Computational and Experimental Analysis of Redundancy in the Central Metabolism of Geobacter sulfurreducens

Abstract

Previous model-based analysis of the metabolic network of Geobacter sulfurreducens suggested the existence of several redundant pathways. Here, we identified eight sets of redundant pathways that included redundancy for the assimilation of acetate, and for the conversion of pyruvate into acetyl-CoA. These equivalent pathways and two other sub-optimal pathways were studied using 5 single-gene deletion mutants in those pathways for the evaluation of the predictive capacity of the model. The growth phenotypes of these mutants were studied under 12 different conditions of electron donor and acceptor availability. The comparison of the model predictions with the resulting experimental phenotypes indicated that pyruvate ferredoxin oxidoreductase is the only activity able to convert pyruvate into acetylCoA. However, the results and the modeling showed that the two acetate activation pathways present are not only active, but needed due to the additional role of the acetyl-CoA transferase in the TCA cycle, probably reflecting the adaptation of these bacteria to acetate utilization. In other cases, the data reconciliation suggested additional capacity constraints that were confirmed with biochemical assays. The results demonstrate the need to experimentally verify the activity of key enzymes when developing in silico models of microbial physiology based on sequence-based reconstruction of metabolic networks.

Highly conserved genes in Geobacter species with expression patterns indicative of acetate limitation

“…The presence of multiple copies of putative acetate transporters in the genomes of the Geobacter species suggests not only functional redundancy, but also an adaptation to the nutrient-deprived status that micro-organisms often encounter in their natural environment. Such gene redundancy is common for essential metabolic pathways in this specialized organism (Mahadevan et al, 2006;Segura et al, 2008). Although the role of the putative acetate transporters could not be definitively confirmed with genetic approaches, the finding that the Group I transporters are more highly expressed under acetate-limiting conditions suggests that the expression of these genes may aid in diagnosing the metabolic state of Geobacter species in subsurface environments.…”

“…Similarly, introduction of aplC : : cm and aplB : : kn markers in aplA : : gm mutants failed as well. It is worth mentioning here that the sequential introduction of antibiotic-marked mutations in G. sulfurreducens has been successful in other studies (Risso et al, 2008;Segura et al, 2008). To attempt to overcome this problem we tried a different strategy whereby the whole chromosomal region comprising aplA and aplB was replaced with a single antibiotic marker via a single step as previously described (Kim et al, 2006).…”

Laboratory evolution of Geobacter sulfurreducens for enhanced growth on lactate via a single-base-pair substitution in a transcriptional regulator

“…G. sulfurreducens oxidizes acetate via the tricarboxylic acid cycle (Galushko and Schink, 2000;Mahadevan et al, 2006). Acetate is activated to acetyl-CoA for introduction into the tricarboxylic acid cycle via succinyl-CoA:acetate-CoA-transferase (Galushko and Schink, 2000;Segura et al, 2008). Succinyl-CoA synthetase is not required for the conversion of succinyl CoA to succinate during growth on acetate and previous studies did not detect succinyl-CoA synthetase activity in acetategrown cells (Galushko and Schink, 2000;Segura et al, 2008).…”

“…Acetate is activated to acetyl-CoA for introduction into the tricarboxylic acid cycle via succinyl-CoA:acetate-CoA-transferase (Galushko and Schink, 2000;Segura et al, 2008). Succinyl-CoA synthetase is not required for the conversion of succinyl CoA to succinate during growth on acetate and previous studies did not detect succinyl-CoA synthetase activity in acetategrown cells (Galushko and Schink, 2000;Segura et al, 2008). However, when lactate is the electron donor, acetyl-CoA is produced directly from pyruvate and succinyl-CoA synthetase is required to Figure 2 The single-base-pair mutations discovered in the five lactate-adapted replicates after 23 transfers in lactate media.…”

The mechanisms of extracellular electron transfer

“…There have been a number of studies that report details of the TCA cycle of S. oneidensis [131,132,224,225] and G sulfurreducens [189,226,227]. However, there are no studies that employ quantitative proteomics to observe the TCA cycle under different environmental or metabolic conditions.…”