Molecular Biomarkers For Respiration In Dehalococcoides Ethenogenes And Methanospirllum Hungatei: Comparing Protein And Messenger-Rna Abundance In Anaerobes
One of the major limitations in environmental microbiology and bioremediation is the inability to confidently monitor microbial processes in situ. The detection of molecular biomarkers, molecules of biological origin that are indicative of these processes, is one promising approach. In an anaerobic, dehalorespiring and methanogenic mixed culture, biomarkers for respiration of two organisms, Dehalococcoides ethenogenes sp. and Methanospirillum hungatei sp., have been identified. Both microbes utilize hydrogen as an electron donor. Targeted absolute quantification assays of mRNA and protein biomarkers for specific respiratory enzymes-the hydrogenases HupL and FrcA, the oxidoreductase MvrD, and the reductive dehalogenases TceA, PceA , DET1559 and DET1545-have been developed and used to quantify these molecules over an array of experimental conditions. To derive transcript-respiration trends, various donors and chloroethene acceptors were continuously fed to sub-cultures at different ratios and rates. These experiments induced pseudo-steady-state respiration and mRNA biomarker levels that could then be correlated. In both Dehalococcoides and Methanospirillum, linear correlations across mRNA biomarker levels and respiration (1 - 150 [MICRO SIGN]eeq/L-hr) were observed in the following targets: MvrD and FrcA for Methanospirillum, and HupL and TceA for Dehalococcoides. Other empirical trends were observed for Dehalococcoides biomarkers, including trends that saturate (the reductive dehalogenases PceA and DET1559) or decline (the dehalogenases DET1545) above a respiration rate of 5 [MICRO SIGN]eeq/L-hr. Insight into how mRNA expression levels affect translation of proteins was gained through quantification of peptide biomarkers. Differences in absolute abundance of proteins-per-genome for Dehalococcoides and Methanospirillum suggest that mRNA abundance is a poor predictor of protein abundance across targets within an organism and across organisms. In Dehalococcoides, more TceA proteins were generated per transcript (0.4 - 1.2 proteins/mRNA-hr) than other monitored biomarker targets (0.03 - 0.17 proteins/ mRNA-hr). Protein decay rates for individual enzymes were indistinguishable from cell decay, suggesting that translation rates, rather than decay rates, are controlling the differences in protein abundance. Quantification of protein abundances allowed for the calculation of enzyme-specific rate constants for enzymes of known function in Dehalococcoides: TceA and PceA. These in vivo parameters could be utilized for predicting in situ process rates from protein abundance and metabolite levels. In addition, these data support the utility of both mRNA and protein biomarkers, especially for inferring process rates. However, they also highlight potential problems with inferring protein abundance from mRNA data alone and emphasize the need for strong empiricallyderived correlations for any newly-discovered mRNA biomarker.
Dehalococcoides; Methanogen; Biomarker
Richardson, Ruth E.
Gossett, James Michael; Zinder, Stephen H
Ph.D. of Microbiology
Doctor of Philosophy
dissertation or thesis