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C -type cytochromes (cyt c ) are proteins that covalently bind heme and are integral to electron transport chains. A growing body of evidence suggests that cyt c play a vital role in both intra- and extra-cellular electron transfer processes in Archaea, especially in members that metabolize methane and other short chain alkanes. Elaborate mechanisms for the biogenesis of cyt c are known in Bacteria and Eukarya but this process remains largely uncharacterized in Archaea. Here, we have used the model methanogenic archaeon Methanosarcina acetivorans to characterize a distinct form of the system I cyt c maturation machinery (referred to as the Ccm machinery henceforth) that is broadly distributed in members of the Archaea. Phenotypic analyses of M. acetivorans mutants deficient in essential components of the Ccm machinery reveal that cyt c are broadly important for growth and methanogenesis, but the magnitude of their impact can vary substantially depending on the growth substrate. Heterologous expression of a synthetic operon with the Ccm machinery (CcmABCEF) from M. acetivorans is both necessary and sufficient for cyt c biogenesis in a non-native host ( M. barkeri Fusaro) that is incapable of cyt c biogenesis. Even though components of the Ccm machinery are universally conserved across the Archaea, our evolutionary analyses indicate that different clades of Archaea acquired this pathway through multiple independent horizontal gene transfer events from different groups of Bacteria. Overall, we have demonstrated the convergent evolution of a novel Archaea-specific Ccm machinery for cyt c biogenesis and its role in methane metabolism.
Microorganisms belonging to the domain Archaea play an especially important role in regulating atmospheric methane levels. Specifically, methanogens are the primary source of biogenic methane and an aerobic me thanotrophic archaea (ANME) consume a substantial proportion of methane released in marine sediments. Genomic studies have implicated a class of electron-transfer proteins called c -type cytochromes as being crucial in mediating archaeal methane metabolism in the environment. However, neither the biogenesis nor the role of c -type cytochromes in methane metabolism has ever been investigated. Here, we have used a model methanogen, Methanosarcina acetivorans, to characterize a distinct pathway for maturation of c -type cytochromes that seems to be uniformly conserved across the Archaea and have also identified substrate-specific functional roles for c -type cytochromes during methanogenesis.
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This paper will be of interest to scientists interested in archaea and the field of bioenergetics. Using a series of elegant experiments the authors show that that archaea have a streamlined and functional cytochrome c biogenesis machinery.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
Reviewer #1 (Public Review):
Within this manuscript the authors set out to determine the biogenesis of c-type cytochromes in methane metabolism. Compared to the bacterical cytochrome c assembly system, genes like ccmD, ccmH and ccmI are not found in archaea that contain a functional cytochrome Cs. They show that the proteins encoded within the ccmABCEF operon of Methanosarcina acetivorans are both essential and sufficient for cyt c biogenesis. They also show the substrate specific role of the mmcA cytochrome C. The authors do this using a combination of genetic, molecular, and physiological and biochemical analyses.
The manuscript is well describes a clear set of experiments and the authors are successful in determining the biogenesis of c-type cytochromes in methane metabolism. The manuscript is well written and is easy to read.
Reviewer #2 (Public Review):
The manuscript by Gupta et al investigates the cytochrome c maturation proteins in archaea, more specifically in methanogens. The authors demonstrate with different deletion mutant and overexpression studies that the genes ccmABCEF are required for cytochrome c maturation. By using phylogenomics, they provide evidence for several horizontal gene transfer events from bacteria to archaea giving rise to the cytochrome c maturation machinery in different clades of archaea. The manuscript is interesting and well written. The experiments are well documented and support the claims made by the authors.