Assistant Professor of Microbiology
Research Interests: Anaerobic one-carbon metabolism in methanogens and Gram-positive bacteria
Methylamine dependent methanogenesis:
Methanosarcina acetivorans is a methylotrophic methanogenic archaeon capable of growth in many environments and on a variety of substrates. Research on this and related organisms has revealed the existence of analogous pathways of methanol- and methylamine-dependent methanogenesis involving homologous corrinoid (vitamin B12) binding proteins and non-homologous methanol and methylamine methyltransferases. In close proximity to the genes encoding the methylamine specific methyltransferases and corrinoid proteins there have been genes encoding putative permeases: mtmP, mtbP, and mttP. The precise role of these putative permease genes has never been demonstrated. One of the goals of my laboratory is to demonstrate the role of these genes in methylamine dependent methanogenesis by generating knock-out mutants in M. acetivorans and by functional expression of the genes. We have successfully knocked out the mtmP gene from this organism and see a decrease in both the maximum OD and the growth rate of the culture on methylamine (MMA) as the substrate suggesting a role for this enzyme in MMA catabolism. We are currently working towards complementing this mutation by re-introducing the mtmP gene into the mutant and examining the ability of the mutant to grow on MMA. We also plan to examine the ability of the mutant to take up labeled MMA to explore its predicted role as a MMA permease. We predict that the corrinoid dependent methyltransferases specific for each of these substrates will interact with its cognate methylamine permease to form a catabolic complex for efficient utilization of the substrate upon entry to the cell. Future experiments will explore this model of interaction.
C-1 metabolism in Desulfitobacterium hafniense:
The genes encoding the methylamine methyltransferases MttB, MtbB, and MtmB all contain an in-frame amber UAG codon. This UAG codon encodes the 22nd genetically encoded amino acid pyrrolysine. The genes necessary for the translation of the UAG codon are found within the pylTSBCD operon in methylotrophic methanogens capable of growth on methylamines. A Gram-positive bacterium called Desulfitobacterium hafniense is one of at least nine species of bacteria to also possess the Pyl operon within its genome. We have begun multiple lines of study in this organism to examine its ability to catabolize one-carbon (C-1) compounds via anaerobic respiration and the role of its Pyl operon and predicted Pyl containing MttB methyltransferase. We have shown that this organism is a much more robust C-1 metabolizer than previously thought. We have expanded the known growth substrates of this organism to include simple methylamines such as mono-, di-, and trimethylamine (MMA, DMA, and TMA) as well as choline and methylated glycine derivatives such as glycine betaine (trimethylglycine or TMG), dimethylglycine (DMG), and sarcosine (monomethylglycine or MMG).
We are currently working towards expressing the amber codon containing mttB gene from D. hafniense in M. acetivorans to exploit the ability of the methanogen to translate the amber codon. Upon production and purification of this enzyme we will examine its ability to catalyze methyl transfer from trimethylamine to either free or enzyme bound corrinoid. Successful demonstration of the activity of this enzyme will be the first of its kind in bacteria.
D. hafniense also contains homologs of the mttB gene that contain no amber codon and therefore encode enzymes that lack the Pyl residue. There are actually a large number of mttB homologs in the database from anaerobic organisms and these genes are part of the MTTB superfamily. Thus far, only the pyl containing MttB enzymes have known functions and yet these enzymes constitute a small portion of the superfamily. It is known that the Pyl residue is essential for activity of these enzymes as TMA:corrinoid methyltransferases. Therefore most MttB-like enzymes are of unknown function due to their lack of this key residue. One such example is the DSY3156 enzyme from D. hafniense strain Y51. We have experiments nearing completion in which we have demonstrated the function of this enzyme and its role in C-1 metabolism in the organism. This work will be submitted for publication in the near future. We are also examining the other mttB homologs from this organism as well as the enzymes and corrinoid binding proteins we predict are also involved in C-1 metabolism of this organism.
I currently have two graduate students and four undergraduate researchers in my laboratory. One of my graduate students is likely to graduate in 2014 and I am therefore seeking applications from qualified students for graduate school. I am also interested in interviewing well-qualified and highly motivated Miami University undergraduates to undertake independent study projects in my lab. Feel free to contact me if interested.
- Ferguson,DJ Jr, Longstaff DG and Krzycki JA. 2011. Assay of methylotrophic methyltransferases from methanogenic archaea. Meth Enzymol. vol 494. pp. 139-158.
- Gong W, Hao B, Wei Z, Ferguson DJ Jr, Tallant T, Krzycki J, and Chan M. Structure of the α2ε2 Ni-CODH component of the Methanosarcina barkeri ACDS complex. Proc Nat Acad Sci. (USA) 2008 Jul 15;105(28):9558-63.
- Ferguson DJ Jr, Gorlatova N, Grahame DA, Krzycki JA. constitution of dimethylamine:coenzyme M methyl transfer with a discrete corrinoid protein and two methyltransferases purified from Methanosarcina barkeri. J Biol Chem. 2000 Sep 15;275(37):29053-60.
- Paul L, Ferguson, DJ Jr, Krzycki JA. The trimethylamine methyltransferase gene and multiple dimethylamine methyltransferase genes of Methanosarcina barkeri contain in-frame and read-through amber codons. J Bacteriol. 2000 May;182(9):2520-9.
- Ferguson DJ Jr, Krzycki JA. Reconstitution of trimethylamine-dependent coenzyme M methylation with the trimethylamine corrinoid protein and the isozymes of methyltransferase II from Methanosarcina barkeri. J Bacteriol. 1997 Feb;179(3):846-52.
- Ferguson DJ Jr, Krzycki JA, Grahame DA. Specific roles of methylcobamide:coenzyme M methyltransferase isozymes in metabolism of methanol and methylamines in Methanosarcina barkeri. J Biol Chem. 1996 Mar 1;271(9):5189-94.