An Archaea-specific c-type cytochrome maturation machinery is crucial for methanogenesis in Methanosarcina acetivorans
- Department of Molecular and Cell Biology, University of California, Berkeley, United States
- Department of Plant and Microbial Biology, University of California, Berkeley, United States
Figures
Figure 1 with 2 supplements
Experimental assays to measure the production and maturation of the diagnostic c-type Cytochrome, MmcA.
(a) The design of diagnostic constructs to use the native heptaheme c-type cytochrome, MmcA, to map the cytochrome c biogenesis pathway in Methanosarcina acetivorans. (i) The control construct (pM) …
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Figure 1—source data 1
Raw gel and blot images.
- https://cdn.elifesciences.org/articles/76970/elife-76970-fig1-data1-v2.pdf
Figure 1—figure supplement 1
The Ccm Machinery in Bacteria and Archaea.
(a) Chromosomal organization of the System I c-type cytochrome (cyt c) maturation (Ccm) machinery genes in a few representative bacteria and the archaeon, Methanosarcina acetivorans. In bacteria …
Figure 1—figure supplement 2
MmcA is a heptaheme c-type cytochrome associated with the Rnf complex in Methanosarcina acetivorans.
(a) Amino acid sequence of the c-type cytochrome (cyt c) MmcA from Methanosarcina acetivorans. The predicted signal peptide is shown in blue, five canonical heme-binding motifs (CXXCH) are shown in …
Figure 2 with 1 supplement
Identifying the role of Ccm genes in c-type cytochrome maturation in Methanosarcina acetivorans.
(a) Chromosomal organization of the System I cytochrome c maturation machinery (Ccm) genes in Methanosarcina acetivorans. Double dashed lines indicate that the genes are located more than 110 kbp …
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Figure 2—source data 1
Raw gel and blot images.
- https://cdn.elifesciences.org/articles/76970/elife-76970-fig2-data1-v2.pdf
Figure 2—figure supplement 1
Alignment of the CcmF amino acid sequence from Escherichia coli with the concatenated CcmF2-CcmF1 amino acid sequences from Methanosarcina acetivorans.
Based on this alignment, the CcmF2 sequence (in blue) from M. acetivorans is homologous to N-terminus of CcmF from bacteria and the CcmF1 (in purple) sequence of M. acetivorans is homologous to the …
Figure 3 with 1 supplement
CcmABC is required for the heme transport to form holo-CcmE in Methanosarcina acetivorans.
(a) The formation of heme-bound holo-CcmE in bacteria containing the System I Ccm machinery is facilitated by the CcmABCD complex. The ATP-dependent CcmABC complex translocates heme b from the …
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Figure 3—source data 1
Raw gel and blot images.
- https://cdn.elifesciences.org/articles/76970/elife-76970-fig3-data1-v2.pdf
Figure 3—figure supplement 1
Expression of CcmE with a C-terminal 1× FLAG-1× Strep tag from a plasmid (pE) in the parent strain of Methanosarcina acetivorans (WWM60) and the ∆ccmE background leads to the production of heme-bound holo-MmcA as detected by a heme peroxidase assay of whole cell lysates (lanes 1, 3 of heme-stained gel).
Thus, the C-tagged CcmE is functional and facilitates the maturation of native c-type cytochromes (cyt c) like MmcA. In contrast, expression of C-tagged CcmE in the ∆ccmABC background does not …
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Figure 3—figure supplement 1—source data 1
Raw gel and blot images.
- https://cdn.elifesciences.org/articles/76970/elife-76970-fig3-figsupp1-data1-v2.pdf
Figure 4 with 2 supplements
Identifying the heme-binding residue in CcmE from Methanosarcina acetivorans.
(a) Alignment of the heme-binding domain in CcmE sequences derived from representative bacteria and archaea. The heme-binding residue in bacteria can vary: some species like Escherichia coli …
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Figure 4—source data 1
Raw gel and blot images.
- https://cdn.elifesciences.org/articles/76970/elife-76970-fig4-data1-v2.pdf
Figure 4—figure supplement 1
Expression of the C120H mutant of CcmE from Methanosarcina acetivorans with a C-terminal 1× FLAG-1× Strep tag in the ∆ccmE background does not lead to the production of any CcmE protein that can be detected by heme peroxidase assays (top gel) or Western blot using an anti-Flag antibody (bottom gel) in the Strep-enriched membrane fraction (lane 1) or the soluble fraction (lane 2).
In contrast, expression of the wild-type (WT) sequence CcmE from M.acetivorans with a C-terminal 1× FLAG-1× Strep tag in the ∆ccmE background leads to the production of protein that can be detected …
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Figure 4—figure supplement 1—source data 1
Raw gel and blot images.
- https://cdn.elifesciences.org/articles/76970/elife-76970-fig4-figsupp1-data1-v2.pdf
Figure 4—figure supplement 2
Expression of the wild-type (WT) sequence of CcmE from Methanosarcina acetivorans with a C-terminal 1× FLAG-1× Strep tag (C-tagged CcmE) in the ∆ccmE background leads to the production of heme-bound holo-MmcA as detected by a heme peroxidase assay of whole cell lysates (lane 1 of heme-stained gel).
Thus, the C-tagged WT sequence of CcmE is functional and facilitates the maturation of native c-type cytochromes (cyt c) like MmcA. In contrast, expression of C-tagged C120H or C120A mutants of CcmE …
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Figure 4—figure supplement 2—source data 1
Raw gel and blot images.
- https://cdn.elifesciences.org/articles/76970/elife-76970-fig4-figsupp2-data1-v2.pdf
Figure 5 with 1 supplement
Heterologous expression of the Ccm machinery and the diagnostic c-type cytochrome MmcA in Methanosarcina barkeri Fusaro.
(a) The design of constructs to express a synthetic operon comprised of the ccmABCEF1F2 genes from Methanosarcina acetivorans and/or the mmcA coding sequence from M. acetivorans with a C-terminal …
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Figure 5—source data 1
Raw gel and blot images.
- https://cdn.elifesciences.org/articles/76970/elife-76970-fig5-data1-v2.pdf
Figure 5—figure supplement 1
Measuring c-type cytochrome production in cell lysates from Methanosarcina acetivorans and Methanosarcina barkeri.
(Left) Heme peroxidase assays to detect proteins that covalent bind heme (including c-type cytochromes) in whole cell lysates of Methanosarcina acetivorans (lanes 1, 2, 3) and Methanosarcina barkeri …
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Figure 5—figure supplement 1—source data 1
Raw gel and blot images.
- https://cdn.elifesciences.org/articles/76970/elife-76970-fig5-figsupp1-data1-v2.pdf
Figure 6 with 1 supplement
Growth curves of the parent strain (WWM60; referred to as wild-type [WT]) (in blue circles), the ∆mmcA mutant (in red diamonds), the ∆ccmABC mutant (in olive triangles), and the ∆ccmE mutant (in green inverted triangles) in high-salt minimal medium with (a) 50 mM trimethylamine hydrochloride (TMA), (b) 125 mM methanol, (c) 40 mM sodium acetate (acetate), and (d) 20 mM dimethyl sulfide (DMS) as the sole carbon and energy source.
Four replicates were used for growth assays on TMA and methanol and three replicates were used for growth assays on acetate and DMS.
Figure 6—figure supplement 1
Different modes of methanogenic growth in Methanosarcina acetivorans.
(a) Methanogenic metabolism of trimethylamine (TMA) in Methanosarcina acetivorans. Green arrows represent TMA, dimethylamine (DMA), monomethylamine (MMA) specific methyltransferase reactions that …
Figure 7 with 1 supplement
Distribution of the Ccm machinery genes in members of the Methanosarcina Genus.
(a) A phylogenetic tree for strains belonging to the genus Methanosarcina in the Genome Taxonomy Database (GTDB) was obtained from AnnoTree (Mendler et al., 2019). The presence or absence of genes …
Figure 7—figure supplement 1
A phylogenomic reconstruction of the relationship between various genera (depicted by different colors along the circumference of the radial tree) within the family Methanosarcinaceae from AnnoTree (Mendler et al., 2019).
Purple lines depict strains that encode homologs of ccmABCEF1F2 in their genome whereas black lines depict strains that lack homologs of ccmABCEF1F2 in their genome. Based on the pattern of …
Figure 8 with 6 supplements
Maximum-likelihood phylogenetic trees of 500 (a) CcmE and (b) CcmF2 sequences obtained from the NCBI non-redundant (nr) protein database using the corresponding sequence from Methanosarcina acetivorans as the search query.
Sequences belonging to certain functional groups or evolutionarily related groups of archaea and bacteria are shown in colors denoted in the legend at the bottom. These gene trees are indicative of …
Figure 8—figure supplement 1
An unrooted maximum-likelihood tree of 500 CcmA sequences retrieved from the NCBI non-redundant protein sequence database using the corresponding sequence from Methanosarcina acetivorans as the search query.
Branch labels indicate support values.
Figure 8—figure supplement 2
An unrooted maximum-likelihood tree of 500 CcmB sequences retrieved from the NCBI non-redundant protein sequence database using the corresponding sequence from Methanosarcina acetivorans as the search query.
Branch labels indicate support values.
Figure 8—figure supplement 3
An unrooted maximum-likelihood tree of 500 CcmC sequences retrieved from the NCBI non-redundant protein sequence database using the corresponding sequence from Methanosarcina acetivorans as the search query.
Branch labels indicate support values.
Figure 8—figure supplement 4
An unrooted maximum-likelihood tree of 500 CcmE sequences retrieved from the NCBI non-redundant protein sequence database using the corresponding sequence from Methanosarcina acetivorans as the search query.
Branch labels indicate support values.
Figure 8—figure supplement 5
An unrooted maximum-likelihood tree of 500 CcmF1 sequences retrieved from the NCBI non-redundant protein sequence database using the corresponding sequence from Methanosarcina acetivorans as the search query.
Branch labels indicate support values.
Figure 8—figure supplement 6
An unrooted maximum-likelihood tree of 500 CcmF2 sequences retrieved from the NCBI non-redundant protein sequence database using the corresponding sequence from Methanosarcina acetivorans as the search query.
Branch labels indicate support values.
Additional files
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Supplementary file 1
List of mutations in CRISPR-edited mutant strain DDN029 containing a ∆ccmABC in-frame deletion mutation.
- https://cdn.elifesciences.org/articles/76970/elife-76970-supp1-v2.docx
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Supplementary file 2
Growth data of Methanosarcina acetivorans strains shown in Figure 6.
- https://cdn.elifesciences.org/articles/76970/elife-76970-supp2-v2.docx
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Supplementary file 3
List of target sequences used in this study.
- https://cdn.elifesciences.org/articles/76970/elife-76970-supp3-v2.docx
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Supplementary file 4
List of plasmids used in this study.
- https://cdn.elifesciences.org/articles/76970/elife-76970-supp4-v2.docx
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Supplementary file 5
List of primers used in this study.
- https://cdn.elifesciences.org/articles/76970/elife-76970-supp5-v2.docx
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Supplementary file 6
List of Methanosarcina strains used in this study.
- https://cdn.elifesciences.org/articles/76970/elife-76970-supp6-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/76970/elife-76970-transrepform1-v2.pdf
