Genome-scale comparison and constraint-based metabolic reconstruction of the facultative anaerobic Fe(III)-reducer Rhodoferax ferrireducens

Abstract

Background: Rhodoferax ferrireducens is a metabolically versatile, Fe(III)-reducing, subsurface microorganism that is likely to play an important role in the carbon and metal cycles in the subsurface. It also has the unique ability to convert sugars to electricity, oxidizing the sugars to carbon dioxide with quantitative electron transfer to graphite electrodes in microbial fuel cells. In order to expand our limited knowledge about R. ferrireducens, the complete genome sequence of this organism was further annotated and then the physiology of R. ferrireducens was investigated with a constraint-based, genome-scale in silico metabolic model and laboratory studies.

Results: The iterative modeling and experimental approach unveiled exciting, previously unknown physiological features, including an expanded range of substrates that support growth, such as cellobiose and citrate, and provided additional insights into important features such as the stoichiometry of the electron transport chain and the ability to grow via fumarate dismutation. Further analysis explained why R. ferrireducens is unable to grow via photosynthesis or fermentation of sugars like other members of this genus and uncovered novel genes for benzoate metabolism. The genome also revealed that R. ferrireducens is well-adapted for growth in the subsurface because it appears to be capable of dealing with a number of environmental insults, including heavy metals, aromatic compounds, nutrient limitation and oxidative stress.

Conclusion: This study demonstrates that combining genome-scale modeling with the annotation of a new genome sequence can guide experimental studies and accelerate the understanding of the physiology of under-studied yet environmentally relevant microorganisms.

Figure 1

Graphic representation of the Rhodoferax ferrireducens genome. The first two outer circles represent the positions of genes in the Rhodoferax chromosome (Circle 1- plus strand, Circle 2- minus strand). Circles 3-6 represent Rhodoferax genes with a bidirectional best match to Polaromonas JS666, and they are grouped according to percent identity of each BLAST match. Circle 7 represents rRNA genes (green ticks), tRNA genes (blue ticks) and sRNA genes (red ticks).

Figure 2

R. ferrireducens BLAST matches to fully sequenced genomes. Curves summarizing BLAST matches for Rhodoferax genes sorted by matching organism and binned according to percent identity of each BLAST match. Each curve represents a different matching organism (see color code in key), and only the top 10 fully sequenced genomes are shown. The x-axis coordinate is binned according to the percent identity for a group of BLAST matches, and the y-axis coordinate indicates the number of Rhodoferax genes in a particular group.

Figure 3

Functional classification of metabolic reactions in the R. ferrireducens model.

Figure 4

Determination of energy parameters. A) Diagram of the algorithm utilized in the determination of proton translocation stoichiometry and NGAM. A range of possible H⁺/2e^- values for both NADH dehydrogenase and cytochrome reductase and NGAM values are iterated by the algorithm so that the "error" (E) between predicted and experimentally determined yields (Y) is minimized. The model was constrained with a fixed GAM and growth rates ≤ 0.04 h^-1 to match actual conditions. B) Representation of errors between model predictions and experimental data. The lowest errors highlighted in red. C) Comparison between predicted (black bars) and experimental (gray bars) yields. Values are presented as ratios, with experimental yields set at 100%. Yields were obtained from cultures in the following conditions: 10 mM acetate/40 mM fumarate; 56 mM ferric citrate; 20 mM acetate/10 mM Fe(III)NTA. The patterned columns represent the validation data by growth on fumarate as the sole substrate (30 mM). Values are presented as ratios, with experimental yield set at 100%.

Figure 5

Fumarate dismutation. A) Growth on fumarate as the sole substrate. Inset: production of succinate and acetate as a result of fumarate consumption. Each point is the average of triplicate cultures with standard deviations.

Figure 6

Growth of R. ferrireducens with citrate as electron donor and Fe(III) as electron acceptor. A) Possible pathways of citrate utilization: by-products differ depending on the availability of electron acceptor Fe(III). The different stoichiometries are summarized. B) R. ferrireducens is predicted to utilize a combination of different pathways during growth with limited Fe(III) availability. The numbers in red represent predicted fluxes.

Figure 7

Cluster of genes involved in the aerobic "hybrid" benzoate degradation pathway.

Figure 8

Analysis of substrate efficiency. Eight representative electron donors (acetate, glycolate, lactate, fumarate, benzoate, citrate, glucose, and cellobiose) were used in simulations of R. ferrireducens growth under donor-limiting conditions with Fe(III) as electron acceptor. The biomass yield to electron donor (gdw/mol substrate) and the Fe(III):substrate ratio from simulation results of R. ferrireducens growth on acetate were set at 1. The biomass yields to other donors and the ratios of Fe(III) to other electron donors are expressed in comparison to that of acetate.

Figure 9

Growth on nitrate as electron acceptor and the sole N source. A) Growth on 0.1% glucose and 15 mM nitrate. Ammonia (0.25 g/L) was either added or omitted from the medium. B) Production of nitrite. The medium contained 0.1% lactate and 20 mM nitrate, ammonia was omitted. C) Experimental vs. predicted growth rate ratios with and without added ammonia. D) Experimental vs. predicted ratio of nitrate converted to nitrite.

Figure 10

Schematic representation of relevant metabolic and physiological features.