NIF-type iron-sulfur cluster assembly system is duplicated and distributed in the mitochondria and cytosol of Mastigamoeba balamuthi

Abstract

In most eukaryotes, the mitochondrion is the main organelle for the formation of iron-sulfur (FeS) clusters. This function is mediated through the iron-sulfur cluster assembly machinery, which was inherited from the α-proteobacterial ancestor of mitochondria. In Archamoebae, including pathogenic Entamoeba histolytica and free-living Mastigamoeba balamuthi, the complex iron-sulfur cluster machinery has been replaced by an ε-proteobacterial nitrogen fixation (NIF) system consisting of two components: NifS (cysteine desulfurase) and NifU (scaffold protein). However, the cellular localization of the NIF system and the involvement of mitochondria in archamoebal FeS assembly are controversial. Here, we show that the genes for both NIF components are duplicated within the M. balamuthi genome. One paralog of each protein contains an amino-terminal extension that targets proteins to mitochondria (NifS-M and NifU-M), and the second paralog lacks a targeting signal, thereby reflecting the cytosolic form of the NIF machinery (NifS-C and NifU-C). The dual localization of the NIF system corresponds to the presence of FeS proteins in both cellular compartments, including detectable hydrogenase activity in Mastigamoeba cytosol and mitochondria. In contrast, E. histolytica possesses only single genes encoding NifS and NifU, respectively, and there is no evidence for the presence of the NIF machinery in its reduced mitochondria. Thus, M. balamuthi is unique among eukaryotes in that its FeS cluster formation is mediated through two most likely independent NIF machineries present in two cellular compartments.

Iron-sulfur (FeS) clusters are among the most ancient protein cofactors and are essential for the function of a wide variety of FeS proteins in the Bacteria, Archaea, and Eukarya domains (1). Although FeS clusters spontaneously assemble into apoproteins in vitro, the formation of FeS clusters in living cells is a catalyzed process (1, 2). There are three distinct systems of bacterial origin that are responsible for the formation of iron-sulfur clusters: the ISC (iron-sulfur cluster), SUF (sulfur mobilization), and NIF (nitrogen fixation) systems (2). ISC and SUF are highly complex, multicomponent machineries that include two core proteins: cysteine desulfurase (IscS or SufS), which releases sulfur from cysteine to produce alanine, and a scaffold protein (IscU or SufB) that provides a platform for the assembly of transient FeS clusters (2). Both machineries are involved in the formation of FeS clusters in housekeeping proteins, although the SUF machinery is more important for FeS cluster formation and repair under oxygen stress (3). The NIF system is less complex, comprising the cysteine desulfurase NifS and scaffold protein NifU (4). The NIF system is generally thought to be specialized for the nitrogenase FeS cluster assembly of nitrogen-fixing bacteria (5).

Eukaryotes gained both the ISC and SUF machineries during eukaryogenesis from bacterial endosymbionts (6, 7). The ISC machinery present in mitochondria is related to the α-proteobacterial ancestor of these organelles (8). Similarly, the SUF machinery in plastids is related to a homologous system in cyanobacteria, which is the bacterial group from which these organelles originated (6, 9). The formation of FeS clusters through the activity of the ISC machinery is the only essential function of mitochondria identified thus far and is required not only for the maturation of mitochondrial FeS proteins but also for the formation of FeS clusters in other cell compartments (2, 10). It has been proposed that the maturation of cytosolic FeS proteins is dependent on the export of an unknown compound from the mitochondria, which is then used by the cytosolic FeS assembly (CIA) machinery (2).

Several lineages of unicellular eukaryotes possess highly modified forms of mitochondria that are adapted to function under anaerobic conditions (11). Hydrogenosomes, which are present in Trichomonas vaginalis and other anaerobic protists, generate hydrogen with the concomitant synthesis of ATP through substrate-level phosphorylation (11). This pathway of extended glycolysis depends on the activities of several FeS proteins, such as pyruvate:ferredoxin oxidoreductase (PFO), and hydrogenase (11). Similar to mitochondria, hydrogenosomes contain ISC machinery that catalyzes FeS cluster assembly (12). Giardia intestinalis, microsporidia, and Cryptosporidium parvum possess mitochondrion-derived organelles known as mitosomes, which completely lack energy metabolism and other metabolic pathways (13–15). The FeS cluster assembly mediated through the ISC machinery is the only known mitochondrial function retained in these organelles (13–15).

The free-living protist Mastigamoeba balamuthi and its parasitic relative Entamoeba histolytica are members of the Archamoebae. Unique among eukaryotes studied so far, these anaerobic organisms possess genes encoding the two components of bacterial NIF machinery: NifS and NifU (16, 17). Phylogenetic analyses have suggested that the amoebic NIF machinery was inherited through a common ancestor of Mastigamoeba and Entamoeba from an ε-proteobacterium via lateral gene transfer (LGT). This transfer is hypothesized to have occurred after the split of the anaerobic Archamoebae and aerobic Mycetozoa (e.g., Dictyostelium), which possess conventional ISC machinery (16).

E. histolytica possess mitosomes that do not generate ATP as do those in other mitosome-harboring organisms (11). However, these organelles do not possess the ISC machinery, and no component of either the ISC or SUF machinery has been identified in the Entamoeba genome. NIF components have been predominantly identified in the cytosol of Entamoeba (18, 19). However, it remains controversial whether these components are present exclusively in the cytosol or also operate in the mitosomes (18–20).

M. balamuthi is a more basal member of the Archamoebae group (16). The mitochondrion-related organelles of M. balamuthi have been suggested to represent an intermediate stage between “classic” mitochondria and the reduced mitosomes of E. histolytica (16). Although aerobic respiration is absent in the modified mitochondria of M. balamuthi, they may have retained some components of the tricarboxylic acid cycle (16). Interestingly, the expressed sequence tag survey revealed partial sequences encoding the NifS and NifU components, and these proteins have been predicted to be cytosolic in M. balamuthi (16). However, it remains unknown how FeS clusters are formed in the mitochondria of M. balamuthi or if the ISC machinery is present in these organelles. Therefore, we analyzed the M. balamuthi genome to identify and characterize the FeS cluster assembly machineries of this species and their corresponding substrates.

Results

Bacterial NIF Machinery Is Duplicated in M. balamuthi.

Searches for components of the bacterial NIF machinery in the M. balamuthi genome sequence revealed two paralogs for each component, i.e., the cysteine desulfurase NifS (MbNifS) and the scaffold protein NifU (MbNifU) (Table S1). The protein sequences for the two MbNifS and MbNifU paralogs displayed high sequence identity (76.5% and 61.8%, respectively), except at their N-termini. Although the first copy of the cysteine desulfurase (MbNifS-C) gene was almost collinear with its bacterial and E. histolytica orthologs, the second copy (MbNifS-M) possessed a 16-amino acid extension at the amino terminus (Fig. 1). Similarly, a 55-amino acid N-terminal extension was identified in the MbNifU-M scaffold protein paralog, but this extension was absent in MbNifU-C. These extensions show features similar to mitochondrial targeting sequences, including predictable cleavage sites for the mitochondrial processing peptidase, with an arginine residue typically located at the −2 position (Fig. 1).

Fig. 1.

Comparison of the amino-terminal sequences of M. balamuthi NifS/NifU paralogs. MTSs were predicted using PSORTII and MITOPROT servers. The predicted cleavage sites are labeled with arrows. The MITOPROT-predicted targeting sequences are underlined. (A) Paralogs of M. balamuthi NifS (MbNifS-M and MbNifS-C) and NifS orthologs of E. histolytica (EhNifS), Campylobacter fetus (CamfNifS), and Azotobacter vinelandii (AzovNifS). (B) NifU paralogs of M. balamuthi (MbNifU-M and MbNifU-C), and NifU orthologs of E. histolytica (EhNifU), C. fetus (CamfNifU), and Arcobacter nitrofigilis (ArcnNifU).

Phylogenetic analysis of the Mastigamoeba and Entamoeba NifS and NifU sequences suggested that a common ancestor of these two lineages possessed cytosolic NifS and NifU proteins, which were obtained via LGT from an ε-proteobacterium. The branching order within the Archamoebal group revealed that both Nif components were ancestrally duplicated in the common ancestor. The analysis further suggested that Entamoeba had retained the mitochondrial version of the Nif machinery, but the cytosolic Nif components were lost. However, the statistical support for the relative branching order was too weak to exclude other scenarios (Fig. S1). Searches for the components of the mitochondrial ISC assembly machinery in the M. balamuthi genome were negative; however, a single gene encoding [2Fe2S] ferredoxin (MbFdx), an electron carrier that provides reducing equivalents for FeS cluster assembly in the mitochondria, was identified. Similar to the NIF components, MbFdx possesses a putative N-terminal targeting sequence (Fig. S2). Components of the ISC export machinery [Atm1 (a mitochondrial ATP-binding cassette transporter) and Erv1 (a sulfhydryl oxidase)] were also not present. Interestingly, we identified all components of the CIA machinery, except electron transport proteins Tah18 and Dre2 (Table S1).

N-Terminal Mitochondrial Targeting Sequences Deliver MbNifS-M and MbNifU-M to the Mitochondria in Saccharomyces cerevisiae.

Because there is no tractable system for protein expression in M. balamuthi, we used S. cerevisiae, which is a well-studied model organism belonging together with Amoebozoa to Unikonta (21). We first expressed the MbNifU-M fused to GFP at carboxyl terminus with and without the N-terminal mitonchondrial targeting sequences (MTS). The experiments showed that the targeting of MbNifU-M-GFP to the yeast mitochondria was entirely MTS-dependent. We obtained the same results for MbNifS-M-GFP. Both NIF paralogs that lacked the amino-terminal extensions (MbNifU-C and MbNifS-C) were localized to the yeast cytosol. Next, we examined whether MbNifU-M and MbNifS-M were translocated into the yeast mitochondria or remained attached to the surface of the membrane. The immunoblot signals for the two proteins were not significantly affected when isolated mitochondria were treated with trypsin, but the signals for both proteins disappeared after trypsin treatment when the organellar membranes were disintegrated with detergent (Fig. 2). These experiments confirmed that MbNifS-M and MbNifU-M were targeted and translocated into the yeast mitochondria through their MTSs.

Fig. 2.

N-terminal presequence-dependent targeting of M. balamuthi NifS-M and NifU-M into S. cerevisiae mitochondria. (A) The proteins were expressed with GFP tags in S. cerevisiae (green). Mitochondria were costained with Mitotracker (red). MTSs were deleted in ∆MbNifS-M and ∆MbNifU-M. MbNifS-C, and MbNifS-C paralogs did not contain the N-terminal MTSs. DIC, differential interference contrast. (Magnification: A, 1,000×.) (B) Protease protection assays. Isolated mitochondria (control) were treated with trypsin alone or together with Triton X-100. Antibodies against entamoebal NifS and NifU as well as yeast Tom20 (outer-membrane marker) and Tim17 (inner-membrane marker) were used for the immunoblot analysis.

Dual Localization of the NIF Machinery in M. balamuthi.

The localization of MbNifS and MbNifU in the subcellular fractions of M. balamuthi revealed that distinct NIF components were present in the cytosol and mitochondrion-enriched fractions. MbNifS was visualized as a double band in the whole-cell lysate, but only single bands of different sizes corresponding to MbNifS-C and MbNifS-M were observed in the cytosol and mitochondria, respectively. A similar pattern was obtained using an antibody that recognizes MbNifU-C and MbNifU-M. The localization of MbNifS-M and MbNifU-M to the mitochondria was confirmed through a protease protection assay performed on the mitochondria-enriched fractions (Fig. 3).

Fig. 3.

Localization of the NIF components in M. balamuthi cellular fractions. (A) Immunoblot analysis of whole cell lysates (Lys), mitochondrial fractions (Mito), and cytosol (Cyto). NifS-M, NifS-C, NifU-M, and NifU-C were visualized using E. histolytica αNifS and αNifU antibodies that recognize both forms. Pyruvate formate lyase (PFL), malate dehydrogenase (MDH), HydE, and SdhB were recognized using homologous polyclonal antibodies. (B) Protease protection assay. Control, mitochondrial fractions without treatment; Trypsin+TX-100, treatment with trypsin and Triton X-100. The mitochondria were probed using antibodies against E. histolytica NifS, NifU, and M. balamuthi MDH.

Next, we measured the activity of cysteine desulfurase based on the detection of sulfur formation. The specific activity of cysteine desulfurase in the cytosol was 1.130 ± 0.177 nmol·min⁻¹·mg⁻¹ (n = 8). Considerably lower specific activity was detected in the mitochondrial fraction (0.146 ± 0.062 nmol·min⁻¹·mg⁻¹; n = 8). However, a 67% increase in activity was observed after disintegration of the organellar membranes using detergent (0.443 ± 0.116 nmol·min⁻¹·mg⁻¹; n = 8) (Table S2). The latency of cysteine desulfurase activity indicated an intraorganellar localization for this protein. The organellar activity of NifS was further confirmed based on alanine production that was comparable with the sulfur formation (0.365 nmol·min⁻¹·mg⁻¹; n = 8) (Fig. S3). Finally, we determined whether the cytosolic and mitochondrial fractions catalyze the formation of FeS clusters in recombinant apo-MbFdx using S³⁵-cysteine. Reconstitution of FeS centers was observed with time-dependent incorporation of S³⁵ into holo-MbFdx when either the cytosolic or mitochondrial fraction was added to the reaction mixture (Fig. 4). Taken together, these results indicate that the NIF machinery catalyzes independent FeS cluster assembly in two cellular compartments, the cytosol and the mitochondria.

Fig. 4.

Reconstitution of [FeS] clusters in M. balamuthi apoferredoxin catalyzed in mitochondrial (Mito) and cytosolic fractions. Recombinant apoferredoxin was incubated with [³⁵S]cysteine, Fe-ascorbate, and cellular fractions for the indicated time period and analyzed using native gel electrophoresis followed by autoradiography.

Mitochondria Contain NIF Machinery Together with Substrate FeS Proteins.

Searches of the M. balamuthi genome database revealed two complete gene sequences encoding candidate FeS proteins: succinate dehydrogenase subunit B (SdhB) and hydrogenase maturase E (HydE) (Table S1). Immunofluorescence microscopy revealed colocalization of SdhB with the signal for HydE in numerous round organelles corresponding to mitochondria (Fig. 5). Importantly, NifU and NifS staining was observed in the cytosol and in vesicles colabeled with the anti-SdhB antibody. The presence of mitochondrial NIF components together with HydE and SdhB in the same compartment was further confirmed through immunoblot analysis of the subcellular fractions (Fig. 3).

Fig. 5.

Localization of FeS assembly machinery (NifS and NifU) and FeS proteins (SdhB and HydE) in M. balamuthi. The MbSdhB mitochondrial FeS protein (green) colocalized with the hydrogenase maturase HydE (red). The signal for NifS and NifU (red) showed the double localization of proteins in the organelles and cytosol. The SdhB colocalized with organellar NifS and NifU. DAPI (blue); DIC, differential interference contrast. (Scale bars: 5 μm.)

Next, we tested the enzymatic activities of two FeS enzymes for which the corresponding genes were identified in the Mastigamoeba genome: a hydrogenase, and PFO (Table S1). High activity of both PFO and hydrogenase was detected in the cytosol (Table S2). However, ∼2% of the activity of both the enzymes was associated with the mitochondrial fraction (Table S2). As in the case of cysteine desulfurase, PFO displayed 77% latency in the mitochondrial fraction, indicating an intraorganellar localization (Table S2). Accordingly, two hydrogenases and two PFOs possessed predictable MTSs that were absent in the other paralogs (Fig. S2 and Table S1). The activities of malate dehydrogenase and NADH oxidase were used as mitochondrial and cytosolic enzyme markers, respectively (Table S2). These data indicate that the NIF machinery is present in mitochondria, together with proteins whose maturation is dependent on FeS cluster assembly.

Components of E. histolytica NIF Machinery Do Not Possess MTSs and Are Found in the Cytosol.

In contrast to M. balamuthi, single-copy genes encode E. histolytica NifS and NifU that do not possess a recognizable amino-terminal MTS (Fig. 1). The expression of both E. histolytica NIF components in S. cerevisiae revealed that these proteins are not targeted to the mitochondria but instead remain in the cytosol (Fig. 6). Subcellular fractionation of E. histolytica (Eh) revealed that the signals for EhNifS and EhNifU appear predominantly in the soluble fraction. A faint band for EhNifS was observed in the organellar fraction when large amounts of protein were loaded. However, this band disappeared upon trypsin treatment of the organelles (Fig. S4). These results do not support previous report suggesting that NIF components function in the mitosomes in addition to the cytosol of E. histolytica (18).

Fig. 6.

Expression of E. histolytica NifS and NifU in S. cerevisiae. (A) Proteins were expressed with a GFP-tag at the carboxyl terminus in S. cerevisiae (green). (Magnification: A, 1,000×.) (B) Immunoblot analysis of mitochondrial and cytosolic fractions. The recombinant proteins were detected using anti-NifS and -NifU antibodies.

Discussion

There are three unique features of the FeS cluster assembly machinery in M. balamuthi: (i) the archetypal mitochondrial ISC machinery that is present in virtually all eukaryotes has been replaced with ε-bacterial NIF components; (ii) the genes encoding the NIF machinery proteins have been duplicated, with one system being present in the cytosol and the second system targeted to the mitochondria; and (iii) both the cytosolic and mitochondrial cellular fractions independently catalyze the formation of FeS clusters.

In the majority of eukaryotes, the mitochondrial ISC machinery is essential for the maturation of cytosolic FeS proteins (10). This phenomenon has been functionally demonstrated in evolutionarily distant organisms, such as S. cerevisiae (10) and Trypanosoma brucei (22). The typical mitochondrial metabolic pathways have been lost during the course of reductive evolution in the mitosomes of Giardia and microsporidia (11). However, the mitosomes of these species have retained the functional mitochondrial ISC machinery, underscoring the essential function of this pathway in eukaryotic cells (13, 23). Maturation of extramitochondrial FeS proteins requires the export of a still unknown compound from the mitochondria to the cytosol, where it is used by the CIA machinery (2). It has been speculated that this compound contains a sulfur moiety or a preassembled FeS cluster that is transferred to apoproteins via the components of the CIA machinery (2). However, M. balamuthi does not follow this general scheme. The present results show that Mastigamoeba contains two distinct NIF-type FeS cluster assembly machineries that most likely function independently in the cytosol and mitochondria. The components of the cytosolic and mitochondrial machineries, including NifS and NifU, are encoded by distinct paralogs, and the presence of the corresponding gene products in both cellular compartments of M. balamuthi was confirmed through subcellular fractionation and immunofluorescence microscopy. Importantly, biochemical studies have demonstrated that both the cytosol and the mitochondria possess cysteine desulfurase activity and can catalyze the formation of FeS clusters on apoferredoxin. The dual localization of the NIF machinery observed in M. balamuthi is reminiscent of how some components of the ISC machinery are localized in human cells, where IscS, IscU, and Nfu are found predominantly in the mitochondria, although small amounts of these proteins are also detected in the cytosol and the nucleus. Unlike Mastigamoeba, human mitochondrial and cytosolic/nuclear proteins are synthesized from a single transcript through alternative translation or splicing (24). Although the involvement of cytosolic/nuclear ISC components in the de novo formation of FeS clusters is currently a matter of discussion (24, 25), it has been shown that depletion of cytosolic/nuclear IscU causes defects in cytosolic FeS cluster assembly (26). However, there is no information regarding the possible relationship between the cytosolic ISC components and the human CIA machinery. Thus, future studies should clarify whether the cytosolic NIF found in Mastigamoeba and the human ISC machineries function independently or whether the cytosolic NIF/ISC provide any substrate for the CIA machinery that is conserved in all eukaryotes, including Mastigamoeba.

The substitution of a multicomponent mitochondrial ISC system with simple NIF machinery was a unique evolutionary event in eukaryotes, but it is not unprecedented elsewhere in nature. In bacteria, the NIF system has been identified in nonnitrogen-fixing anaerobic ε-proteobacteria, such as Helicobacter, in which the NifS and NifU components function in FeS cluster assembly, whereas the ISC and SUF machineries are absent (27). Interestingly, the expression of Helicobacter NifS/NifU and E. histolytica EhNifS/EhNifU in Escherichia coli, in which both the isc and suf operons have been deleted, supports the growth of mutated bacteria. However, full complementation of the ISC and SUF systems is observed only under anaerobic conditions (17, 28). Thus, it seems plausible that the substitution of the ISC machinery with the oxygen-sensitive NIF machinery that occurred in the Mastigamoeba ancestor would have been possible only in anaerobic niches, where this species exists together with NIF-possessing ε-proteobacteria (16). The oxygen sensitivity of the NIF system might also explain why the NIF machinery has not been identified in any aerobic organism.

The replacement of the ISC assembly machinery in mitochondria with the NIF system requires the acquisition of mitochondrial signals to mediate the targeting and translocation of NifS and NifU to these organelles. There are two types of MTSs: (i) amino-terminal cleavable presequences, and (ii) inner signals embedded within the structure of the mature protein (29). In the present study, we showed that both NifS-M and NifU-M possess amino-terminal presequences that are strictly required for protein delivery to the yeast mitochondria, and deletion of these extensions results in the mislocalization of the NIF proteins to the cytosol. The strict presequence-dependent targeting of NifS-M and NifU-M to the mitochondria indicates that inner signals are absent from these NIF components. Hence, it is likely that gene duplication and acquisition of the N-terminal signals were essential for the targeting of the NIF components to these organelles upon LGT from ε-proteobacteria.

The dual localization of the NIF machinery observed in M. balamuthi is consistent with the dual localization of the proteins that require the assembly of FeS clusters for maturation. We demonstrated that the Mastigamoeba mitochondria contain several FeS proteins, including the SdhB subunit of the succinyl dehydrogenase complex, the hydrogenase maturase HydE and, most likely, [2Fe2S] ferredoxin. Interestingly, the activity of two typical FeS enzymes involved in extended anaerobic glycolysis, PFO and hydrogenase, was identified in both compartments, although this activity was predominantly localized to the cytosol. Hydrogenase is also encoded in the E. histolytica genome, and episomally expressed entamoebic hydrogenase has been detected in the cytosol of transformed cells (30). However, hydrogenase activity has not been observed in nontransfected entamoebas, and rather low activity has been identified in transfected cells (0.0035 µmol·min·mg) (31). In this context, the activity of hydrogenase in the cytosol of M. balamuthi (1.351 ± 0.176 µmol·min·mg) is unusually high.

The presence of aerobic mitochondria in Mycetozoa, which is a sister taxon of the anaerobic Archamoebae (32), indicates that the mitochondria of M. balamuthi represent secondarily adapted and reduced forms of these organelles and that reductive evolution has progressed further in entamoebids, which retain mitochondria in the form of mitosomes. In contrast to the mitochondria of M. balamuthi, there is no evidence for the presence of any protein with classic multiple iron FeS clusters in E. histolytica mitosomes. Therefore, the predominant cytosolic distribution of the NIF machinery should be sufficient to accommodate the cellular requirements for FeS cluster biogenesis in this organism. Surprisingly, dual localization of the NIF machinery in the cytosol and the mitosomes of E. histolytica has also been suggested, and FeS cluster biogenesis has been proposed as one of the fundamental functions of entamoebal mitosomes (18). However, following proteomic analysis of E. histolytica mitosomes has not confirmed the presence of NIF machinery (20). In our cell localization analyses, we detected components of the E. histolytica NIF machinery, particularly in the cytosolic fraction, which had been suggested previously (19). Moreover, the E. histolytica NIF components were found to be encoded by single copy genes, which lack the mitochondrial targeting signals. Neither the cytosolic NIF components of M. balamuthi nor the NIF components of E. histolytica were imported into the mitochondria in yeast, thereby suggesting an absence of inner mitochondrial targeting signals in these proteins. However, we cannot exclude the potential recognition of a small amount of NifS and NifU through the translocation machinery of the mitosomes, which are considerably modified compared with yeast mitochondria (19), although the present experiments do not provide support for this possibility.

In conclusion, based on the present and previous findings (16, 17), we propose the following scenario for the evolution of FeS cluster assembly in Archamoebae: (i) a common ancestor of Mastigamoeba and Entamoeba that inhabited anaerobic niches gained the NIF machinery from ε-proteobacteria through LGT; (ii) the genes encoding the NIF machinery were ancestrally duplicated and the components that acquired MTSs replaced the ISC machinery, as observed in Mastigamoeba; and (iii) in Entamoeba, the mitochondrial NIF machinery may have lost its organellar targeting sequences and replaced the cytosolic NIF version, as suggested by our phylogenetic analysis. However, the low resolution of the Archamoeba tree cannot exclude a more parsimonious scenario, in which the mitochondrial NIF machinery was lost and the cytosolic version was retained. Although the characteristics of the NIF system that were advantageous over the ISC system under anaerobic conditions remain enigmatic, the presence of the NIF system in Archamoebae highlights the diversity involved in the adaptation of eukaryotes to anaerobic niches, which has repeatedly occurred in distinct eukaryotic lineages (11).

Materials and Methods

Cell Cultivation and Fractionation.

M. balamuthi (ATCC 30984), E. histolytica (strain HM-1:IMSS), and YPH499 S. cerevisiae strain were maintained axenically, as previously described (33–35). The preparation of subcellular fractions of M. balamuthi and E. histolytica through differential centrifugation of the cell homogenate is described in SI Materials and Methods. The yeast mitochondria were obtained according to previously described methods (36).

Genome Sequencing.

The partial genome sequence of M. balamuthi was assembled from sequences generated using Illumina and 454 pyrosequencing. Based on the estimated genome size of 50 Mb, the genome coverage is 15× for long 454 reads and 20× for short paired-end Illumina reads (SI Material and Methods). The preliminary assembly of M. balamuthi genome and raw sequencing data have been submitted to EMBL (www.ebi.ac.uk/ena/data/view/PRJEB1507).

Phylogenetic Analysis.

Maximum-likelihood and Bayesian phylogenetic analyses of selective NifS and NifU proteins from epsilon proteobacteria along with the homologs from the archamoebae species were performed as decribed in SI Materials and Methods.

Gene Searches and Cloning.

The local M. balamuthi genome database was searched using the TBLASTN algorithm with protein sequences of known orthologs in E. histolytica and T. vaginalis (Table S3). The PCR primers were designed based on identified genomic sequences (Table S4) for the amplification of coding sequences using M. balamuthi cDNA as a template. The genes encoding MbNifS-M, MbNifU-M, MbNifS-C and MbNifU-C (the accession numbers are provided in Table S1) were cloned and expressed as described in SI Materials and Methods.

Protease Protection Assay.

The topology of the mitochondrial proteins was tested using trypsin treatment as previously described (37). Details of the procedure are given in SI Materials and Methods. Rabbit polyclonal antibodies against E. histolytica NifS, NifU, and Cpn60 (a kind gift from T. Nozaki, National Institute of Infectious Diseases, Tokyo, Japan), and S. cerevisiae Tom20 and Tim17 (a kind gift from T. Lithgow and K. Gabriel, Monash University, Melbourne, Australia) were used for immonoblot analyses.

Immunofluorescent Microscopy.

Image acquisition was performed using standard techniques (38) (SI Materials and Methods).

Enzyme Assays.

The PFO, hydrogenase, NADH oxidase, and malate dehydrogenase activities were assayed as previously described (39). The cysteine desulfurase activity was monitored through the production of sulfide (40) or alanine (SI Materials and Methods).

Reconstitution of FeS Clusters.

Recombinant MbFdx was expressed in BL21 E. coli (DE3) using the pET42b⁺ vector (Novagen) and purified under native conditions (Qiagen). The reconstitution of FeS clusters in apoMbFdx was performed as previously described (12).