Figure 1.
Composition of the Dehalococcoides pan-genome.
Core, dispensable, and unique genomes are represented by blue, green, and orange, respectively. Genes in these genomes are also categorized as metabolic (spotted pattern), non-metabolic (plain), and hypothetical (grid pattern) on the basis of various bioinformatic analyses (see text for details).
Figure 2.
Distribution of dispensable and unique metabolic genes in different Dehalococcoides strains.
Colors are assigned to further categorize the genes according to their function identified from annotation and verified by different bioinformatic analyses. Each color except black signifies the presence of a corresponding metabolic gene while black indicates the absence of the corresponding gene. Genes belonging to amino acid metabolism, lipid metabolism and nucleotide metabolism are small in number; hence, included in ‘other’ category. This heat map essentially describes the differences among Dehalococcoides strains from the context of metabolic genes.
Table 1.
General features of Dehalococcoides metabolic network (iAI549).
Table 2.
Composition of the in silico minimal medium of Dehalococcoides.
Table 3.
Comparison of various in silico genome-scale models with iAI549.
Figure 3.
The reconstructed TCA-cycle and CO2 fixation pathway of Dehalococcoides.
The arrows show the directionality of the reactions. (A) Grey: citrate synthase gene currently not identified in iAI549, but the pathway is suggested to be present by Tang et al. [84]; Orange: pathways for which homologous putative genes (∼30% amino acid sequence identity) were tentatively identified in Dehalococcoides, but are suggested to be absent by Tang et al. [84]; Red: pathways for which putative genes are confirmed to be present by both iAI549 and Tang et al. [84]. In all cases, the TCA-cycle of Dehalococcoides is not closed which explains their inability to use acetate as an energy source. (B) Dehalococcoides' requirement of CO2 in addition to acetate for their in silico growth. The numbers are flux values in mmol.gDCW−1.h−1. During pyruvate synthesis, Dehalococcoides require 67% carbon (molar basis) from acetate and 33% (molar basis) from CO2. Thus, Dehalococcoides fix carbon via the pyruvate-ferredoxin oxidoreductase or pyruvate synthase (POR) pathway.
Figure 4.
Analysis of the citrate synthase (CS) reaction on Dehalococcoides growth.
(A) In the absence of the CS reaction, the TCA-cycle operates reductively via succinyl-CoA synthetase and 2-oxoglutarate synthase for producing biomass precursors for Dehalococcoides to grow. (B) The oxidative TCA-cycle operates when the CS reaction is present, but succinyl-CoA synthetase and 2-oxoglutarate synthase are absent, as suggested by the carbon labeling experiments [84]. However, Dehalococcoides growth remains almost unchanged with and without the CS reaction (0.0137 h−1 vs. 0.014 h−1) as represented by the flux values obtained from the growth simulations of iAI549.
Figure 5.
Reconstructed cobalamin biosynthesis pathway of Dehalococcoides.
Dashed orange lines indicate cell membrane, grey lines indicate missing pathways, and red lines indicate existing pathways, putative genes of which are identified in Dehalococcoides during the reconstruction of iAI549. The arrows are denoting the directionality of the reactions. Since the genomes encode a putative cobalamin transporter, Dehalococcoides may salvage vitamin B12 either in the form of cobinamide or cobalamin from the environment as indicated by ‘cobinamide transport’ and ‘cobalamin transport’ reactions in the figure. The adenosylcobalamin, which is the end product of the entire pathway, is a biomass constituent and is assumed to take part in Dehalococcoides cell formation.
Figure 6.
Influence of cobalamin on the growth rate and yield of Dehalococcoides.
(A) Growth rate of Dehalococcoides is simulated as a function of both cobalamin salvage rate and cobalamin fraction in the biomass equation. It shows the role of cobalamin in limiting the growth rate of Dehalococcoides. Clearly, the cobalamin uptake or salvage rate at which Dehalococcoides growth is limiting increases with the increase of cobalamin fraction in the biomass. (B) The cost of de novo cobalamin synthesis in terms of Dehalococcoides growth yield is compared (see text for details). The predicted yield of Dehalococcoides with and without the de novo cobalamin synthesis pathway remains almost identical for the reported maximum cobalamin fraction in the biomass. However, the predicted yield decreased only by 4% (from 0.72 gDCW/eeq to 0.69 gDCW/eeq) with 10 fold increase of cobalamin fraction in the biomass indicating the low cost of de novo cobalamin synthesis.
Figure 7.
Effect of carbon and energy sources on the growth yield of Dehalococcoides.
(A) The experimental growth yield of Dehalococcoides in the minimal medium (0.69 gDCW/eeq) is compared with increased growth yields achieved by allowing unlimited fluxes of all amino acids at a H2 flux of 10 mmol.gDCW−1.h−1 (corresponding to the experimental dechlorination rate), as well as doubling the H2 flux (20 mmol.gDCW−1.h−1). It shows that unlimited flux of amino acids (carbon source) increased the in silico growth yield of Dehalococcoides by 55%, whereas doubling the H2 flux (electron donor or energy source) alone enhanced the yield by 65%. (B) Analysis of the energy limited growth of Dehalococcoides. Since the growth yield of Dehalococcoides varies linearly with the energy transfer efficiency, their yield can be improved by increasing the flux of their energy source or electron donor to generate more ATP per electron. However, the variation in acetate fluxes has no effect on growth yields. Red and green arrows show growth yields and corresponding efficiencies for Dehalococcoides growth in mixed and pure cultures, respectively. ‘MM’ = minimal medium; ‘Tyr’ = tyrosine; ‘Glu’ = glutamate; ‘Gln’ = glutamine; ‘Gly’ = glycine; ‘Ala’ = alanine; ‘Thr’ = threonine; ‘Asp’ = aspartate; ‘All AA’ = all amino acids; ‘2X H2 flux’ = 20 mmol H2.gDCW−1.h−1.