D. Gupta, K. E. Shalvarjian, D.D. Nayak. An Archaea-specific c-type cytochrome maturation machinery is crucial for methanogenesis in Methanosarcina acetivorans. eLife 11:e76970 (2022).
* Archaea encode a unique, streamlined form of the System I pathway for c-type cytochrome biogenesis with three unusual features that distinguish it from the Bacterial/Eukaryotic System I pathway.
* C-type cytochromes are important for the growth and fitness of methanogens like Methanosarcina acetivorans but their role in methanogenesis varies substantially in a substrate-specific manner.
* The Archaea-specific System I pathway for c-type cytochrome biogenesis has been acquired by horizontal gene transfer (HGT) events from Bacteria and undergone convergent evolution within the domain.
K.E. Shalvarjian, D.D. Nayak*. Transcriptional Regulation of Methanogenic Metabolism in Archaea. Current Opinion in Microbiology, 60: 8—15 (2021). Review.
* Transcriptional regulation in Archaea is distinct from that of Bacteria or Eukarya.
* Methanogens present a unique opportunity to study archaeal regulation.
* Methanogenesis is regulated in response to substrate availability.
* Post-transcriptional regulation contributes to gene expression in methanogens.
D.D. Nayak, A. Liu, N. Agarwal, R. Rodriguez-Carrero, S.H. Dong, D.A. Mitchell, S.K. Nair, W.W.Metcalf. Functional Interactions Between Posttranslationally Modified Amino Acids of Methyl-Coenzyme M Reductase in Methanosarcina acetivorans. PloS Biology, 18(2): e3000507 (2020).
* We found a dedicated S-adenosylmethionine (SAM) dependent methyltransferase installs a S-methylcysteine post-translational modification (PTM) in Methyl-Coenzyme M Reductase (MCR).
* We observe that mutants of M. acetivorans that lack three different PTMs in MCR (S-methylcysteine, 5-methylarginine, thioglycine) in all possible combinations are viable.
* We show that interactions between the three different PTMs are important for the structure and function of MCR in vivo and in vitro.
D.D. Nayak*, W. W. Metcalf. Methylamine-specific Methyltransferase Paralogs in Methanosarcina are Functional Distinct Despite Frequent Gene Conversion. ISME Journal, 13(9): 2173-2182 (2019).
* Most Methanosarcina strains encode at least two copies of the methylamine-specific methyltransferase that are functionally distinct.
* We show that one copy of the methylamine-specific methyltransferase is specialized for carbon metabolism whereas the other is used for nitrogen metabolism.
* Our evolutionary analyses reveal that the coding sequence of methylamine-specific methyltransferases undergo gene conversion and the gene specific function is conferred by 5’ regulatory elements.
D.D. Nayak, W.W. Metcalf. Genetic Techniques for Studies of Methyl-Coenzyme M Reductase from Methanosarcina acetivorans. Methods in Enzymology, 613:325-247 (2018).
D.D. Nayak, N. Mahanta, D.A. Mitchell, W.W. Metcalf. Post-translational Thioamidation of Methyl-Coenzyme M Reductase, a Key Enzyme in Methanogenic and Methanotrophic Archaea. eLife 6:e29218 (2017).
* Methyl-coenzyme M reductase (MCR) is found in methanogenic and methanotrophic archaea and catalyzes the reversible production and consumption of methane
* A highly unusual thioglycine modification, in which the peptide amide bond is converted to a thioamide, is present in the α-subunit of all characterized MCRs and is implicated to play a critical role in catalysis
* Using our Cas9-mediated genome editing tools, we show that mutants of the methanogen Methanosarcina acetivorans with deletions in the ycaO-tfuA operon are viable and lack the thioglycine modification in MCR
* Our analyses support a role for thioglycine in stabilizing the protein secondary structure near the active site of MCRz
D.D. Nayak, W. W. Metcalf. Cas9 Mediated Genome Editing in the Methanogenic Archaeaon, Methanosarcina acetivorans. Proceedings of the National Academy of Sciences. 114:2976-2981 (2017).
Press and other mentions:
“Finally, Archaea Get Their CRISPR-Cas Toolbox”, spotlight article in Trends in Microbiology, Volume 25, Issue 6, June 2017, Pages 430-432.
Abstract: “The majority of archaea encode CRISPR-Cas systems but only a few CRISPR-Cas-based genetic tools have been developed for organisms from this domain. Nayak and Metcalf have harnessed a bacterial Cas9 protein for genome editing in Methanosarcina acetivorans, enabling efficient gene deletion and replacement.”
* We report the development of the first Cas9-mediated genome editing tool for an archaeon, the slow-growing methanogen Methanosarcina acetivorans.
* Introduction of both insertions and deletions by homology directed repair (HDR) is efficient and precise (~ 20% of transformation efficiency) without any detectable off-target activity.
* Multiple single guide RNAs (sgRNAs) can be expressed in the same transcript and allows simultaneous construction of double mutants with high efficiency.
* Co-expression of the non-homologous end joining (NHEJ) machinery from the closely related archaeon, Methanocella paludicola, allows for efficient Cas9-mediated genome editing without the need for a repair template.
D.D. Nayak, D. Agashe, M.C. Lee, C.J. Marx. Selection Maintains Apparently Degenerate Metabolic Pathways Due to Tradeoffs in Using Methylamine for Carbon Versus Nitrogen. Current Biology. 26: 1416-1426. (2016).
D. D. Nayak, C.J. Marx. Experimental Horizontal Gene Transfer of Methylamine Dehydrogenases Mimics Prevalent Exchange in Nature and Overcomes the Methylamine Growth Constraints Posed by the Sub-Optimal N-Methylglutamate Pathway. Microorganisms. 3: 60-79 (2015).
D.D. Nayak, C.J. Marx. Methylamine Utilization via the N-Methylglutamate Pathway in Methylobacterium extorquens PA1 Involves a Novel Flow of Carbon through C1 Assimilation and Dissimilation Pathways. Journal of Bacteriology. 196: 4130-4139 (2014).
D. D. Nayak, C.J. Marx. Genetic and Phenotypic Comparison of Facultative Methylotrophy between Methylobacterium strains PA1 and AM1. PLoS One. 9: e107887 (2014).
All members of the Nayak lab are underlined and manuscripts with Dipti Nayak as the corresponding author are indicated with an *.
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