambient O2 levels (months at 4 °C in pH 7 buffer). However, when diluted into buffers with pH values

below 3, multiple spectral changes occurred (Figure 4a). The α-band at 572 nm was replaced by a split α-band with maxima at 575 and 585 nm. The ratio of the two sets of α-bands was pH

dependent, with the split α-band predominating at low pH. At pH⩽1.8, partial oxidation of Cyt572 occurred, as observed by the appearance of a new Soret band at 419 nm. Although partial

oxidation of the cytochrome occurs at pH 2.6, addition of excess Fe(II) caused full reduction of the cytochrome and the appearance of the symmetric α-band at 572 nm (data not shown).

Addition of Fe(III) to fully reduced Cyt572 caused complete oxidation of the heme, reflected in loss of the 572 nm peak and a shift of the 435 nm Soret band to 419 nm (Figure 4b). The

oxidized state of Cyt572 was unstable, and removal of excess iron from the protein by dialysis or desalting resulted in partial reduction of Cyt572. To study the reactions of oxidized Cyt572

with Fe(II), it was necessary to minimize the quantity of Fe(III) used to generate oxidized Cyt572, then add a molar excess of reductant. Based on the reappearance of the symmetric 572 nm

peak, Cyt572 was re-reduced by the addition of excess Fe(II). Reduction of oxidized Cyt572 occurred with equal efficiency between pH 0.95 and 3.4; however, at pH⩽1.4, the split α-band at 575

and 585 nm was observed instead of the 572 nm band for reduced Cyt572 (data not shown). Buffer composition and presence of additional DM did not influence these results: at the same pH in

different buffers, spectral changes upon oxidation and reduction of Cyt572 were the same. STRUCTURAL PROPERTIES OF CYT572 Chromatographic experiments indicated that Cyt572 is a multimer or

complexed with other proteins under the solution conditions used (Figure 1A). To investigate the nature of the Cyt572 complex, we used 2D Blue-Native PAGE, an electrophoresis technique

specifically designed for identifying membrane protein complexes and their subunits (Schagger et al., 1994). Protein complexes bind to Coomassie dye, which is negatively charged, and migrate

in a predictable relationship according to their native molecular weights. The first dimension, non-denaturing gel was calibrated with protein standards (Supplementary Figure S3). This was

used to estimate the molecular weight of the Cyt572 complex run under the same conditions (Figure 5). The second (denaturing) dimension indicated that the two apparent complex protein

species were composed primarily of Cyt572, identified by the prominent (saturated) silver-staining at approximately 60 kDa. _In situ_ tryptic digests and MS analysis of the first dimension

blue-stained bands at 210 and 260 kDa confirmed this assignment. The relative intensities of Coomassie-stained bands in both Blue-Native and denaturing gels indicated that the 260 kDa band

represents a minor species, possibly due to a distribution of oligomeric forms, and that ⩾90% of the Cyt572 in the sample was found in the 210 kDa band. A protein identified as

peptidoglycan-associated lipoprotein is often found with Cyt572 complexes by this analysis. In standard SDS-PAGE, the purified Cyt572 protein contains minor protein species of higher

molecular weight, estimated at 114, 170 and 226 kDa (data not shown). These mass values are almost exactly what would be expected for dimeric, trimeric and tetrameric Cyt572, suggesting that

some oligomeric species persist under denaturing conditions. We tested this further with chemical crosslinking, and found that the dimeric and tetrameric species were specifically enhanced

(Supplementary Figure S4). Higher oligomeric forms are possible, which is one possible explanation for the different mass values calculated for Cyt572 in 2D Blue-Native PAGE and size

exclusion chromatography. The requirement of detergent to solubilize Cyt572 indicates that it is an integral membrane protein. Based on examination of the 630-6 protein sequence by the

PRED-TMBB program (Bagos et al., 2004), a mostly β-strand structure is predicted with up to 20 transmembrane strands, a prediction that is consistent with an outer membrane localization. To

experimentally test these folding characteristics, we used circular dichroism spectroscopy to analyze purified Cyt572 (Supplementary Figure S5). These results revealed a dominance of

β-strand structure (43%), similar to that predicted by PRED-TMBB (38%), and a low proportion of α-helical structure (7%). DISCUSSION We found an abundant and unusual cytochrome by extracting

and purifying proteins from natural, low-diversity microbial biofilm samples. The protein, Cyt572, was purified as a homo-multimeric complex from membranes and identified by MS as a gene

product of _Lepto_II, matching a protein of unknown function documented in our previous proteogenomic data (Ram et al., 2005). The prosthetic heme group of this cytochrome is novel,

ascertained by both the visible absorbance spectrum and pyridine hemochrome spectrum of purified Cyt572. We speculate that the observed spectral red shift of the α-absorption band in Cyt572

relative to conventional _c_-type cytochromes may be caused by oxidation of the organic portion of the heme, which would raise the cytochrome midpoint potential (Zhuang et al., 2006). An

elevated potential for Cyt572 may be essential to efficiently oxidize Fe(II) in the low pH regime of AMD. Cyt572 also displays unusual pH-dependent spectral properties. At higher pH

(>2.6) the 572 nm absorbance peak predominates in the spectrum of reduced Cyt572, but at low pH, a split band replaces this band with maxima at 575 nm and 585 nm. Similar split α-bands

have been observed for _c_-type cytochromes, especially at low temperature (77 K), and have been attributed to changes in the heme binding pocket (Reddy et al., 1996). This observation,

along with alteration of this pH-dependence in the presence of excess Fe(II), suggests that the heme binding site is sensitive to pH and may have important implications for the biological

activity of Cyt572. Current studies are focused on obtaining sufficient Cyt572 from the acidophilic biofilms for further biophysical characterization. Although no archaeal homologs of Cyt572

were found in the database searches, an absorption band at 572/573 nm has been observed as a feature of the visible spectra of cell extracts from the acidophilic archaea _Sulfolobus

metallicus_, _Metallosphaera sedula_ and _Acidanus brierlyi_ when grown on soluble Fe(II) or pyrite (Blake et al., 1993; Kappler et al., 2005; Bathe and Norris, 2007). These observations

suggest that the heme found in Cyt572 may be a specialized prosthetic group in microbes that catalyze Fe(II) oxidation under acidic conditions. Despite minimal sequence similarity, Cyt572

shares many properties with Cyc2, the putative iron oxidase localized to the outer membrane of _A. ferrooxidans._ Both proteins have structures that are primarily composed of β-strands, are

monoheme cytochromes with heme binding sequences that begin 12 amino acids from the N-terminus of the mature protein and oxidize Fe(II) readily at low pH. Cyc2 undergoes a partial digestion

upon treatment of whole cells of _A. ferrooxidans_ with proteinase K, indicating that domains of this protein are exposed to the exterior of the cell (Yarzabal et al., 2002). Experiments are

currently underway to determine whether portions of Cyt572 are exposed to the exterior of _Lepto_II cells in Richmond Mine biofilms. A small soluble cytochrome with unusual absorbance

characteristics similar to Cyt572, Cytochrome 579 (Cyt579), has also been purified directly from Richmond Mine biofilms (Singer _et al._, manuscript in preparation). Biochemical studies of

Cyt579 are most consistent with it serving as a periplasmic electron transfer protein that shuttles electrons derived from Fe(II) oxidation to protein complexes localized on the cytoplasmic

membrane. Future biochemical studies will focus on determining if Cyt572 oxidizes Fe(II) at the surface of _Lepto_II cells and donates electrons to Cyt579 as part of an effort to reconstruct

the Fe(II)-dependent respiration pathway of _Lepto_II in the Richmond Mine biofilms. Environmental genomic data obtained from the 5-way site of the Richmond Mine contained five variant

sequences that result in the composite 630-6 gene of the original data set (Tyson et al., 2004), and an additional 12 variants of Cyt572 were identified in the reconstructed genomes of UBA

_Lepto_II (Lo et al., 2007) and 5-way _Lepto_III. The high level of variation in genes encoding Cyt572 is striking because of the protein abundance and its iron oxidation activity. The

extent of sequence diversity and the effect of this variation on the biochemical characteristics of Cyt572 will be determined by examining multiple environmental samples. The high peptide

coverage of the Cyt572 by the combination of pepsin digestion and 2D LC-MS/MS (Figure 3a) will enable us to distinguish the presence of these variants. Of particular interest is whether

strain variation affects the midpoint potential of Cyt572, which we are currently investigating in an ecological context. We postulate that a high level of recombination in a relevant

genomic region (Lo et al., 2007) leads to sequence variation and duplication of Cyt572 genes. If Cyt572 is confirmed as an iron oxidase in _Lepto_II, this will be a notable example of

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using designed heme proteins. _Biochemistry_ 45: 12530–12538. Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Brett Baker and others in the Banfield group for

field sample collections; Mona Hwang and Stephanie Wong for laboratory assistance; Drs Yongqin Jiao and Korin Wheeler for helpful comments on the manuscript and Drs Jason Raymond and Adam

Zemla for insightful discussions on phylogenetics and protein structure. We also thank Mary Ann Gawinowicz at the Columbia University Protein Core Facility for protein sequence analyses.

This work was performed under the auspices of the US Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344, and was funded by the DoE Office of

Science, Genomics: GTL Program Grant DE-FG02-05ER64134 to JF Banfield, RL Hettich and MP Thelen. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Chemistry Directorate, Lawrence Livermore

National Laboratory, Livermore, CA, USA Chris Jeans, Steven W Singer & Michael P Thelen * Department of Earth and Planetary Sciences, University of California, Berkeley, CA, USA Clara S

Chan & Jillian F Banfield * Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA Nathan C VerBerkmoes & Robert L Hettich * Life Sciences Division, Oak Ridge

National Laboratory, Oak Ridge, TN, USA Manesh Shah Authors * Chris Jeans View author publications You can also search for this author inPubMed Google Scholar * Steven W Singer View author

publications You can also search for this author inPubMed Google Scholar * Clara S Chan View author publications You can also search for this author inPubMed Google Scholar * Nathan C

VerBerkmoes View author publications You can also search for this author inPubMed Google Scholar * Manesh Shah View author publications You can also search for this author inPubMed Google

Scholar * Robert L Hettich View author publications You can also search for this author inPubMed Google Scholar * Jillian F Banfield View author publications You can also search for this

author inPubMed Google Scholar * Michael P Thelen View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Michael P

Thelen. ADDITIONAL INFORMATION Supplementary Information accompanies the paper on The ISME Journal website (http://www.nature.com/ismej) SUPPLEMENTARY INFORMATION SUPPLEMENTARY METHODS (PDF

763 KB) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Jeans, C., Singer, S., Chan, C. _et al._ Cytochrome 572 is a conspicuous membrane protein with

iron oxidation activity purified directly from a natural acidophilic microbial community. _ISME J_ 2, 542–550 (2008). https://doi.org/10.1038/ismej.2008.17 Download citation * Received: 22

November 2007 * Revised: 14 January 2008 * Accepted: 14 January 2008 * Published: 28 February 2008 * Issue Date: May 2008 * DOI: https://doi.org/10.1038/ismej.2008.17 SHARE THIS ARTICLE

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by the Springer Nature SharedIt content-sharing initiative KEYWORDS * Fe(II) oxidation * heme * _c_-type cytochrome * biofilm * acid mine drainage * membrane protein