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Key Enzymes for Anaerobic Lactate Metabolism in Geobacter sulfurreducens.

ABSTRACT: Growth of Geobacter sulfurreducens PCA on lactate was enhanced by laboratory adaptive evolution. The enhanced growth was considered to be attributed to increased expression of the sucCD genes, encoding a succinyl-coenzyme A (CoA) synthetase. To further investigate the function of the succinyl-CoA synthetase, the sucCD genes were deleted from G. sulfurreducens The mutant showed defective growth on lactate but not on acetate. Introduction of the sucCD genes into the mutant restored the full potential to grow on lactate. These results verify the importance of the succinyl-CoA synthetase in growth on lactate. Genome analysis of Geobacter species identified candidate genes, GSU1623, GSU1624, and GSU1620, for lactate dehydrogenase. Deletion mutants of the identified genes for d-lactate dehydrogenase (ΔGSU1623 ΔGSU1624 mutant) or l-lactate dehydrogenase (ΔGSU1620 mutant) could not grow on d-lactate or l-lactate but could grow on acetate and l- or d-lactate, respectively. Introduction of the respective genes into the mutants allowed growth on the corresponding lactate stereoisomer. These results suggest that the identified genes were essential for d- or l-lactate utilization. The lacZ reporter assay demonstrated that the putative promoter regions were more active during growth on lactate than during growth on acetate, indicating that the genes for the lactate dehydrogenases were expressed more during growth on lactate than during growth on acetate. The gene deletion phenotypes and the expression profiles indicate that there are metabolic switches between lactate and acetate. This study advances the understanding of anaerobic lactate utilization in G. sulfurreducens IMPORTANCE Lactate is a microbial fermentation product as well as a source of carbon and electrons for microorganisms in the environment. Furthermore, lactate is a common amendment for stimulation of microbial growth in environmental biotechnology applications. However, anaerobic metabolism of lactate has been poorly studied for environmentally relevant microorganisms. Geobacter species are found in various environments and environmental biotechnology applications. By employing genomic and genetic approaches, succinyl-CoA synthetase and lactate dehydrogenase were identified as key enzymes in anaerobic metabolism of lactate in Geobacter sulfurreducens, a representative Geobacter species. Differential gene expression during growth on lactate and acetate was observed, demonstrating that G. sulfurreducens could metabolically switch to adapt to available substrates in the environment. The findings provide new insights into basic physiology in lactate metabolism as well as cellular responses to growth conditions in the environment and can be informative for the application of lactate in environmental biotechnology.

SUBMITTER: Ueki T

PROVIDER: S-EPMC7783348 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Geobacter sulfurreducens is a ubiquitous iron-reducing bacterium in soils, and in engineered systems, it can respire an electrode to produce measurable electric current. Its unique metabolism, heavily dependent on an extensive network of cytochromes, requires a unique cell composition. In this work, we used metallomics, cell fraction and elemental analyses, and transcriptomics to study and analyze the cell composition of G. sulfurreducens. Elemental composition studies (C, H, O, N, and ash content) showed high C:O and H:O ratios of approximately 1.7:1 and 0.25:1, indicative of more reduced cell composition that is consistent with high lipid content. Our study shows that G. sulfurreducens cells have a large amount of iron (2 ± 0.2 μg/g dry weight) and lipids (32 ± 0.5% dry weight/dry weight) and that this composition does not change whether the cells are grown with a soluble or an insoluble electron acceptor. The high iron concentration, higher than similar microorganisms, is attributed to the production of cytochromes that are abundant in transcriptomic analyses in both solid and soluble electron acceptor growth. The unique cell composition of G. sulfurreducens must be considered when growing this microorganism for lab studies and commercial applications. IMPORTANCE Geobacter sulfurreducens is an electroactive microorganism. In nature, it grows on metallic minerals by transferring electrons to them, effectively "breathing" metals. In a manmade system, it respires an electrode to produce an electric current. It has become a model organism for the study of electroactive organisms. There are potential biotechnological applications of an organism that can bridge the gap between biology and electrical signal and, as a ubiquitous iron reducer in soils around the world, G. sulfurreducens has an impact on the global iron cycle. We measured the concentrations of metals, macromolecules, and basic elements in G. sulfurreducens to define this organism's composition. We also used gene expression data to discuss which proteins those metals could be associated with. We found that G. sulfurreducens has a large amount of lipid and iron compared to other bacteria-these observations are important for future microbiologists and biotechnologists working with the organism.

Project description:Biomineralization of Cu has been shown to control contaminant dynamics and transport in soils. However, very little is known about the role that subsurface microorganisms may play in the biogeochemical cycling of Cu. In this study, we investigate the bioreduction of Cu(II) by the subsurface metal-reducing bacterium Geobacter sulfurreducens Rapid removal of Cu from solution was observed in cell suspensions of G. sulfurreducens when Cu(II) was supplied, while transmission electron microscopy (TEM) analyses showed the formation of electron-dense nanoparticles associated with the cell surface. Energy-dispersive X-ray spectroscopy (EDX) point analysis and EDX spectrum image maps revealed that the nanoparticles are rich in both Cu and S. This finding was confirmed by X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) analyses, which identified the nanoparticles as Cu2S. Biomineralization of CuxS nanoparticles in soils has been reported to enhance the colloidal transport of a number of contaminants, including Pb, Cd, and Hg. However, formation of these CuxS nanoparticles has only been observed under sulfate-reducing conditions and could not be repeated using isolates of implicated organisms. As G. sulfurreducens is unable to respire sulfate, and no reducible sulfur was supplied to the cells, these data suggest a novel mechanism for the biomineralization of Cu2S under anoxic conditions. The implications of these findings for the biogeochemical cycling of Cu and other metals as well as the green production of Cu catalysts are discussed.IMPORTANCE Dissimilatory metal-reducing bacteria are ubiquitous in soils and aquifers and are known to utilize a wide range of metals as terminal electron acceptors. These transformations play an important role in the biogeochemical cycling of metals in pristine and contaminated environments and can be harnessed for bioremediation and metal bioprocessing purposes. However, relatively little is known about their interactions with Cu. As a trace element that becomes toxic in excess, Cu can adversely affect soil biota and fertility. In addition, biomineralization of Cu nanoparticles has been reported to enhance the mobilization of other toxic metals. Here, we demonstrate that when supplied with acetate under anoxic conditions, the model metal-reducing bacterium Geobacter sulfurreducens can transform soluble Cu(II) to Cu2S nanoparticles. This study provides new insights into Cu biomineralization by microorganisms and suggests that contaminant mobilization enhanced by Cu biomineralization could be facilitated by Geobacter species and related organisms.

Project description:Geobacter sulfurreducens is a well-studied representative of the Geobacteraceae, which play a critical role in organic matter oxidation coupled to Fe(III) reduction, bioremediation of groundwater contaminated with organics or metals, and electricity production from waste organic matter. In order to investigate G. sulfurreducens central metabolism and electron transport, a metabolic model which integrated genome-based predictions with available genetic and physiological data was developed via the constraint-based modeling approach. Evaluation of the rates of proton production and consumption in the extracellular and cytoplasmic compartments revealed that energy conservation with extracellular electron acceptors, such as Fe(III), was limited relative to that associated with intracellular acceptors. This limitation was attributed to lack of cytoplasmic proton consumption during reduction of extracellular electron acceptors. Model-based analysis of the metabolic cost of producing an extracellular electron shuttle to promote electron transfer to insoluble Fe(III) oxides demonstrated why Geobacter species, which do not produce shuttles, have an energetic advantage over shuttle-producing Fe(III) reducers in subsurface environments. In silico analysis also revealed that the metabolic network of G. sulfurreducens could synthesize amino acids more efficiently than that of Escherichia coli due to the presence of a pyruvate-ferredoxin oxidoreductase, which catalyzes synthesis of pyruvate from acetate and carbon dioxide in a single step. In silico phenotypic analysis of deletion mutants demonstrated the capability of the model to explore the flexibility of G. sulfurreducens central metabolism and correctly predict mutant phenotypes. These results demonstrate that iterative modeling coupled with experimentation can accelerate the understanding of the physiology of poorly studied but environmentally relevant organisms and may help optimize their practical applications.

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Key Enzymes for Anaerobic Lactate Metabolism in Geobacter sulfurreducens.

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