Diagnosis of In Situ Metabolic State and Rates of Microbial Metabolism During In Situ Uranium Bioremediation with Molecular Techniques

The goal of these projects was to develop molecule tools to tract the metabolic activity and physiological status of microorganisms during in situ uranium bioremediation. Such information is important in able to design improved bioremediation strategies. As summarized below, the research was highly successful with new strategies developed for estimating in situ rates of metabolism and diagnosing the physiological status of the predominant subsurface microorganisms. This is a first not only for groundwater bioremediation studies, but also for subsurface microbiology in general. The tools and approaches developed in these studies should be applicable to the study of microbial communities in a diversity of soils and sediments

Interference with histidyl-tRNA synthetase by a CRISPR spacer sequence as a factor in the evolution of Pelobacter carbinolicus

BACKGROUND: Pelobacter carbinolicus, a bacterium of the family Geobacteraceae, cannot reduce Fe(III) directly or produce electricity like its relatives. How P. carbinolicus evolved is an intriguing problem. The genome of P. carbinolicus contains clustered regularly interspaced short palindromic repeats (CRISPR) separated by unique spacer sequences, which recent studies have shown to produce RNA molecules that interfere with genes containing identical sequences. RESULTS: CRISPR spacer #1, which matches a sequence within hisS, the histidyl-tRNA synthetase gene of P. carbinolicus, was shown to be expressed. Phylogenetic analysis and genetics demonstrated that a gene paralogous to hisS in the genomes of Geobacteraceae is unlikely to compensate for interference with hisS. Spacer #1 inhibited growth of a transgenic strain of Geobacter sulfurreducens in which the native hisS was replaced with that of P. carbinolicus. The prediction that interference with hisS would result in an attenuated histidyl-tRNA pool insufficient for translation of proteins with multiple closely spaced histidines, predisposing them to mutation and elimination during evolution, was investigated by comparative genomics of P. carbinolicus and related species. Several ancestral genes with high histidine demand have been lost or modified in the P. carbinolicus lineage, providing an explanation for its physiological differences from other Geobacteraceae. CONCLUSIONS: The disappearance of multiheme c-type cytochromes and other genes typical of a metal-respiring ancestor from the P. carbinolicus lineage may be the consequence of spacer #1 interfering with hisS, a condition that can be reproduced in a heterologous host. This is the first successful co-introduction of an active CRISPR spacer and its target in the same cell, the first application of a chimeric CRISPR construct consisting of a spacer from one species in the context of repeats of another species, and the first report of a potential impact of CRISPR on genome-scale evolution by interference with an essential gene

Final Report Coupling in silico microbial models with reactive transport models to predict the fate of contaminants in the subsurface.

This project successfully accomplished its goal of coupling genome-scale metabolic models with hydrological and geochemical models to predict the activity of subsurface microorganisms during uranium bioremediation. Furthermore, it was demonstrated how this modeling approach can be used to develop new strategies to optimize bioremediation. The approach of coupling genome-scale metabolic models with reactive transport modeling is now well enough established that it has been adopted by other DOE investigators studying uranium bioremediation. Furthermore, the basic principles developed during our studies will be applicable to much broader investigations of microbial activities, not only for other types of bioremediation, but microbial metabolism in diversity of environments. This approach has the potential to make an important contribution to predicting the impact of environmental perturbations on the cycling of carbon and other biogeochemical cycles

Nanowires, Capacitors, and Other Novel Outer-Surface Components Involved in Electron Transfer to Fe(III) Oxides in Geobacter Species

The overall goal of this project was to better understand the mechanisms by which Geobacter species transfer electrons outside the cell onto Fe(III) oxides. The rationale for this study was that Geobacter species are often the predominant microorganisms involved in in situ uranium bioremediation and the growth and activity of the Geobacter species during bioremediation is primarily supported by electron transfer to Fe(III) oxides. These studies greatly expanded the understanding of electron transfer to Fe(III). Novel concepts developed included the potential role of microbial nanowires for long range electron transfer in Geobacter species and the importance of extracytoplasmic cytochromes functioning as capacitors to permit continued electron transfer during the hunt for Fe(III) oxide. Furthermore, these studies provided target sequences that were then used in other studies to tract the activity of Geobacter species in the subsurface through monitoring the abundance of gene transcripts of the target genes. A brief summary of the major accomplishments of the project is provided

Molecular Analysis of Rates of Metal Reduction andMetabolic State of Geobacter Species During in situ Uranium Bioremediation

This report summarizes progress from June 2004 through April 2005. Research focused on monitoring the in situ rates of metabolism and the metabolic state of Geobacteraceae during in situ bioremediation of uranium at the field study site in Rifle, Colorado. As detailed below, it was demonstrated for the first time that it is possible to quantify in situ levels of transcripts for key metabolic genes and from this information infer not only rates of electron transfer to metals, but also nutrient limitations which might be limiting this process

In Silico Modeling of Geobacter Species.

This project employed a combination of in silico modeling and physiological studies to begin the construction of models that could predict the activity of Geobacter species under different environmental conditions. A major accomplishment of the project was the development of the first genome-based models of organisms known environmental relevance. This included the modeling of two Geobacter species and two species of Pelobacter. Construction of these models required increased sophistication in the annotation of the original draft genomes as well as collection of physiological data on growth yields, cell composition, and metabolic reactions. Biochemical studies were conducted to determine whether proposed enzymatic reactions were in fact expressed. During this process we developed an Automodel Pipeline process to accelerate future model development of other environmentally relevant organisms by using bioinformatics techniques to leverage predicted protein sequences and the Genomatica database containing a collection of well-curated metabolic models. The Automodel Pipeline was also used for iterative updating of the primary Geobacter model of G. sulfurreducens to expand metabolic functions or to add alternative pathways. Although each iteration of the model does not lead to another publication, it is an invaluable resource for hypothesis development and evaluation of experimental data. In order to develop a more accurate G. sulfurreducens model, a series of physiological studies that could be analyzed in the context of the model were carried out. For example, previous field trials of in situ uranium bioremediation demonstrated that Geobacter species face an excess of electron donor and a limitation of electron acceptor near the point of acetate injection into the groundwater. Therefore, a model-based analysis of electron acceptor limitation physiology was conducted and model predictions were compared with growth observed in chemostats. Iterative studies resulted in the model accurately predicting acetate oxidation and electron acceptor reduction. The model also predicted that G. sulfurreducens must release hydrogen under electron-accepting conditions in order to maintain charge and electron balance. This prediction was borne out by subsequent hydrogen measurements. Furthermore, changes in gene expression were consistent with model predictions of flux changes around central metabolism. The model revealed multiple redundant pathways in central metabolism suggesting an apparent versatility unusual in microbial metabolism. The computational analysis led to the identification of 32 reactions that participated in eight sets of redundant pathways. The computational results guided the design of strains with mutations in key reactions to elucidate the role of the alternate pathways and obtain information on their physiological function. A total of seven strains with mutations in genes encoding five metabolic reactions were constructed and their phenotypes analyzed in 12 different environments. This analysis revealed several interesting insights on the role of the apparent redundant pathways. 13C labeling approaches were developed for further elucidation of metabolic pathways with model-driven interpretation. For example, the model was used to calculate the optimal acetate 13C labeling ratio for distinguishing flux through various pathways based on amino acid isotopomer distributions. With this method it was possible to elucidate the pathways for amino acid biosynthesis. Surprisingly, the labeling pattern of isoleucine deviated significantly from what was predicted by the metabolic reconstruction. Detailed analysis of the labeling patterns with the model led to the discovery that there are two pathways for leucine biosynthesis, including a novel citramalate pathway that was subsequently confirmed with biochemical analysis. In summary, the combined computational and experimental studies have been instrumental in further characterizing the central metabolism of members of the Geobacteraceae. Furthermore, the methods developed in these studies provide a strategy for the genome-based study of the physiology of other understudied, but environmentally significant organisms

Biotransformation involved in sustained reductive removal of uranium in contaminant aquifers

This report summarizes progress made from June 2003 to July 2004. During this period research focused on further understanding the factors controlling the growth and activity of dissimilatory metal reducers in subsurface environments and the application of these findings to better design of strategies for in situ bioremediation of uranium

Nanowires, Capacitors, and Other Novel Outer-Surface Components Involved in Electron Transfer to Fe(III) Oxides in Geobacter Species

In the past year studies have primarily focused on elucidating the role of pili in electron transport to Fe(III) oxide in Geobacter sulfurreducens. As summarized in last year's report, it was previously found that pili are specifically expressed during growth on Fe(III) oxide and that Fe(III) oxide reduction is inhibited if the gene for the structural pilin protein is deleted. However, it was also found that a pilin-deficient mutant of G. sulfurreducens could attached to Fe(III) oxide as well as wild type

Growth with high planktonic biomass in Shewanella oneidensis fuel cells

Shewanella oneidensis MR-1 grew for over 50 days in microbial fuel cells, incompletely oxidizing lactate to acetate with high recovery of the electrons derived from this reaction as electricity. Electricity was produced with lactate or hydrogen and current was comparable to that of electricigens which completely oxidize organic substrates. However, unlike fuel cells with previously described electricigens, in which cells are primarily attached to the anode, at least as many of the S. oneidensis cells were planktonic as were attached to the anode. These results demonstrate that S. oneidensis may conserve energy for growth with an electrode serving as an electron acceptor and suggest that multiple strategies for electron transfer to fuel cell anodes exist

Evolution of electron transfer out of the cell: comparative genomics of six Geobacter genomes

Background: Geobacter species grow by transferring electrons out of the cell - either to Fe(III)-oxides or to manmade substances like energy-harvesting electrodes. Study of Geobacter sulfurreducens has shown that TCA cycle enzymes, inner-membrane respiratory enzymes, and periplasmic and outer-membrane cytochromes are required. Here we present comparative analysis of six Geobacter genomes, including species from the clade that predominates in the subsurface. Conservation of proteins across the genomes was determined to better understand the evolution of Geobacter species and to create a metabolic model applicable to subsurface environments. Results: The results showed that enzymes for acetate transport and oxidation, and for proton transport across the inner membrane were well conserved. An NADH dehydrogenase, the ATP synthase, and several TCA cycle enzymes were among the best conserved in the genomes. However, most of the cytochromes required for Fe(III)-reduction were not, including many of the outer-membrane cytochromes. While conservation of cytochromes was poor, an abundance and diversity of cytochromes were found in every genome, with duplications apparent in several species. Conclusions: These results indicate there is a common pathway for acetate oxidation and energy generation across the family and in the last common ancestor. They also suggest that while cytochromes are important for extracellular electron transport, the path of electrons across the periplasm and outer membrane is variable. This combination of abundant cytochromes with weak sequence conservation suggests they may not be specific terminal reductases, but rather may be important in their heme-bearing capacity, as sinks for electrons between the inner-membrane electron transport chain and the extracellular acceptor