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c -Type cytochromes (cyt c ) are proteins that undergo post-translational modification to covalently bind heme, which allows them to facilitate redox reactions in electron transport chains across all domains of life. Genomic evidence suggests that cyt c are involved in electron transfer processes among the Archaea, especially in members that produce or consume the potent greenhouse gas methane. However, neither the maturation machinery for cyt c in Archaea nor their role in methane metabolism has ever been functionally characterized. Here, we have used CRISPR-Cas9 genome editing tools to map a distinct pathway for cyt c biogenesis in the model methanogenic archaeon Methanosarcina acetivorans, and have also identified substrate-specific functional roles for cyt c during methanogenesis. Although the cyt c maturation machinery from M. acetivorans is universally conserved in the Archaea, our evolutionary analyses indicate that different clades of Archaea acquired this machinery through multiple independent horizontal gene transfer events from different groups of Bacteria. Overall, we demonstrate the convergent evolution of a novel Archaea-specific cyt c maturation machinery and its physiological role during methanogenesis, a process which contributes substantially to global methane emissions.
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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.