Preferential Reduction of Fe(III) over Fumarate by Geobacter sulfurreducens

The presence of Fe(III), but not that of Fe(II), resulted inca. 20-fold-lower levels of mRNA for fumarate reductase, inhibitingfumarate reduction and favoring utilization of fumarate as anelectron donor in chemostat cultures of Geobacter sulfurreducens,despite the fact that growth yield with fumarate was 3-foldhigher than with Fe(III).

INTRODUCTION

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Introduction

The study of Fe(III) reduction has been greatly facilitatedby the finding that microorganisms in the family Geobacteraceaeare the predominant Fe(III)-reducing microorganisms in a diversityof subsurface environments (1, 3, 6, 11) and the fact that Geobacteraceaesimilar to those that predominate in these environments canbe recovered in pure culture. Geobacter sulfurreducens, whichcommonly serves as the model organism for the Geobacteraceaein subsurface environments, can use both Fe(III) and fumarateas an electron acceptor (2). Understanding the relative utilizationof Fe(III) and fumarate as electron acceptors could aid in developingstrategies to best promote the growth of Geobacter species forbioremediation applications, such as the reductive precipitationof uranium from contaminated groundwater (1). In previouslystudied microorganisms, the regulation of electron acceptorutilization favors the reduction of the electron acceptor thatcan yield the most energy to support cell growth (5, 7, 8, 12).However, it is not clear whether the same trend applies to microorganismsin subsurface environments.

Inhibition of fumarate respiration in the presence of Fe(III).

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Inhibition of fumarate...

In order to evaluate potential controls on fumarate and Fe(III)respiration, G. sulfurreducens was grown in continuous culture(A. Esteve-Núñez, M. Rothermich, M. Sharma, andD. R. Lovley, submitted for publication) with the electron donor,acetate (5.5 mM), which limited growth at a dilution rate of0.05 h^–1. This culture condition was designed to simulatethe most prevalent conditions in anoxic soils and subsurfaceenvironments, in which acetate is the key electron donor forFe(III) reduction (10). When fumarate (30 mM) was provided asthe electron acceptor, there was a steady-state accumulationof succinate resulting from fumarate reduction (Fig. 1). Thesteady-state biomass was 45.5 ± 5 (mean ± standarddeviation of results from three replicate experiments) mg ofprotein per liter. When cultures were grown with the same acetateinput, but with 60 mM Fe(III) citrate as the electron acceptor,the culture density was 15.5 ± 3 mg of protein per liter.Thus, fumarate reduction provided significantly more energyto support growth than Fe(III) reduction.

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FIG. 1. A steady-state culture of G. sulfurreducens growing at a dilution rate of 0.05 h^–1 with fumarate as the terminal electron acceptor was pulsed with 10 mM Fe(III) citrate (arrow). The response of the cell to the pulse was monitored by analyzing the respective respiration products, succinate (filled circles) and Fe(II) (open circles). All the Fe(II) produced was finally washed out of the chemostat as expected in a continuous culture system. Cells were harvested before the Fe(III) pulse, after the Fe(III) pulse (6 h), and when all the Fe(III) was fully reduced to Fe(II) (14 h). RNA was isolated from those cells, and Northern blot hybridization was performed with a specific oligoprobe for frdC (fumarate reductase). The detected transcript corresponds with the 3.3 kb of the frdCAB operon. As a control, parallel cultures were pulsed with the chelating agent citrate, and Northern analysis was performed as described for the Fe(III) citrate pulse.

In order to examine the interaction between fumarate and Fe(III)reduction, Fe(III) citrate (10 mM) was added to steady-statecultures of cells growing with fumarate as the electron acceptor(Fig. 1). There was an immediate decrease in succinate concentrationconcurrent with an accumulation of Fe(II) resulting from Fe(III)reduction. The inhibition of fumarate reduction was associatedwith a dramatic decline in levels of mRNA for the fumarate reductaseoperon, frdCAB (Fig. 1), measured by Northern analysis (theprobe was generated with PCR using the primers frdCfor [5'-GTTCGGTATCCAGCTGAG-3']and frdCrev [5'-CTTTCAGAATGCCGGTGACG-3']). Six hours after theaddition of Fe(III), the mRNA levels for frdCAB were 17-foldlower than in cells not exposed to Fe(III) (Fig. 1). Furthermore,at this time fumarate reductase-specific activity in the membranefraction of Fe(III)-amended cultures was fourfold lower thanin unamended cultures (13 ± 1 versus 52 ± 4 nmolmin^–1 mg of protein^–1). Once the Fe(III) was completelyreduced to Fe(II), the level of mRNA for the fumarate reductasegenes returned to the level detected prior to the addition ofFe(III), even though there was still a significant quantityof Fe(II) in the culture (Fig. 1). The finding that the expressionof the fumarate reductase was down-regulated in the presenceof Fe(III), but not Fe(II), represents a novel instance in whichdiscrimination between iron redox states seems essential forregulating processes that might have an impact on the pathwayfor electron transfer.

In order to determine whether Fe(III) had a similar effect onfumarate respiration over longer periods of time in which G.sulfurreducens would have time to adapt to the simultaneouspresence of high quantities of Fe(III) and fumarate, G. sulfurreducenswas grown in continuous culture with acetate as the electrondonor and the limiting growth substrate, with both fumarate(35 mM) and Fe(III) (70 mM) as potential electron acceptors(Fig. 2). As was observed in the short-term studies, fumaratereduction was inhibited and levels of fumarate reductase mRNAwere lower in the presence of Fe(III) and fumarate than in controlcultures growing on fumarate alone. The steady-state biomassconcentration in the presence of both fumarate and Fe(III) wasslightly higher than that with Fe(III) alone but significantlylower than when fumarate was the sole electron acceptor.

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FIG. 2. (A) Effect of terminal electron acceptors on the growth yield. Cells of G. sulfurreducens were cultured in chemostats in the presence of fumarate, fumarate and Fe(III), or just Fe(III) as the terminal electron acceptor. The results shown are the means of results from triplicate cultures for each treatment. (B) Effect of the terminal electron acceptor on fumarate reductase (frdCAB) expression. Northern blot hybridizations on isolated RNA were performed with a specific oligoprobe for frdC. fum, fumarate.

During bacterial growth on fumarate, the succinate that is producedfrom fumarate reduction is not oxidized in the tricarboxylicacid cycle (4), but it is possible that in the presence of Fe(III),small amounts of fumarate were used as an electron donor orcarbon source in a pathway in which fumarate was converted toacetyl coenzyme A via malate and pyruvate with the formationof formate (Fig. 3A). In the presence of Fe(III), high concentrationsof malate accumulated, and there was an increased rate of formateproduction (Fig. 3B). The high levels of malate indicated thatconversion of malate to pyruvate might be the limiting stepin fumarate utilization. Once Fe(III) was depleted and fumaratereduction resumed, malate accumulation and formate productionstopped (Fig. 3B).

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FIG. 3. (A) Scheme of fumarate transformation by G. sulfurreducens in the presence of Fe(III). Fumarate is transformed into malate when fumarate respiration is inhibited. Malate is subsequently converted to acetyl coenzyme A (CoA) via pyruvate with the release of formate. TCA, tricarboxylic acid; e^–, electron. (B) Malate (filled circles) and formate (open triangles) formation after a pulse of 10 mM Fe(III) citrate. The reduction of Fe(III) is plotted as Fe(II) production (open circles).

Implications.

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Implications.

The respiration in G. sulfurreducens is clearly not optimizedfor maximal growth when high concentrations of fumarate as wellas Fe(III) are available. However, the down-regulation of fumaraterespiration in the presence of Fe(III) may represent an effectiveadaptation to conditions found in most subsurface environments.Generally, the scarcity of readily metabolizable organic matterseverely limits microbial growth in the subsurface. In contrast,Fe(III) is often the most abundant electron acceptor availablefor microbial respiration (9). Therefore, in typical anoxicsubsurface environments in which Geobacter species predominate,if any fumarate becomes available it may be more beneficialto funnel it toward catabolic reactions rather than to use itas an electron acceptor. Thus, although the regulation of respirationin G. sulfurreducens does not fit the paradigm that the mostenergetically favorable electron acceptors are reduced first,this finding may represent the most adaptive response to theenvironmental conditions that Geobacter species typically encounter.

ACKNOWLEDGMENTS

This research was funded by the Genomes to Life Program, U.S.Department of Energy (grant DE-FC02-02ER63446). A.E.-N. wasthe recipient of a postdoctoral fellowship from the Secretaríade Estado de Educación y Universidades (Madrid, Spain),cofunded by the European Social Fund. In addition, C.N. wasthe recipient of a DGAPA/UNAM postdoctoral fellowship.

FOOTNOTES

* Corresponding author. Mailing address: University of Massachusetts, Dept. of Microbiology, Morrill Science Center IVN, Amherst, MA 01003. Phone: (413) 545-9782. Fax: (413) 545-1578. E-mail: aesteve{at}microbio.umass.edu.

REFERENCES

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