Research

Project 1: Unraveling the role of iron-sulfur clusters in RNA polymerase

Recently, the RpoD subunit of RNA polymerase (RNAP) in several archaea, including methanogens, and the corresponding Rpb3 subunit of RNAP in several eukaryotes were shown or predicted to contain a [4Fe-4S] cluster. The function of the [4Fe-4S] cluster is unknown.  RpoD forms a heterodimer with RpoL, which is the first step in assembly of multi-subunit RNAP in Archaea. The clusters are postulated to regulate the interaction of RpoD with RpoL or other subunits to control RNAP assembly and activity in response to oxidants. M. acetivorans RpoD is predicted to bind two [4Fe-4S] clusters within a domain that is likely not required for interaction with RpoL (Fig. 1). We are using a combination of in vivo mutational analyses and native and recombinant RpoDL/RNAP purification to elucidate the importance of the clusters to RNAP assembly and activity (4). This project is currently funded by the NSF.

Project 2: Understanding the role of metal cofactors as molecular switches in the MdrA family of disulfide reductases 

We have identified a novel iron-sulfur cluster containing disulfide reductase (MdrA) from M. acetivorans (2).The iron-sulfur clusters in MdrA are postulated to function as a molecular switch to control disulfide reductase activity. MdrA may function to repair of oxidatively-damaged iron-sulfur clusters (Fig. 2). We are using a combination of genetics and biochemistry to determine the precise physiological role(s) of MdrA. Through collaboration with Dr. Suresh Kumar (Dept. of Chemistry & Biochemistry) we are using biophysical techniques (NMR, CD, ITC, and DSC) to examine the influence of the clusters, and other metals, on the structure and properties of MdrA. This project is currently supported by the Arkansas Biosciences Institute and the NIH COBRE center.

Project 3: Genetic engineering of methanogens

We are generating strains of M. acetivorans with improved characteristics, using genetic engineering and directed evolution approaches. We have recently produced a strain of M. acetivorans with a hybrid methanogenesis pathway that confers the ability to grow and produce methane with non-native substrates (Fig. 3) (3). We are currently constructing strains with increased oxygen tolerance and metabolic capabilities. 

Publications

1.         Lessner, D. 2009. Methanogenesis: Biochemistry, Encyclopedia of Life Sciences. Nature Publishing Group.

2.         Lessner, D. J., and J. G. Ferry. 2007. The Archaeon Methanosarcina acetivorans Contains a Protein Disulfide Reductase with an Iron-Sulfur Cluster. J Bacteriol 189:7475-84.

3.         Lessner, D. J., L. Lhu, C. S. Wahal, and J. G. Ferry. 2010. An engineered methanogenic pathway derived from the domains Bacteria and Archaea. mBio 1:e000243-10.

4.         Lessner, F. H., M. E. Jennings, A. Hirata, E. C. Duin, and D. J. Lessner. 2012. Subunit D of RNA polymerase from Methanosarcina acetivorans contains two oxygen-labile [4Fe-4S] clusters: implications for regulation of transcription in archaea. J Biol. Chem. 287:18510-18523.