Formation of m2G6 in Methanocaldococcus jannaschii tRNA catalyzed by the novel methyltransferase Trm14

The modified nucleosides N2-methylguanosine and N22-dimethylguanosine in transfer RNA occur at five positions in the D and anticodon arms, and at positions G6 and G7 in the acceptor stem. Trm1 and Trm11 enzymes are known to be responsible for several of the D/anticodon arm modifications, but methylases catalyzing post-transcriptional m2G synthesis in the acceptor stem are uncharacterized. Here, we report that the MJ0438 gene from Methanocaldococcus jannaschii encodes a novel S-adenosylmethionine-dependent methyltransferase, now identified as Trm14, which generates m2G at position 6 in tRNACys. The 381 amino acid Trm14 protein possesses a canonical RNA recognition THUMP domain at the amino terminus, followed by a γ-class Rossmann fold amino-methyltransferase catalytic domain featuring the signature NPPY active site motif. Trm14 is associated with cluster of orthologous groups (COG) 0116, and most closely resembles the m2G10 tRNA methylase Trm11. Phylogenetic analysis reveals a canonical archaeal/bacterial evolutionary separation with 20–30% sequence identities between the two branches, but it is likely that the detailed functions of COG 0116 enzymes differ between the archaeal and bacterial domains. In the archaeal branch, the protein is found exclusively in thermophiles. More distantly related Trm14 homologs were also identified in eukaryotes known to possess the m2G6 tRNA modification

Physiology and Genetics of Biogenic Methane-Production from Acetate

Biomass conversion catalyzed by methanogenic consortia is a widely available, renewable resource for both energy production and waste treatment. The efficiency of this process is directly dependent upon the interaction of three metabolically distinct groups of microorganisms; the fermentative and acetogenic Bacteria and the methanogenic Archaea. One of the rate limiting steps in the degradation of soluble organic matter is the dismutation of acetate, a predominant intermediate in the process, which accounts for 70 % or more of the methane produced by the methanogens. Acetate utilization is controlled by regulation of expression of carbon monoxide dehydrogensase (COdh), which catalyzes the dismutation of acetate. However, physiological and molecular factors that control differential substrate utilization have not been identified in these Archaea. Our laboratory has identified sequence elements near the promoter of the gene (cdh) encoding for COdh and we have confirmed that these sequences have a role in the in vivo expression of cdh. The current proposal focuses on identifying the regulatory components that interact with DNA and RNA elements, and identifying the mechanisms used to control cdh expression. We will determine whether expression is controlled at the level of transcription or if it is mediated by coordinate interaction of transcription initiation with other processes such as transcription elongation rate and differential mRNA stability. Utilizing recently sequenced methanosarcinal genomes and a DNA microarray currently under development genes that encode regulatory proteins and transcription factors will be identified and function confirmed by gene disruption and subsequent screening on different substrates. Functional interactions will be determined in vivo by assaying the effects of gene dosage and site-directed mutagenesis of the regulatory gene on the expression of a cdhAÂ::lacZ operon fusion. Results of this study will reveal whether this critical catabolic pathway is controlled by mechanisms similar to those employed by the Bacteria and Eukarya, or by a regulatory paradigm that is unique to the Archaea. The mechanism(s) revealed by this investigation will provide insight into the regulatory strategies employed by the aceticlastic methanogenic Archaea to efficiently direct carbon and electron flow in anaerobic consortia during fermentative processes

Physiology and Genetics of Biogenic Methane-Production from Acetate

Biomass conversion catalyzed by methanogenic consortia is a widely available, renewable resource for both energy production and waste treatment. The efficiency of this process is directly dependent upon the interaction of three metabolically distinct groups of microorganisms; the fermentative and acetogenic Bacteria and the methanogenic Archaea. One of the rate limiting steps in the degradation of soluble organic matter is the dismutation of acetate, a predominant intermediate in the process, which accounts for 70 % or more of the methane produced by the methanogens. Acetate utilization is controlled by regulation of expression of carbon monoxide dehydrogensase (COdh), which catalyzes the dismutation of acetate. However, physiological and molecular factors that control differential substrate utilization have not been identified in these Archaea. Our laboratory has identified sequence elements near the promoter of the gene (cdh) encoding for COdh and we have confirmed that these sequences have a role in the in vivo expression of cdh. The current proposal focuses on identifying the regulatory components that interact with DNA and RNA elements, and identifying the mechanisms used to control cdh expression. We will determine whether expression is controlled at the level of transcription or if it is mediated by coordinate interaction of transcription initiation with other processes such as transcription elongation rate and differential mRNA stability. Utilizing recently sequenced methanosarcinal genomes and a DNA microarray currently under development genes that encode regulatory proteins and transcription factors will be identified and function confirmed by gene disruption and subsequent screening on different substrates. Functional interactions will be determined in vivo by assaying the effects of gene dosage and site-directed mutagenesis of the regulatory gene on the expression of a cdhAÂ::lacZ operon fusion. Results of this study will reveal whether this critical catabolic pathway is controlled by mechanisms similar to those employed by the Bacteria and Eukarya, or by a regulatory paradigm that is unique to the Archaea. The mechanism(s) revealed by this investigation will provide insight into the regulatory strategies employed by the aceticlastic methanogenic Archaea to efficiently direct carbon and electron flow in anaerobic consortia during fermentative processes

Development of a Plasmid-Mediated Reporter System for In Vivo Monitoring of Gene Expression in the Archaeon Methanosarcina acetivorans

A plasmid-based gene reporter system has been developed to construct lacZ gene fusions for monitoring intrinsic promoter expression in Methanosarcina acetivorans. Constructs transform with high efficiency that can be readily screened by color selection on plates and exhibit a consistent copy number on different substrates negating the need for gene copy normalization. Expression of the CO dehydrogenase-acetyl coenzyme A synthase promoter fusion to lacZ revealed 18- to 54-fold down-regulation in cells grown on methylotrophic substrates compared with acetate-grown cells, which is up to an order of magnitude greater than the range of regulation previously reported by enzyme activity assays. This system complements and expands the current techniques for studying genetics of the methanosarcinal Archaea by providing a rapid method for monitoring and quantifying gene expression

Evaluation of PCB dechlorination pathways in anaerobic sediment microcosms using an anaerobic dechlorination model

A detailed quantitative analysis of anaerobic dechlorination (AD) pathways of polychlorinated biphenyls (PCBs) in sediment microcosms was performed by applying an anaerobic dechlorination model (ADM). The purpose of ADM is to systematically analyze changes in a contaminant profile that result from microbial reductive dechlorination according to empirically determined dechlorination pathways. In contrast to prior studies that utilized modeling tools to predict dechlorination pathways, ADM also provides quantification of individual pathways. As only microbial reductive dechlorination of PCBs occurred in the modeled laboratory microcosms, extensive analysis of AD pathways was possible without the complicating effect of concurrent physico-chemical or other weathering mechanisms. The results from this study showed: (1) ninety three AD pathways are active; (2) tetra- to hepta-chlorobiphenyl (CB) congeners were common intermediates in several AD pathways, penta-CBs being the most frequently observed; (3) the highest rates of dechlorination were for penta-CB homologs during the initial 185 days; (4) the dominant terminal products of AD were PCB 32(26-4), 49(24-25), 51(24-26), 52(25-25), 72(25-35), 73(26-35) and 100(246-24), (5) potential toxicity of the sediment was reduced. ADM serves as a powerful tool not only for a thorough analysis of AD pathways, but also for providing necessary input for numerical fate models (as a degradation term) that investigate dechlorination products or outcome of natural attenuation, or bioremediation/bioaugmentation of PCB-impacted sediments