Monitoring the Metabolic Status of Geobacter Species in Contaminated Groundwater by Quantifying Key Metabolic Proteins with Geobacter-Specific Antibodies

Department of Microbiology, University of Massachusetts, Amherst, Massachusetts, USA.
Applied and Environmental Microbiology (Impact Factor: 3.67). 07/2011; 77(13):4597-602. DOI: 10.1128/AEM.00114-11
Source: PubMed


Simple and inexpensive methods for assessing the metabolic status and bioremediation activities of subsurface microorganisms are required before bioremediation practitioners will adopt molecular diagnosis of the bioremediation community as a routine practice for guiding the development of bioremediation strategies. Quantifying gene transcripts can diagnose important aspects of microbial physiology during bioremediation but is technically challenging and does not account for the impact of translational modifications on protein abundance. An alternative strategy is to directly quantify the abundance of key proteins that might be diagnostic of physiological state. To evaluate this strategy, an antibody-based quantification approach was developed to investigate subsurface Geobacter communities. The abundance of citrate synthase corresponded with rates of metabolism of Geobacter bemidjiensis in chemostat cultures. During in situ bioremediation of uranium-contaminated groundwater the quantity of Geobacter citrate synthase increased with the addition of acetate to the groundwater and decreased when acetate amendments stopped. The abundance of the nitrogen-fixation protein, NifD, increased as ammonium became less available in the groundwater and then declined when ammonium concentrations increased. In a petroleum-contaminated aquifer, the abundance of BamB, an enzyme subunit involved in the anaerobic degradation of mono-aromatic compounds by Geobacter species, increased in zones in which Geobacter were expected to play an important role in aromatic hydrocarbon degradation. These results suggest that antibody-based detection of key metabolic proteins, which should be readily adaptable to standardized kits, may be a feasible method for diagnosing the metabolic state of microbial communities responsible for bioremediation, aiding in the rational design of bioremediation strategies.

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    • "Proteins involved in growth Exposure to U(VI) slightly reduced the growth rate of G. sulfurreducens (Fig. 2a) and many proteins associated with the central metabolism were in lower abundance in cells exposed to U(VI). For example, the expression of citrate synthase (GltA, GSU1106) that catalyses the condensation of acetyl-CoA and oxaloacetate to citric acid in the TCA cycle and, therefore, directly correlated with metabolic rates of G. sulfurreducens (Holmes et al., 2005; Wilkins et al., 2011; Yun et al., 2011), was lower in the presence of U(VI) compared with the untreated control (Table 1), suggesting that metabolism was slower in the presence of U(VI). Phosphoenolpyruvate synthase (PpsA, GSU0803), which activates pyruvate to phosphoenolpyruvate (Mahadevan et al., 2006), and two subunits of ATP synthase (GSU0108 and GSU0111) were also less abundant when U(VI) was present (Table 1). "
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    ABSTRACT: Geobacter species often play an important role in the in situ bioremediation of uranium-contaminated groundwater, but little is known about how these microbes avoid uranium toxicity. To evaluate this further, the proteome of G. sulfurreducens exposed to 100 µM U(VI) acetate was compared with control cells not exposed to U(VI). Of the 1363 proteins detected from these cultures, 203 proteins had higher abundance during exposure to U(VI) compared to the control cells and 148 proteins had lower abundance. U(VI)-exposed cultures expressed lower levels of proteins involved in growth, protein and amino acid biosynthesis, as well as key central metabolism enzymes as a result of the deleterious effect of U(VI) in the growth of G. sulfurreducens. In contrast, proteins involved in detoxification, such as several efflux pumps belonging to the RND family, and protection of membrane and proteins, such as chaperons and proteins involved in secretion systems, were in higher abundance in cells exposed to U(VI). Exposing G. sulfurreducens to U(VI) resulted in higher abundance of many proteins associated with the oxidative stress response, such as superoxide dismutase and superoxide reductase. A strain in which the gene for superoxide dismutase was deleted grew slower than the wild-type strain in the presence U(VI), but not in its absence. The results suggest that there is not one specific mechanism for uranium detoxification. Rather, multiple general stress responses are induced, which presumably enable Geobacter species to tolerate high uranium concentrations.
    Full-text · Article · Oct 2014 · Microbiology
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    • "ies on the respiratory mechanisms of Fe(III) oxide (Kim et al., 2005; Lloyd et al., 2003; Lovley et al., 2011; Rollefson et al., 2009; Yun et al., 2011), microbial fuel cells (MFC) (Bond and Lovley, 2003), development of genetic manipulation techniques (Coppi et al., 2001; Mahadevan et al., 2011; Park and Kim, 2011; Ueki and Lovley, 2010), and the first genome analysis within the genus Geobacter (Methe et al., 2003). "
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    ABSTRACT: An ethanol-utilizing Fe(III)-reducing bacterial strain, OSK2A(T), was isolated from a lotus field in Aichi, Japan. Phylogenetic analysis of the 16S rRNA gene sequences of OSK2A(T) and related strains placed it within Geobacter sulfurreducens PCA(T). Strain OSK2A(T) was shown to be a Gram-negative, motile, rod-shaped bacterium, strictly anaerobic, 0.76-1.65 µm long and 0.28-0.45 μm wide. Its growth occurred at 20-40℃, pH 6.0-8.1, and it tolerated up to 1% NaCl. The G+C content of the genomic DNA was 61.2 mol% and DNA-DNA hybridization value with Geobacter sulfurreducens PCA(T) was 60.7%. The major respiratory quinone was MK-8. The major fatty acids were 16:1 ω7c, 16:0, 14:0, 15:0 iso, 16:1 ω5c, and 18:1 ω7c. Strain OSK2A(T) could utilize H2, ethanol, acetate, lactate, pyruvate, and formate as substrates with Fe(III)-citrate as electron acceptor. Amorphous Fe(III) hydroxide, Fe(III)-NTA, fumarate, malate, and elemental sulfur were utilized as electron acceptors with either acetate or ethanol as substrates. Results obtained from physiological, DNA-DNA hybridization, and chemotaxonomic tests support genotypic and phenotypic differentiation of strain OSK2A(T) from its closest relative. The isolate is assigned as a novel subspecies with the name Geobacter sulfurreducens subsp. ethanolicus, subsp. nov. (type strain OSK2A(T)=DSMZ 26126(T)=JCM 18752(T)).
    Full-text · Article · Nov 2013 · The Journal of General and Applied Microbiology
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    ABSTRACT: Geobacter species specialize in making electrical contacts with extracellular electron acceptors and other organisms. This permits Geobacter species to fill important niches in a diversity of anaerobic environments. Geobacter species appear to be the primary agents for coupling the oxidation of organic compounds to the reduction of insoluble Fe(III) and Mn(IV) oxides in many soils and sediments, a process of global biogeochemical significance. Some Geobacter species can anaerobically oxidize aromatic hydrocarbons and play an important role in aromatic hydrocarbon removal from contaminated aquifers. The ability of Geobacter species to reductively precipitate uranium and related contaminants has led to the development of bioremediation strategies for contaminated environments. Geobacter species produce higher current densities than any other known organism in microbial fuel cells and are common colonizers of electrodes harvesting electricity from organic wastes and aquatic sediments. Direct interspecies electron exchange between Geobacter species and syntrophic partners appears to be an important process in anaerobic wastewater digesters. Functional and comparative genomic studies have begun to reveal important aspects of Geobacter physiology and regulation, but much remains unexplored. Quantifying key gene transcripts and proteins of subsurface Geobacter communities has proven to be a powerful approach to diagnose the in situ physiological status of Geobacter species during groundwater bioremediation. The growth and activity of Geobacter species in the subsurface and their biogeochemical impact under different environmental conditions can be predicted with a systems biology approach in which genome-scale metabolic models are coupled with appropriate physical/chemical models. The proficiency of Geobacter species in transferring electrons to insoluble minerals, electrodes, and possibly other microorganisms can be attributed to their unique "microbial nanowires," pili that conduct electrons along their length with metallic-like conductivity. Surprisingly, the abundant c-type cytochromes of Geobacter species do not contribute to this long-range electron transport, but cytochromes are important for making the terminal electrical connections with Fe(III) oxides and electrodes and also function as capacitors, storing charge to permit continued respiration when extracellular electron acceptors are temporarily unavailable. The high conductivity of Geobacter pili and biofilms and the ability of biofilms to function as supercapacitors are novel properties that might contribute to the field of bioelectronics. The study of Geobacter species has revealed a remarkable number of microbial physiological properties that had not previously been described in any microorganism. Further investigation of these environmentally relevant and physiologically unique organisms is warranted.
    Full-text · Article · Jan 2011 · Advances in Microbial Physiology
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