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

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.

Figure 1

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).

Figure 2

Geochemical profiles for U(VI) and Fe(II) in well D07 during the 2007 biostimulation experiment. Heat‐map abundances are shown for both the (A) conserved ‘biomarker’ peptides and (B) unique peptides matching CS proteins in all three samples.

Figure 3

Geochemical profiles for U(VI), Fe(II) and S²⁻ in well D04 during the 2008 biostimulation experiment. Heat‐map abundances are shown for both the (A) conserved ‘biomarker’ peptides and (B) unique peptides matching CS proteins in all nine samples. Light blue zones show time periods of acetate injection. Uranium rebounds starts at ∼day 20.

Figure 4

A. Non‐metric multidimensional scaling diagram showing clustering of 2008 samples using both conserved ‘biomarker’ and unique peptide abundances. Environmental vectors have been added illustrating the correlations between the clustering patterns of the biomass samples and geochemical gradients.
B. Principal components analysis bi‐plot using 2007 unique peptides, illustrating the relative clustering of three biomass samples recovered during the 2007 biostimulation experiment. Four technical replicates are shown per sample. The points are shown as arrows, illustrating the sample‐specific nature of the unique peptide abundances. Peptides only detected in sample D07_day9 are defined by red arrows, those only detected in D05_day15 by green arrows, and those only detected in D07_day21 by blue arrows.

Figure 5

A. A neighbour‐joining tree constructed from aligned citrate synthase sequences for which unique peptides were detected in the 2007 data. Isolate sequences are included for reference. Peptides were detected for those isolate sequences marked by an asterisk ‘*’. Red and blue labels indicate sequences for which unique peptides were detected in only one of the samples (Red = D07_day9, Blue = D07_day21). Isolate CS identifications are as follows; G. bemidjiensis (1), gi145617433; G. bemidjiensis (2), gi145620657; Strain M21 (1), gm829064; Strain M21 (2), gm826531; Strain FRC32 (1), gi110599265; Strain FRC32 (2), gi110600278; G. lovleyi, gi118746732; G. sulfurreducens, gi39996208; G. uraniireducens, gi148263639; G. metallireducens (1), gi78222340; G. metallireducens (2), gi78223885.
B. Peptide % abundances for all unique peptides matched against the citrate synthase database in samples D07_day9 and D07_day21 from the 2007 experiment.
C. A neighbour‐joining tree constructed from aligned citrate synthase sequences detected in the 2008 data. Isolate sequences are included for reference. Peptides were detected for those isolate sequences marked by an asterisk ‘*’. Geobacter CS identifications are as above.
D. Peptide % abundances for all unique peptides matched against the citrate synthase database in samples D04_day5 through D04_day20 from the 2008 experiment. Saccharomyces cerevisaeae was used as an out‐group for the trees. The scale bar indicates the number of changes inferred as having occurred along each branch.