Progress 10/01/03 to 09/30/09
Outputs
OUTPUTS: One of the most promising strategies for remediating metal contaminants in the subsurface is bioremediation with dissimilatory metal-reducing microorganisms. Species of Geobacter are known in many cases to be the dominant organism in this process in the environment. Recently, Geobacter sulfurreducens has become amenable to molecular approaches enabling it to become a model organism to study this type of anaerobic bioremediation in the laboratory. Geobacter sulfurreducens can also produce electricity. Pili are extracellular appendages. Recent evidence suggests that the electricity produced by Geobacter flows along these pili. We wanted to further test this idea by finding mutations of pilA, the gene that codes for pilin protein that makes up the pili. PARTICIPANTS: Steven J. Sandler is a professor working at The University of Massachusetts/Amherst. Lubna Al-Challah was the graduate student working on project so there was also training and professional development. Derek Lovley was a collaborator. TARGET AUDIENCES: People interested in basic science and in particular microbial production of energy. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have found that the simple in-frame deletion of pilA cannot be simply complemented by a plasmid copy of pilA. To see full complementation downstream sequences (the entire operon) are also needed. We also found that a tyrosine residue at position 61 is modified with a phosphate glycerol group. We have mutated this residue to a phenylalanine and found that the mutant is partially defective in biofilm formation, making thicker films on some substrates than others. It still produces electricity, however, it is slower to maximal output possibly because biofilm formation is slower. The impact from this very basic work is at least two fold. It will serve as the basis for future experimentation in this very important area of research. It also serves by producing energy (electricity) in a carbon neutral way. This is a concern for all. There are two publications that are currently being written for this work.
Publications
- No publications reported this period
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: One of the most promising strategies for remediating metal contaminants in the subsurface is bioremediation with dissimilatory metal-reducing microorganisms. Species of Geobacter are known in many cases to be the dominant organism in this process in the environment. Recently, Geobacter sulfurreducens has become amenable to molecular approaches enabling it to become a model organism to study this type of anaerobic bioremediation in the laboratory. Geobacter sulfurreducens can also produce electricity. Pili are extracellular appendages. Recent evidence suggests that the electricity produced by Geobacter flows along these pili. We wanted to further test this idea by finding mutations of pilA, the gene that codes for pilin protein that makes up the pili. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Results: We have found that the simple in-frame deletion of pilA cannot be simply complemented by a plasmid copy of pilA. This suggests that other downstream sequences are also needed. We also found that a tyrosine residue at position 61 is modified with a phosphate glycerol group. We have mutated this residue to a phenylalanine and found that the mutant is defective in biofilm formation. Impact: The impact from this very basic work is at least two fold. It will serve as the basis for future experimentation in this very important area of research. It also serves communities and individuals by producing energy (electricity) in a carbon neutral way. This is a concern for all.
Publications
- No publications reported this period
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Introduction: One of the most promising strategies for remediating metal contaminants in the subsurface is bioremediation with dissimilatory metal-reducing microorganisms. Species of Geobacter are known in many cases to be the dominant organism in this process in the environment. Recently, Geobacter sulfurreducens has become amenable to molecular approaches enabling it to become a model organism to study this type of anaerobic bioremediation in the laboratory. Geobacter sulfurreducens can also produce electricity. Pili are extracellular appendages. Recent evidence suggests that the electricity produced by Geobacter flows along these pili. We wanted to further test this idea by finding mutations of pilA, the gene that codes for pilin protein that makes up the pili. Results: Results are tangible on several fronts. We have made an in-frame pilA deletion mutant that is not polar on downstream genes. We have cloned the pilA gene to be used for complementation studies and as the
source for pilA mutants on a plamid under control of its own native promoter. We have purified the pilA protein to near homogeneity and are bulking up quantities for iron-binding and structural studies.
PARTICIPANTS: Lubna Al-Challah
Impacts
The impact from this very basic work is at least two fold. It will serve as the basis for future experimentation in this very important area of research. It also serves the needs of many, by producing energy (electricity) in a carbon neutral way.
Publications
- No publications reported this period
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Progress 10/01/05 to 09/30/06
Outputs
Introduction: One of the most promising strategies for remediating metal contaminants in the subsurface is bioremediation with dissimilatory metal-reducing microorganisms. Species of Geobacter are known in many cases to be the dominant organism in this process in the environment. Recently, Geobacter sulfurreducens has become amenable to molecular approaches enabling it to become a model organism to study this type of anaerobic bioremediation in the laboratory. Much information is usually known about the biochemistry and genetics of processes in bacteria, but little on where in the cell do these processes occur and what is their spatial relationship to one another. We will test two different methods to study protein localization using fluorescent microscopy: (1) making fusions with the Green Fluorescence Protein (GFP) to Geobacter proteins of interest and (2) making an addition of a six amino acid sequence (CCPGCC called a tetracysteine motif) to Geobacter proteins of
interest and a chemical called Flash. After identification of useful protocols, we will label proteins of interests (recombination, replication, electron transport and pili) and localize these proteins to begin to build hypotheses of how their localization might dictate their function. Results: Since the use of the Flash compound in Geobacter was problematic, we have refocused our studies back on the E. coli RecA-GFP model system in order to better understand how recombination occurs in vivo. This effort led to several important observations that eventually led to a paper accepted October 06 (Molecular Microbiology in press). The work showed that two proteins: DinI and RecX can influence (helping to stabilize or de-stabilize respectively) the number of RecA-GFP foci in a strain of E. coli .
Impacts
The impact from this very basic work is at least two fold. It will serve as the basis for future experimentation in this very important area of research. It also serves the needs of racial/ethic minority groups, underserved communities and individuals in at least one way: it studies the localization of proteins important for remediation of DNA damage. This is a concern for all.
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/05
Outputs
Introduction: One of the most promising strategies for remediating metal contaminants in the subsurface is bioremediation with dissimilatory metal-reducing microorganisms. Species of Geobacter are known in many cases to be the dominant organism in this process in the environment. Recently, Geobacter sulfurreducens has become amenable to molecular approaches enabling it to become a model organism to study this type of anaerobic bioremediation in the laboratory. Much information is usually known about the biochemistry and genetics of processes in bacteria, but little on where in the cell do these processes occur and what is their spatial relationship to one another. We will test two different methods to study protein localization using fluorescent microscopy: (1) making fusions with the Green Fluorescence Protein (GFP) to Geobacter proteins of interest and (2) making an addition of a six amino acid sequence (CCPGCC called a tetracysteine motif) to Geobacter proteins of
interest and a chemical called FlAsh. After identification of useful protocols, we will label proteins of interests (recombination, replication, electron transport and pili) and localize these proteins to begin to build hypotheses of how their localization might dictate their function. Results: Since the use of the Flash compound in Geobacter was problematic, we have refocused our studies back on the E. coli RecA-GFP model system in order to better learn how to quantitate and interpret the images that we see. This effort led to several important observations that eventually led to a paper published last summer (Molecular Microbiology 57:1074-1085). The work showed that RecA-GFP resides in two different kinds of structures in the cell. Ones that are on the DNA and ones that are not. These can be distinguished through the use of DAPI stain. The structures on the DNA localize to portions of the cell where the replication factory should be.
Impacts
This research studies localization of proteins important for remediation of toxic compounds. The inability to properly dispose of toxic compounds presents a health hazard to all communities.
Publications
- Renzette, N., Gumlaw, N., Nordman, J.T., Krieger, M., Yeh, S.P., Long, E., Centore, R., Boonsombat, R., and Sandler, S.J. (2005) Localization of RecA in Escherichia coli K-12 using RecA-GFP. Mol Microbiol 57: 1074-1085.
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Progress 10/01/03 to 09/30/04
Outputs
Introduction: One of the most promising strategies for remediating metal contaminants in the subsurface is bioremediation with dissimilatory metal-reducing microorganisms. Species of Geobacter are known in many cases to be the dominant organism in this process in the environment. Recently, Geobacter sulfurreducens has become amenable to molecular approaches enabling it to become a model organism to study this type of anaerobic bioremediation in the laboratory. Much information is usually known about the biochemistry and genetics of processes in bacteria, but little on where in the cell do these processes occur and what is their spatial relationship to one another. We will test two different methods to study protein localization using fluorescent microscopy: (1) making fusions with the Green Fluorescence Protein (GFP) to Geobacter proteins of interest and (2) making an addition of a six amino acid sequence (CCPGCC called a tetracysteine motif) to Geobacter proteins of
interest and a chemical called FlAsh. After identification of useful protocols, we will label proteins of interests (recombination, replication, electron transport and pili) and localize these proteins to begin to build hypotheses of how their localization might dictate their function. Results: In this past year, we have tested the GFP technology in Geobacter using a characterized E. coli RecA-GFP protein expressed in Geobacter. Initial studies show that although one can see fluorescence, the reproducibility of this fluorescence is poor. It is not yet possible to draw a conclusion of if this technology will be useful. Therefore current studies are focused on optimization of protocols to test reproducibility. We have also begun to test the Flash technology. Here several outer membrane cytochrome proteins and pili proteins have been fused with the CCPGCC tetracysteine motif. Work is currently underway to express these proteins in Geobacter. We are testing their ability to complement
known mutations in the genes and their ability to fluoresce (see localization) in the presence of FlAsh.
Impacts
The impact from this very basic work is not yet known. In this experiment we study localization of proteins important for remediation of toxic compounds which is a concern for all. The inability to properly dispose of toxic compounds presents a health hazard to all communities.
Publications
- No publications reported this period
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