An Archaea-specific c-type cytochrome maturation machinery is crucial for methanogenesis in Methanosarcina acetivorans

Abstract

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

The dual role of a multi-heme cytochrome in methanogenesis: MmcA is important for energy conservation and carbon metabolism in Methanosarcina acetivorans

“…In addition, the MHC subunit MmcA is vital for the functionality of the Rnf complex. Our growth data for the Δ mmcA mutant on acetate agree with previous work from our group, but are in contrast a previous study where a mmcA deletion mutant of M. acetivorans had no growth defect on acetate compared to the corresponding parent strain (22, 24). We suspect that the difference in phenotype observed may pertain to differences in the genetic techniques used for generating deletion mutants, or the presence of a suppressor mutation in the mmcA deletion strain generated in the previous work (22).…”

“…To characterize the in vivo function of the Rnf complex, we obtained a mutant that has a markerless in-frame deletion in the M. acetivorans chromosome spanning mmcA through rnfX as described in (23) (Figure 2b). To understand the function of the MmcA gene in the Rnf complex, we generated a markerless in-frame deletion in the mmcA gene as described previously using Cas9-mediated genome editing (24, 25) (Figure 2c). We sequenced the genome of the Δ mmcA mutant and, relative to the parent strain (WWM60), did not detect mutations elsewhere on the chromosome (Supplementary Table 1).…”

“…The empty vector contains the β-glucuronidase gene, uidA , under the control of a tetracycline inducible P mcrB ( tetO4 ) promoter described previously in (60). The complementation vector contains mmcA under the control of a tetracycline inducible P mcrB ( tetO4 ) promoter described previously in (24). Three replicate tubes of each strain were used for growth assays and tetracycline was added to a final concentration of 100 ug/mL in the growth medium.…”

“…The Rnf complex as well as MmcA are essential for growth on acetate via acetoclastic methanogenesis (18, 23, 24). We observed no growth of the Δ rnf or Δ mmcA mutants on acetate, while WWM60 with a fully functional Rnf complex was viable (Figure 3a, Table 1).…”

The dual role of a multi‐heme cytochrome in methanogenesis: MmcA is important for energy conservation and carbon metabolism in Methanosarcina acetivorans

“…To characterize the in vivo function of the Rnf complex, we obtained a ∆ mmcA‐rnf mutant that has a markerless in‐frame deletion in the M. acetivorans chromosome spanning mmcA through rnfX as described in Mand (2018) (Figure 2b). To understand the function of the MmcA gene in the Rnf complex, we generated a markerless in‐frame deletion in the mmcA gene as described previously using Cas9‐mediated genome editing (Gupta et al, 2022; Nayak & Metcalf, 2017) (Figure 2c). We sequenced the genome of the ∆ mmcA mutant and, relative to the parent strain (WWM60), did not detect mutations elsewhere on the chromosome (Table S1).…”