Archaea are single-celled organisms that were discovered over half a century ago. Recently, there has been a renewed interest in these microbes because theyplay a key role in climate change by controlling greenhouse gas emissions, like methane. Indeed, methane-producing Archaea generate nearly 70% of the methane gas released into the atmosphere.

A group of proteins called c-type cytochromes are essential to energy generation in several methane-producing archaea. However, it is a mystery how Archaea assemble their c-type cytochromes. In fact, genomic studies suggest that Archaea are missing some of the c-type cytochrome assembly machinery that bacteria use. This has led scientists to suspect that Archaea have an alternate mechanism for building these essential components.

To solve this mystery, Gupta, Shalvarjian, and Nayak used CRISPR-Cas9 gene-editing tools to characterize which proteins are essential for c-type cytochrome production in Methanosarcina acetivorans, a species of Archaea that produces methane. These experiments showed that M. acetivorans discarded a few parts of the process used by bacteria to generate c-type cytochromes, streamlining the assembly of these proteins. By comparing the genes of different Archaeal species, Gupta, Shalvarjian and Nayak were able to determine that Archaea acquired the genes for producing c-type cytochromes from bacteria via horizontal gene transfer, a process in which genes move directly from one organism into another. The streamlining of the process took place later, as different Archaeal species evolved independently, but losing the same parts of the process.

Gupta Shalvajiran and Nayak’s experiments also showed that c-type cytochromes are essential for the growth and fitness of methane-producing Archaea like M. acetivorans. The role of c-type cytochromes in methane production varies in different species of Archaea depending on their growth substrate or where they live. These results provide vital information about how Archaea produce methane, and the tools and techniques developed will aid further investigation of the role of Archaea in climate change.

C-type cytochromes (cyt(s) c) are found across the tree of life, and are critical to electron transfer processes ranging from aerobic respiration in the mitochondrion of Eukaryotes to intra/extracellular electron transport in Bacteria and Archaea (Richardson, 2000; Bertini et al., 2006; Shi et al., 2016; Gupta et al., 2020). The production of a holo-cyt c, that is capable of electron transfer, requires complex post-translational processing to covalently attach iron-protoporphyrin IX (heme b) to an unfolded apo-cyt c protein (Kranz et al., 2009; Sanders et al., 2010). Heme attachment occurs on the extracellular face of the cytoplasmic membrane, and, thus also requires the translocation of the apoprotein and heme across the membrane. The apoprotein is translocated via the Sec pathway (Sambongi et al., 1996; Thöny-Meyer and Künzler, 1997; Helde et al., 1997) and a set of proteins collectively known as the cyt c maturation system are involved in energy-dependent heme transport, apoprotein handling, and covalent cofactor attachment (Kranz et al., 2009; Sanders et al., 2010; Kranz et al., 1998). Three distinct systems for cyt c maturation have been identified thus far, and have been studied extensively in bacterial and eukaryotic model systems (Kranz et al., 2009; Sanders et al., 2010). However, despite growing evidence that cyt c play a vital role in electron transport processes across members of the Archaea – particularly in microorganisms that metabolize methane and other alkanes – the biogenesis of cyt c has never been functionally characterized in an archaeon.

Based on genomic surveys (Allen et al., 2006; Kletzin et al., 2015; Chadwick et al., 2021) of sequenced isolates and metagenomics assembled genomes, the vast majority of cyt c containing Archaea encode homologs of the System I cyt c maturation pathway (Ccm pathway) that is widespread in members of the Bacteria and is also present in the mitochondria of some Plants and Protozoa (Kranz et al., 2009; Giegé et al., 2008). The Ccm pathway is best characterized in Gram-negative bacteria, such as Escherichia coli, where nine to ten proteins, encoded by ccmABCDEFGH(I) and ccdA (or dsbD), are involved in cyt c biogenesis (Figure 1—figure supplement 1; Kranz et al., 2009; Sanders et al., 2010). Typically, the ccm genes are encoded in an operon on the chromosome and the ccmH open reading frame in E. coli is often found as two different genes, annotated as ccmH (or ccl2) and ccmI (or cycH), in bacteria like Rhodobacter capsulatus (Kranz et al., 2009). Genetic and biochemical studies of the Ccm pathway have revealed that the membrane-associated CcmABCD complex translocates heme across the cytoplasmic membrane to form holo-CcmE (Figure 1—figure supplement 1). Heme from holo-CcmE is ultimately transferred to the apo-cyt c by the CcmF/H complex (cytochrome synthetase) to form holo-cyt c. CcmG and CcdA (or DsbD) reduce the disulfide bond between cysteine residues of heme-binding motifs (CXXCH) in the apo-cyt c prior to heme attachment (Figure 1—figure supplement 1; Fabianek et al., 2000; Setterdahl et al., 2000; Reid et al., 2001). Curiously, most, if not all, archaeal genomes sequenced thus far lack several ccm genes (namely ccmD, ccmH, ccmI) that have been shown to be essential for cyt c biogenesis in Bacteria like E. coli, R. capsulatus, Paracoccus denitrificans, Bradyrhizobium japonicum, Shewenella oneidensis, and Desulfovibrio desulfuricans (Kranz et al., 1998; Thöny-Meyer, 1997; Goddard et al., 2010; Fu et al., 2015). In bacteria, CcmD facilitates the release of the heme-bound holo-CcmE from the CcmABCD complex (Richard-Fogal et al., 2008), while CcmH and CcmI are a part of the protein complex involved in covalent attachment of heme to the apo-cyt c (Figure 1—figure supplement 1; Kranz et al., 2009; Sanders et al., 2010). As such, based on evidence from studies with bacteria, the streamlined Ccm machinery found in archaeal genomes, comprised only of CcmABCEFG and CcdA, would be insufficient for the biogenesis of cyt c. However, mature cyt c proteins have been identified in many archaeal strains, such as Haloferax volcanii, Ignicoccus hospitalis, Pyrobaculum islandicum, Ferroglobus placidus, and Methanosarcina spp. (Kletzin et al., 2015; Sreeramulu, 2003; Nab et al., 2014; Smith et al., 2015; Feinberg et al., 2008; Wang et al., 2011). Whether Archaea have replaced CcmD, CcmH, and CcmI with non-orthologous proteins or have reconfigured the Ccm machinery such that ccmDHI are no longer essential remains unclear.

In this study, we used the genetically tractable methanogenic archaeon, M. acetivorans, as a model system to functionally characterize the pathway for biogenesis of cyt c in archaea. Most archaea that encode cyt c proteins are either recalcitrant to laboratory cultivation techniques, genetically intractable, or only encode only one cyt c that might be essential for growth (Allen et al., 2006; Kletzin et al., 2015; Chadwick et al., 2021; Arshad et al., 2015). In contrast, M. acetivorans encodes multiple cyt c proteins (Holmes et al., 2019), can be easily cultivated in a laboratory (Sowers et al., 1993), and has state-of-the-art genetic tools (Guss et al., 2008; Kohler and Metcalf, 2012), including inducible gene expression, tests for gene essentiality, and CRISPR-Cas9-based genome editing for complex genetic manipulation (Nayak and Metcalf, 2017). M. acetivorans is a methanogen that can only grow by coupling energy conservation to the production of methane gas using acetate or methylated compounds, like methanol, as a growth substrate. At least five different cyt c proteins are encoded in the M. acetivorans genome and these proteins contain between one and seven heme-binding motifs (Holmes et al., 2019). Recent studies have even proposed distinct roles for some of these cyt c proteins in intracellular electron transport during methanogenesis (Wang et al., 2011; Li et al., 2006; Schlegel et al., 2012; Ferry, 2020), extracellular electron transfer (Holmes et al., 2019), as well as direct interspecies electron transfer between M. acetivorans and bacterial strains like Geobacter metallireducens (Holmes et al., 2021). Thus, M. acetivorans is not only an ideal candidate to dissect the biogenesis of cyt c in Archaea but also to functionally characterize the role of cyt c in various electron transfer processes that have ramifications on global carbon cycling as well as on the formation of microbial communities in anoxic environments.

Here, we have used a combination of genetic, molecular, and biochemical analyses, to show that M. acetivorans and other archaea use a streamlined version of the Ccm machinery that only requires ccmABCEF for cyt c biogenesis. To this end, we have shown that the ccmABCEF from M. acetivorans is sufficient to produce holo-cyt c in a heterologous host, Methanosarcina barkeri Fusaro, that otherwise is incapable of cyt c biogenesis. Our physiological analyses also reveal substrate-specific phenotypes for the cyt c biogenesis pathway and cyt(s) c during growth and methanogenesis in M. acetivorans. A closer inspection of the distribution and synteny of the cyt c biogenesis genes in methane-metabolizing archaea related to Methanosarcina (i.e. belonging to the order Methanosarcinales) suggests that the cyt c biogenesis genes were likely acquired in the last common ancestor of the Methanosarcinales and have been lost in many extant clades that also do not encode any cyt c genes. Although the streamlined Ccm machinery is conserved across Archaea, our evolutionary analyses suggest that the acquisition of cyt c biogenesis in Archaea has occurred through multiple horizontal gene transfer (HGT) events with different members of the Bacteria. Overall, we have used the model methanogenic archaeon, M. acetivorans, as a model system to characterize a streamlined form of the Ccm machinery used for the biogenesis of cyt c in members of Archaea.

Cyt c are widely found in members of the Archaea where they catalyze numerous electron transfer processes that facilitate microbial interactions and fuel biogeochemical cycles. The biogenesis of cyt c is complex and is mediated by a dedicated pathway that can vary substantially across the tree of life. In the vast majority of eukaryotes, a single protein called cyt c heme lyase is involved in the biogenesis of cyt c (Kranz et al., 2009). In contrast, bacteria contain more complex pathways that are fine-tuned to work with lower concentrations of heme (Kranz et al., 2009). The most complex of these pathways, called the Ccm machinery, requires at least nine to ten proteins (CcmABCDEFGH(I) and CcdA/DsbD) that work in concert for the biogenesis of cyt c. The biochemical details of the Ccm machinery in bacteria has been studied in great detail using model systems over the past few decades and the role of each ccm gene is well established at this point (Kranz et al., 2009; Sanders et al., 2010). Previous research has shown that archaea encode some components of the bacterial Ccm machinery (Allen et al., 2006; Kletzin et al., 2015), however, orthologs of at least three proteins (CcmD, CcmH, and CcmI) that are absolutely critical for cyt c maturation in bacteria are universally absent in archaeal genomes. Thus, it was unclear how the Ccm machinery is being repurposed (if at all) for cyt c biogenesis in archaea. In this study, we use the methanogen M. acetivorans to characterize a streamlined form of the Ccm system comprised of only five proteins (CcmABCEF) that likely serves as the predominant route for cyt c biogenesis in members of the Archaea (Figures 2 and 5).

Based on our findings, there are three key differences between the Ccm machinery in Bacteria and Archaea. While ccmD seems to be uniformally absent in archaeal genomes (Allen et al., 2006), it is universally present in bacterial genomes and is essential for cyt c biogenesis in a number of strains ranging from E. coli and Rhodobacter capsultus to D. desulfuricans (Kranz et al., 2009; Goddard et al., 2010; Richard-Fogal et al., 2008). In bacteria, CcmD is a small (69 aa long) transmembrane protein that associates with CcmABC and facilitates the release of holo-CcmE in the periplasm after heme transport and attachment (Richard-Fogal et al., 2008; Feissner et al., 2006a). In M. acetivorans, the CcmABC complex is capable of forming holo-CcmE and releasing it into the pseudo-periplasm in a CcmD-independent manner (Figure 3). It is unlikely that the ccmD gene has been fused to ccmA, ccmB, or ccmC based on the sequence length and alignment of the archaeal genes with their bacterial counterparts (Figure 8—figure supplements 1–3). It is also unlikely that the CcmD-independent mechanism for the release of holo-CcmE from CcmABC might be linked to the CXXXY heme-binding motif in CcmE because some bacteria, like D. desulfuricans, that encode ccmD and require this gene product for cyt c biogenesis also contain a CXXXY heme-binding motif in CcmE (Figure 4; Goddard et al., 2010). One plausible hypothesis for the absence of CcmD might be the unusual architecture of archaeal membranes. The archaeal cytoplasmic membrane is composed of isoprenoid lipids (phytanyl units), compared to phospholipids in bacterial cytoplasmic membranes. This lipid composition might alter how CcmABC and CcmE are anchored into the membrane and render CcmD inconsequential during the formation and release of holo-CcmE.

Next, the archaeal CcmE universally contains a CXXXY heme-binding motif whereas bacterial CcmE can either have an HXXXY motif or a CXXXY motif depending on the strain (Kranz et al., 2009; Goddard et al., 2010). In E. coli, an H130C allele of CcmE can still bind heme but cannot transfer the heme to the CcmFH complex (Goddard et al., 2010; Enggist and Thöny-Meyer, 2003; Stevens et al., 2003). In contrast, a C120H mutation in M. acetivorans seems to destabilize the protein to such an extent that it can no longer be detected (Figure 4 and Figure 4—figure supplement 1). Similarly, no heme-bound CcmE was detected for the C127H allele of CcmE derived from the bacterium D. desulfuricans (Goddard et al., 2010). Taken together, these data suggest that the Cys and His residues involved in heme-binding CcmE are not interchangeable. In fact, many other as yet uncharacterized features of CcmE, possibly unique to these two groups of CcmE proteins, are likely to be equally important for the formation of holo-CcmE and the transfer of heme to the apo-cyt c via interactions between CcmE and CcmF as well as other proteins. Whether the absence of an HXXXY motif containing CcmE in archaea is just an artifact of historical contingency or due to functional/evolutionary constraints remains unclear.

Finally, the cyt c synthetase complex in bacteria contains two (CcmFH or CcmFI) or three (CcmFHI) proteins depending on the strain whereas the archaeal counterpart only contains CcmF. In M. acetivorans, the CcmF gene is split into two coding sequences (ccmF1 and ccmF2), each of which might be derived from different bacterial groups (Figure 8 and Figure 8—figure supplements 5 and 6). Functionally important domains of the cyt c synthetase complex including the two histidine residues that coordinate the pseudo-periplasmic heme b as well as the highly conserved WWD domain are all present in CcmF2 (Figure 2—figure supplement 1). CcmF1 probably plays a secondary role in cyt c biogenesis, which might explain why a small amount of holo-cyt c can still be detected in the ∆ccmF1 mutant of M. acetivorans (Figure 2). In bacteria and Arabidopsis, CcmH/I have been shown to interact with the apo-cyt c and deliver it to CcmF (Kranz et al., 2009; Setterdahl et al., 2000; Meyer et al., 2005). It is unlikely that M. acetivorans and other archaea have replaced CcmH/I with a non-orthologous protein as heterologous expression of ccmABCEF1F2 in the non-native host, M. barkeri Fusaro, seems to be sufficient for cyt c biogenesis (Figure 5). However, we cannot distinguish between the possibility that M. barkeri Fusaro also encodes this non-ortholgous protein or that the standalone CcmF in M. acetivorans performs the function of CcmFH in E. coli.

Within the Archaea, cyt c are especially enriched in the family Methanosarcinaceae wherein these proteins have likely fostered tremendous metabolic innovation (Mand and Metcalf, 2019). Our work and other studies (Holmes et al., 2019; Ferry, 2020; Holmes et al., 2021; Zhou et al., 2021) show that cyt c like MmcA are intricately coupled to growth under a variety of conditions in the marine methanogen M. acetivorans (Figure 6). Furthermore, membrane-spanning multi-heme cyt c have been shown to facilitate anaerobic methane oxidation by microbial consortia comprised of ANME archaea and sulfate reducing bacteria (Wegener et al., 2015; McGlynn et al., 2015; Scheller et al., 2016; He et al., 2019) and recent studies also suggest an important role for cyt c in facilitating anaerobic alkane metabolism in several members of the Methanosarcinaceae (Borrel et al., 2019). Since ANME and alkane oxidizing archaea are yet to be isolated in pure culture, our ability to manipulate the biogenesis of cyt c also renders M. acetivorans as an ideal platform to perform functional analyses of diverse cyt c across the Methanosarcinaceae. Curiously, even though cyt c are widely present in members of the Methanosarcinaceae, there are many extant strains, such as M. barkeri Fusaro, that lack the Ccm machinery altogether and do not encode any cyt c proteins in their genome. Our evolutionary analyses provide strong evidence that the uneven distribution of the Ccm machinery and cyt c in the Methanosarcinaceae is due to gene loss events rather than independent gene gain events (Figure 7 and Figure 7—figure supplement 1). It is also worth noting that many of the extant lineages that lack the Ccm machinery and cyt c are found in freshwater or anthropogenic sources (like waste digestors); reciprocally, the Ccm machinery and diverse cyt c are especially conserved in lineages derived from marine environments (Figure 7 and Figure 7—figure supplement 1). This pattern is indicative of the likelihood that there might be environment-specific selective pressure to retain or discard cyt c and the associated biogenesis machinery. However, the specific biotic or abiotic environmental cues that underlie this pattern remain unclear at the moment.

Previous studies (Allen et al., 2006; Kletzin et al., 2015) have used some of the unique features that unify the archaeal Ccm machinery, notably the absence of CcmDHI and a conserved CXXXY heme-binding domain in CcmE, to support an ancestral origin of the Ccm machinery in the Archaea. Our phylogenetic analyses (Figure 8 and Figure 8—figure supplements 1–6) contradict this hypothesis and provide strong evidence supporting the hypothesis that there have been many independent HGT events that have cross-pollinated the Ccm machinery between the Archaea and Bacteria. In fact, even the Ccm machinery in one single archaeon, like M. acetivorans, may not have been derived from a single source (Figure 8 and Figure 8—figure supplements 1–6). Taken together, the streamlined Ccm machinery in the Archaea is functionally uniform despite independent evolutionary origins.

All data generated or analysed during this study are included in the manuscript and supporting file; sequencing data have been deposited in the NCBI SRA (Sequence Read Archive) under bioproject number PRJNA800036. Source data files for Figure1b, Figure 2c, Figure 3d, Figure 4c and Figure 3—figure supplement 1, Figure 4—figure supplement 1 and 2, are provided.

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Author details

1. Dinesh Gupta

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9108-9669

2. Katie E Shalvarjian

   Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   Data curation, Formal analysis, Methodology, Validation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4792-8477

3. Dipti D Nayak

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing - review and editing

   For correspondence

   dnayak@berkeley.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3449-3419

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