(699b) Integrated Computational and Experimental Analysis of Redundancy in the Metabolic Network of Geobacter Sulfurreducens

Geobacteraceae have been
shown to be important in bioremediation of uranium contaminated subsurface
environments, and in harvesting electricity from waste organic matter.  These
applications are intricately linked to cellular metabolism, motivating the need
to understand metabolism in these metal-reducing bacteria. An iterative
approach of mathematical modeling followed by experimentation was adopted to
understand metabolism in these organisms.

A genome-scale
metabolic model has been developed using the constraint-based modeling
approach. Model-based analysis has revealed significant insights on the effect
of global proton balance on the physiology of G. sulfurreducens and has
provided explanation for the reduced yields during Fe (III) reduction. The in
silico analysis of the energetics of menaquinone secretion indicated a
substantial reduction in the growth rate and suggested an explanation for why Geobacteraceae
predominate over other bacteria that require such electron shuttles. The
initial metabolic model provided important physiological and ecological
insights on the metabolism of Geobacteraceae. However, the analysis of
metabolism revealed several redundant pathways in central metabolism around
acetate utilization and pyruvate metabolism.

Acetate is the key electron donor
for Geobacter species during in situ uranium bioremediation and
in the conversion of organic matter to electricity. Further analysis of the in
silico metabolic model for G. sulfurreducens identified redundant
pathways for acetate metabolism. . These included  two acetate activation
pathways encoded in the genome, the acetate kinase/phosphate transacetylase
(Ack/Pta) pathway and the acetyl-CoA transferase (Ato), which plays a dual role
in acetate activation and the TCA cycle. There are also two enzymes catalyzing
the synthesis of oxaloacetate, the TCA cycle enzyme, malate dehydrogenase (Mdh)
and pyruvate carboxylase (PC), which catalyzes the conversion of pyruvate to
oxaloacetate. Three reactions are present for the synthesis of acetyl-CoA from
pyruvate: pyruvate dehydrogenase, pyruvate formate lyase and pyruvate
ferredoxin oxidoreductase (Por) and three are possible pathways for synthesis
of PEP involving pyruvate phosphate dikinase (PpdK), PEP synthase (PpS), and
PEP carboxykinase (PpcK). 

To evaluate the role of these
pathways, five knockout mutant strains lacking elements of the various
redundant pathways (Ato, Pta, Por, Mdh, PpcK) were constructed and evaluated
along with the wild type for their ability to grow under twelve distinct
environmental conditions (72 combinations) and the model predictions were
compared to the results of the phenotypic analysis. The model predicted that G.
sulfurreducens would be able to compensate for the absence of Ato by
increasing flux through the Ack/Pta pathway and succinyl-CoA synthetase. 
However, failure of the Ato-knockout mutant to grow on acetate suggested that
the succinyl-CoA synthetase was inactive.  Similar constraints on metabolism
were derived from the comparison of the in vivo phenotypes with the
model predictions. Following the incorporation of these new constraints, the in
silico model now correctly predicts the experimental result in 89% of the
possible conditions providing highly accurate characterization of central
metabolism in G. sulfurreducens.

Comparison of
the in silico and the in vivo phenotypes has lead to additional
information on the role and activity of the central metabolic pathways in G.
sulfurreducens. The combined experimental and computational analysis
clearly highlights the role of pyruvate ferredoxin oxidoreductase as the sole
mechanism of acetyl-CoA to pyruvate conversion.  Furthermore, the role for the
two alternative mechanisms for acetate activation (acetate kinase route for the
low gluconeogenic flux, and acetyl-CoA transferase for the high TCA cycle flux)
was clearly elucidated through the integrated analysis of the PTA and ATO3
mutant's phenotypes.  Such integrated analysis of computational and
experimental data can provide valuable insights on the activity and function of
metabolic pathways in a rapid manner for poorly characterized organisms in
environmental microbiology.