Proteome of Geobacter sulfurreducens grown with Fe(III) oxide or Fe(III) citrate as the electron acceptor

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Abstract

The mechanisms for Fe(III) oxide reduction in Geobacter species are of interest because Fe(III) oxides are the most abundant form of Fe(III) in many soils and sediments and Geobacter species are prevalent Fe(III)-reducing microorganisms in many of these environments. Protein abundance in G. sulfurreducens grown on poorly crystalline Fe(III) oxide or on soluble Fe(III) citrate was compared with a global accurate mass and time tag proteomic approach in order to identify proteins that might be specifically associated with Fe(III) oxide reduction. A total of 2991 proteins were detected in G. sulfurreducens grown with acetate as the electron donor and either Fe(III) oxide or soluble Fe(III) citrate as the electron acceptor, resulting in 86% recovery of the genes predicted to encode proteins. Of the total expressed proteins 76% were less abundant in Fe(III) oxide cultures than in Fe(III) citrate cultures, which is consistent with the overall slower rate of metabolism during growth with an insoluble electron acceptor. A total of 269 proteins were more abundant in Fe(III) oxide-grown cells than in cells grown on Fe(III) citrate. Most of these proteins were in the energy metabolism category: primarily electron transport proteins, including 13 c-type cytochromes and PilA, the structural protein for electrically conductive pili. Several of the cytochromes that were more abundant in Fe(III) oxide-grown cells were previously shown with genetic approaches to be essential for optimal Fe(III) oxide reduction. Other proteins that were more abundant during growth on Fe(III) oxide included transport and binding proteins, proteins involved in regulation and signal transduction, cell envelope proteins, and enzymes for amino acid and protein biosynthesis, among others. There were also a substantial number of proteins of unknown function that were more abundant during growth on Fe(III) oxide. These results indicate that electron transport to Fe(III) oxide requires additional and/or different proteins than electron transfer to soluble, chelated Fe(III) and suggest proteins whose functions should be further investigated in order to better understand the mechanisms of electron transfer to Fe(III) oxide in G. sulfurreducens.

Introduction

The mechanism for Fe(III) oxide reduction in dissimilatory metal-reducing microorganisms is of interest because this is an important biogeochemical process in a variety of soils and sediments [1], [2], [3]. In addition to playing an important role in the oxidation of naturally occurring organic matter [1], Fe(III) reducers can degrade a variety of organic contaminants [4], [5], [6]. Furthermore, Fe(III) reduction can affect the fate of a variety of other metals and inorganic nutrients in pristine and contaminated soils and groundwater [6], [7], [8].

In many environments in which Fe(III) oxide reduction is an important process, members of the family Geobacteraceae are the predominant organisms [9], [10]. Previous studies have suggested that, in the absence of exogenous Fe(III) chelators and/or electron shuttles, such as humic substances [11], [12], Geobacter species have to contact Fe(III) oxides directly in order to reduce them [13]. Biochemical and genetic studies [14], [15], [16], [17] have revealed some components likely to be involved in Fe(III) oxide reduction in Geobacter species. It has been proposed [10] that electrons generated from central metabolism are probably shuttled across the periplasm via c-type cytochromes such as MacA [18] and PpcA [19] to the outer-membrane cytochromes such as OmcB [20], and OmcS [21]. The final electron transfer to Fe(III) oxides may be via pili, which are conductive and have been shown to serve as nanowires that can extend far beyond the cell's outer membrane [17].

Previous global studies on gene expression [22], and protein abundance [23] have examined Geobacter species grown on soluble electron acceptors, such as fumarate and Fe(III) citrate due to the ease of cell growth and processing of samples. However, Fe(III) oxides are the most environmentally relevant electron acceptor [1], [10]. Recent studies have suggested that proteins that are specifically expressed during growth on Fe(III) oxide play an important role in Fe(III) oxide reduction [15], [17], [21], but these studies focused on a limited subset of proteins.

In order to obtain a more extensive list of proteins that are more abundant during growth on Fe(III) oxide containing cultures, and thus potentially important in the reduction of Fe(III) oxide, we employed a global proteomic approach in the study reported here. The accurate mass and time (AMT) tag technique for the quantitative identification of peptides results in high confidence and high throughput [24], [25], [26] and our previous study demonstrated that it is a useful tool to compare protein expression profiles of G. sulfurreducens under various culture conditions [23]. Here we report on a global AMT analysis of protein expression of G. sulfurreducens grown on Fe(III) oxide. The results indicate that there is a substantial subset of proteins that are significantly more abundant during growth on Fe(III) oxide than during growth with soluble, chelated Fe(III) as the electron acceptor.

Section snippets

Chemicals

Dithiothreitol (DTT), iodoacetamide, CHAPS, oxalic acid, ammonium oxalate, and trypsin were purchased from Sigma (St. Louis, MO), protease inhibitor from Roche (Indianapolis, IN), and acetic acid from Promega (Madison, WI).

Bacterial growth

G. sulfurreducens was grown under anaerobic conditions (N2/CO2: 80/20) at 30 °C in batch cultures as previously described [27]. Acetate (10 mM) served as the electron donor and either synthetic poorly crystalline Fe(III) oxide (100 mmol/l) or Fe(III) citrate (60 mM) was the

Results and discussion

G. sulfurreducens was grown with acetate as the electron donor and either Fe(III) oxide or Fe(III) citrate as the electron acceptor. Cells were harvested at late logarithmic phase after 2 and 29 days of growth with Fe(III) citrate and Fe(III) oxide, respectively (Fig. 1). A total of 2991 proteins were detected in cells from these cultures (Supplementary Table). This represents 86% of the genes predicted to encode proteins and is comparable to the percentage of proteins identified in previous

Conclusions

This study suggests that the physiology of cells growing with Fe(III) oxide as the electron acceptor is substantially different than that of cells growing with soluble, Fe(III) citrate. This study not only corroborates some of the previous findings of proteins expressed in higher abundance during growth on Fe(III) oxide, such as pili and some outer-membrane cytochromes, it has identified additional proteins that could be important in Fe(III) oxide reduction. Further investigation of the

Acknowledgement

This research was supported by the Office of Science (BER), U. S. Department of Energy, Grant No. DE-FC02-02ER63446.

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