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Impact of iron reduction on the metabolism of Clostridium acetobutylicum.

ABSTRACT: Iron is essential for most living organisms. In addition, its biogeochemical cycling influences important processes in the geosphere (e.g., the mobilization or immobilization of trace elements and contaminants). The reduction of Fe(III) to Fe(II) can be catalysed microbially, particularly by metal-respiring bacteria utilizing Fe(III) as a terminal electron acceptor. Furthermore, Gram-positive fermentative iron reducers are known to reduce Fe(III) by using it as a sink for excess reducing equivalents, as a form of enhanced fermentation. Here, we use the Gram-positive fermentative bacterium Clostridium acetobutylicum as a model system due to its ability to reduce heavy metals. We investigated the reduction of soluble and solid iron during fermentation. We found that exogenous (resazurin, resorufin, anthraquinone-2,6-disulfonate) as well as endogenous (riboflavin) electron mediators enhance solid iron reduction. In addition, iron reduction buffers the pH, and elicits a shift in the carbon and electron flow to less reduced products relative to fermentation. This study underscores the role fermentative bacteria can play in iron cycling and provides insights into the metabolic profile of coupled fermentation and iron reduction with laboratory experiments and metabolic network modelling.

SUBMITTER: List C

PROVIDER: S-EPMC6852232 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Impact of iron reduction on the metabolism of Clostridium acetobutylicum.

List Cornelia C Hosseini Zhaleh Z Lederballe Meibom Karin K Hatzimanikatis Vassily V Bernier-Latmani Rizlan R

Environmental microbiology 20190516 10

Iron is essential for most living organisms. In addition, its biogeochemical cycling influences important processes in the geosphere (e.g., the mobilization or immobilization of trace elements and contaminants). The reduction of Fe(III) to Fe(II) can be catalysed microbially, particularly by metal-respiring bacteria utilizing Fe(III) as a terminal electron acceptor. Furthermore, Gram-positive fermentative iron reducers are known to reduce Fe(III) by using it as a sink for excess reducing equival ...[more]

PMID: 31020759

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Project description:An operon comprising two genes, CA_P0037 and CA_P0036, that encode proteins of unknown function that were previously shown to be highly expressed in acidogenic cells and repressed in solventogenic and alcohologenic cells is located on the pSOL1 megaplasmid of Clostridium acetobutylicum upstream of adhE2 A CA_P0037::int (189/190s) mutant in which an intron was inserted at position 189/190 in the sense strand of CA_P0037 was successfully generated by the Targetron technique. The resultant mutant showed significantly different metabolic flux patterns in acidogenic (producing mainly lactate, butyrate, and butanol) and alcohologenic (producing mainly butyrate, acetate, and lactate) chemostat cultures but not in solventogenic or batch cultures. Transcriptomic investigation of the CA_P0037::int (189/190s) mutant showed that inactivation of CA_P0037 significantly affected the expression of more than 258 genes under acidogenic conditions. Surprisingly, genes belonging to the Fur regulon, involved in iron transport (CA_C1029-CA_C1032), or coding for the main flavodoxin (CA_C0587) were the most significantly expressed genes under all conditions, whereas fur (coding for the ferric uptake regulator) gene expression remained unchanged. Furthermore, most of the genes of the Rex regulon, such as the adhE2 and ldhA genes, and of the PerR regulon, such as rbr3A-rbr3B and dfx, were overexpressed in the mutant. In addition, the whole CA_P0037-CA_P0036 operon was highly expressed under all conditions in the CA_P0037::int (189/190s) mutant, suggesting a self-regulated expression mechanism. Cap0037 was shown to bind to the CA_P0037-CA_P0036 operon, sol operon, and adc promoters, and the binding sites were determined by DNA footprinting. Finally, a putative Cap0037 regulon was generated using a bioinformatic approach. Clostridium acetobutylicum is well-known for its ability to produce solvents, especially n-butanol. Understanding the regulatory network of C. acetobutylicum will be crucial for further engineering to obtain a strain capable of producing n-butanol at high yield and selectivity. This study has discovered that the Cap0037 protein is a novel regulator of C. acetobutylicum that drastically affects metabolism under both acidogenic and alcohologenic fermentation conditions. This is pioneering work for further determining the regulatory mechanism of Cap0037 in C. acetobutylicum and studying the role of proteins homologous to Cap0037 in other members of the phylum Firmicutes.

Project description:UnlabelledEngineering industrial microorganisms for ambitious applications, for example, the production of second-generation biofuels such as butanol, is impeded by a lack of knowledge of primary metabolism and its regulation. A quantitative system-scale analysis was applied to the biofuel-producing bacterium Clostridium acetobutylicum, a microorganism used for the industrial production of solvent. An improved genome-scale model, iCac967, was first developed based on thorough biochemical characterizations of 15 key metabolic enzymes and on extensive literature analysis to acquire accurate fluxomic data. In parallel, quantitative transcriptomic and proteomic analyses were performed to assess the number of mRNA molecules per cell for all genes under acidogenic, solventogenic, and alcohologenic steady-state conditions as well as the number of cytosolic protein molecules per cell for approximately 700 genes under at least one of the three steady-state conditions. A complete fluxomic, transcriptomic, and proteomic analysis applied to different metabolic states allowed us to better understand the regulation of primary metabolism. Moreover, this analysis enabled the functional characterization of numerous enzymes involved in primary metabolism, including (i) the enzymes involved in the two different butanol pathways and their cofactor specificities, (ii) the primary hydrogenase and its redox partner, (iii) the major butyryl coenzyme A (butyryl-CoA) dehydrogenase, and (iv) the major glyceraldehyde-3-phosphate dehydrogenase. This study provides important information for further metabolic engineering of C. acetobutylicum to develop a commercial process for the production of n-butanol.ImportanceCurrently, there is a resurgence of interest in Clostridium acetobutylicum, the biocatalyst of the historical Weizmann process, to produce n-butanol for use both as a bulk chemical and as a renewable alternative transportation fuel. To develop a commercial process for the production of n-butanol via a metabolic engineering approach, it is necessary to better characterize both the primary metabolism of C. acetobutylicum and its regulation. Here, we apply a quantitative system-scale analysis to acidogenic, solventogenic, and alcohologenic steady-state C. acetobutylicum cells and report for the first time quantitative transcriptomic, proteomic, and fluxomic data. This approach allows for a better understanding of the regulation of primary metabolism and for the functional characterization of numerous enzymes involved in primary metabolism.

Dataset Information

Impact of iron reduction on the metabolism of Clostridium acetobutylicum.

Publications

Impact of iron reduction on the metabolism of Clostridium acetobutylicum.

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