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Development of a biomarker for Geobacter activity and strain composition; proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI).

Wilkins MJ, Callister SJ, Miletto M, Williams KH, Nicora CD, Lovley DR, Long PE, Lipton MS - Microb Biotechnol (2011)

Bottom Line: Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle.Abundances of unique peptides indicated potential differences at the strain level between Fe(III)-reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC.These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.

View Article: PubMed Central - PubMed

Affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99353, USA. michael.wilkins@pnl.gov

ABSTRACT
Monitoring the activity of target microorganisms during stimulated bioremediation is a key problem for the development of effective remediation strategies. At the US Department of Energy's Integrated Field Research Challenge (IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via subsurface acetate addition leads to precipitation of U(VI) from groundwater as U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle. Here, we utilize shotgun proteomic methods to demonstrate that the measurement of gltA peptides can be used to track Geobacter activity and strain evolution during in situ biostimulation. Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in U(VI) concentrations during biostimulation, whereas changing patterns of unique peptide abundances between samples suggested sample-specific strain shifts within the Geobacter population. Abundances of unique peptides indicated potential differences at the strain level between Fe(III)-reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC. These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.

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Alignment of eukaryotic and prokaryotic citrate synthase proteins illustrating certain conserved and more divergent regions associated with Geobacter copies of this protein. Red highlighted text in regions A and B correspond to two of the conserved peptides used as indicators of overall Geobacter activity. Blue highlighted amino acids in area C illustrate more divergent regions where differences occur among Geobacter sequences (e.g. 1 or 2 base pair differences).
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f1: Alignment of eukaryotic and prokaryotic citrate synthase proteins illustrating certain conserved and more divergent regions associated with Geobacter copies of this protein. Red highlighted text in regions A and B correspond to two of the conserved peptides used as indicators of overall Geobacter activity. Blue highlighted amino acids in area C illustrate more divergent regions where differences occur among Geobacter sequences (e.g. 1 or 2 base pair differences).

Mentions: The citrate synthase protein (subsequently referred to as CS in the text), which is responsible for controlling flux into the TCA cycle by catalysing the condensation of acetyl‐CoA and oxaloacetate to produce citric acid, has a number of characteristics that make it a suitable candidate as a Geobacter‐specific peptide‐based biomarker. The amino acid sequence in members of the Geobacteraceae is more closely related to a eukaryotic CS than other prokaryotic sequences (Bond et al., 2005), limiting the potential for false positive identifications from other subsurface species. Within the Geobacteraceae however, certain regions of the protein are highly conserved (Butler et al., 2010) and have the potential to act as biomarkers for general Geobacter abundance and activity (Fig. 1). While other proteins contain highly conserved regions that could potentially act as Geobacter biomarkers, these regions frequently match other closely related species, such as Pelobacter and Desulfuromonas. In addition, the analysis of more divergent regions of the CS protein sequence shows potential as a technique to track strain‐level changes within the Geobacter community. Clear shifts in CS unique peptide abundances and diversity are observed over the duration of biostimulation, and therefore may be used to ‘fingerprint’ the microbial community at a specific period during the biostimulation process. As these community ‘fingerprints’ may be characteristic of certain time points during the biostimulation process, they are potentially useful indicators of changes in the biogeochemistry of the system (Wilkins et al., 2009). Finally, the stimulation of microbial growth in the Rifle subsurface via acetate amendment ensures that proteins involved in the efficient utilization of this substrate (e.g. TCA cycle proteins) are abundant within proteomic samples. Citrate synthase peptides are therefore suitable for roles as biomarkers given that they are easily detected where Geobacter species are active. It is likely that the reduction of contaminants such as U(VI) is tightly linked to respiratory processes (Lovley et al., 1991; Gorby and Lovley, 1992), and therefore, the fluctuating abundance of a protein involved in key metabolic processes such as citrate synthase will act as an effective proxy for Geobacter activity.


Development of a biomarker for Geobacter activity and strain composition; proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI).

Wilkins MJ, Callister SJ, Miletto M, Williams KH, Nicora CD, Lovley DR, Long PE, Lipton MS - Microb Biotechnol (2011)

Alignment of eukaryotic and prokaryotic citrate synthase proteins illustrating certain conserved and more divergent regions associated with Geobacter copies of this protein. Red highlighted text in regions A and B correspond to two of the conserved peptides used as indicators of overall Geobacter activity. Blue highlighted amino acids in area C illustrate more divergent regions where differences occur among Geobacter sequences (e.g. 1 or 2 base pair differences).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815795&req=5

f1: Alignment of eukaryotic and prokaryotic citrate synthase proteins illustrating certain conserved and more divergent regions associated with Geobacter copies of this protein. Red highlighted text in regions A and B correspond to two of the conserved peptides used as indicators of overall Geobacter activity. Blue highlighted amino acids in area C illustrate more divergent regions where differences occur among Geobacter sequences (e.g. 1 or 2 base pair differences).
Mentions: The citrate synthase protein (subsequently referred to as CS in the text), which is responsible for controlling flux into the TCA cycle by catalysing the condensation of acetyl‐CoA and oxaloacetate to produce citric acid, has a number of characteristics that make it a suitable candidate as a Geobacter‐specific peptide‐based biomarker. The amino acid sequence in members of the Geobacteraceae is more closely related to a eukaryotic CS than other prokaryotic sequences (Bond et al., 2005), limiting the potential for false positive identifications from other subsurface species. Within the Geobacteraceae however, certain regions of the protein are highly conserved (Butler et al., 2010) and have the potential to act as biomarkers for general Geobacter abundance and activity (Fig. 1). While other proteins contain highly conserved regions that could potentially act as Geobacter biomarkers, these regions frequently match other closely related species, such as Pelobacter and Desulfuromonas. In addition, the analysis of more divergent regions of the CS protein sequence shows potential as a technique to track strain‐level changes within the Geobacter community. Clear shifts in CS unique peptide abundances and diversity are observed over the duration of biostimulation, and therefore may be used to ‘fingerprint’ the microbial community at a specific period during the biostimulation process. As these community ‘fingerprints’ may be characteristic of certain time points during the biostimulation process, they are potentially useful indicators of changes in the biogeochemistry of the system (Wilkins et al., 2009). Finally, the stimulation of microbial growth in the Rifle subsurface via acetate amendment ensures that proteins involved in the efficient utilization of this substrate (e.g. TCA cycle proteins) are abundant within proteomic samples. Citrate synthase peptides are therefore suitable for roles as biomarkers given that they are easily detected where Geobacter species are active. It is likely that the reduction of contaminants such as U(VI) is tightly linked to respiratory processes (Lovley et al., 1991; Gorby and Lovley, 1992), and therefore, the fluctuating abundance of a protein involved in key metabolic processes such as citrate synthase will act as an effective proxy for Geobacter activity.

Bottom Line: Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle.Abundances of unique peptides indicated potential differences at the strain level between Fe(III)-reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC.These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.

View Article: PubMed Central - PubMed

Affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99353, USA. michael.wilkins@pnl.gov

ABSTRACT
Monitoring the activity of target microorganisms during stimulated bioremediation is a key problem for the development of effective remediation strategies. At the US Department of Energy's Integrated Field Research Challenge (IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via subsurface acetate addition leads to precipitation of U(VI) from groundwater as U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle. Here, we utilize shotgun proteomic methods to demonstrate that the measurement of gltA peptides can be used to track Geobacter activity and strain evolution during in situ biostimulation. Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in U(VI) concentrations during biostimulation, whereas changing patterns of unique peptide abundances between samples suggested sample-specific strain shifts within the Geobacter population. Abundances of unique peptides indicated potential differences at the strain level between Fe(III)-reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC. These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.

Show MeSH
Related in: MedlinePlus