Complete activity profile of<i>Clostridium acetobutylicum</i>[FeFe]-hydrogenase and kinetic parameters for endogenous redox partners

Demuez, Marie; Cournac, Laurent; Guerrini, O; Soucaille, Philippe; Girbal, Laurence

doi:10.1111/j.1574-6968.2007.00868.x

Cited by 74 publications

(61 citation statements)

References 42 publications

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“…Two Fe-only hydrogenases (HydA1 and HydA2) and one putative [NiFe] hydrogenase are encoded in its genome (Demuez et al, 2007;Gorwa et al, 1996). It has been shown previously that iron limitation leads to a significant reduction in hydrogenase activity (Junelles et al, 1988).…”

Section: Discussionmentioning

confidence: 99%

Effect of iron limitation and fur gene inactivation on the transcriptional profile of the strict anaerobe Clostridium acetobutylicum

et al. 2012

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Iron is a nutrient of critical importance for the strict anaerobe Clostridium acetobutylicum, as it is involved in numerous basic cellular functions and metabolic pathways. A gene encoding a putative ferric uptake regulator (Fur) has been identified in the genome of C. acetobutylicum. In this work, we inactivated the fur gene by using insertional mutagenesis. The resultant mutant showed a slow-growing phenotype and enhanced sensitivity to oxidative stress, but essentially no dramatic change in its fermentation pattern. A unique feature of its physiology was the overflowing production of riboflavin. To gain further insights into the role of the Fur protein and the mechanisms for establishment of iron balance in C. acetobutylicum, we characterized and compared the gene-expression profile of the fur mutant and the iron-limitation stimulon of the parental strain. Not surprisingly, a repertoire of iron-transport systems was upregulated in both microarray datasets, suggesting that they are regulated by Fur according to the availability of iron. In addition, iron limitation and inactivation of fur affected the expression of several genes involved in energy metabolism. Among them, two genes, encoding a lactate dehydrogenase and a flavodoxin, were highly induced. In order to support the function of the latter, the ribDBAH operon responsible for riboflavin biosynthesis was also upregulated significantly. Furthermore, the iron-starvation response of C. acetobutylicum involved transcriptional modifications that were not detected in the fur mutant, suggesting that there exist additional mechanisms for adaptation to low-iron environments. Collectively, these results demonstrate that the strict anaerobe C. acetobutylicum senses and responds to availability of iron on multiple levels using a sophisticated system, and that Fur plays an important role in this process.

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Section: Discussionmentioning

confidence: 99%

Effect of iron limitation and fur gene inactivation on the transcriptional profile of the strict anaerobe Clostridium acetobutylicum

et al. 2012

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“…Furthermore, it cannot be excluded at this point that the clostridial protein also serves a protective function in the hydrogen production of this organism. H 2 -evolving hydrogenase A1 (HydA1) of C. acetobutylicum is highly sensitive to oxygen-dependent inactivation (11,20). While low doses of molecular O 2 lead to irreversibly damaged enzymes, CO was previously shown to act as a competitive inhibitor that antagonizes O 2 and can greatly reduce the hydrogenase inactivation state (16,43).…”

Section: Discussionmentioning

confidence: 99%

The Role of PerR in O₂-Affected Gene Expression ofClostridium acetobutylicum

et al. 2009

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In the strict anaerobe Clostridium acetobutylicum, a PerR-homologous protein has recently been identified as being a key repressor of a reductive machinery for the scavenging of reactive oxygen species and molecular O 2 . In the absence of PerR, the full derepression of its regulon resulted in increased resistance to oxidative stress and nearly full tolerance of an aerobic environment. In the present study, the complementation of a Bacillus subtilis PerR mutant confirmed that the homologous protein from C. acetobutylicum acts as a functional peroxide sensor in vivo. Furthermore, we used a transcriptomic approach to analyze gene expression in the aerotolerant PerR mutant strain and compared it to the O 2 stimulon of wild-type C. acetobutylicum. The genes encoding the components of the alternative detoxification system were PerR regulated. Only few other targets of direct PerR regulation were identified, including two highly expressed genes encoding enzymes that are putatively involved in the central energy metabolism. All of them were highly induced when wild-type cells were exposed to sublethal levels of O 2 . Under these conditions, C. acetobutylicum also activated the repair and biogenesis of DNA and Fe-S clusters as well as the transcription of a gene encoding an unknown CO dehydrogenase-like enzyme. Surprisingly few genes were downregulated when exposed to O 2 , including those involved in butyrate formation. In summary, these results show that the defense of this strict anaerobe against oxidative stress is robust and by far not limited to the removal of O 2 and its reactive derivatives.

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“…The best studied is a soluble, monomeric hydrogenase (AAA23248) from C. pasteurianum with an M3 modular structure that is characteristic for all the proteins in the (Clostridium-specific) A2 cluster. These enzymes catalyse hydrogen production by transferring electrons from ferredoxins and flavodoxins to protons (Demuez et al, 2007). The hydrogenase-encoding gene is located downstream from another ORF which encodes a butyrate kinase.…”

Section: The Monomeric [Fefe] Hydrogenases Of Group Amentioning

confidence: 99%

The surprising diversity of clostridial hydrogenases: a comparative genomic perspective

et al. 2010

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Among the large variety of micro-organisms capable of fermentative hydrogen production, strict anaerobes such as members of the genus Clostridium are the most widely studied. They can produce hydrogen by a reversible reduction of protons accumulated during fermentation to dihydrogen, a reaction which is catalysed by hydrogenases. Sequenced genomes provide completely new insights into the diversity of clostridial hydrogenases. Building on previous reports, we found that [FeFe] hydrogenases are not a homogeneous group of enzymes, but exist in multiple forms with different modular structures and are especially abundant in members of the genus Clostridium. This unusual diversity seems to support the central role of hydrogenases in cell metabolism. In particular, the presence of multiple putative operons encoding multisubunit [FeFe] hydrogenases highlights the fact that hydrogen metabolism is very complex in this genus. In contrast with [FeFe] hydrogenases, their [NiFe] hydrogenase counterparts, widely represented in other bacteria and archaea, are found in only a few clostridial species. Surprisingly, a heteromultimeric Ech hydrogenase, known to be an energy-converting [NiFe] hydrogenase and previously described only in methanogenic archaea and some sulfur-reducing bacteria, was found to be encoded by the genomes of four cellulolytic strains: Clostridum cellulolyticum, Clostridum papyrosolvens, Clostridum thermocellum and Clostridum phytofermentans. IntroductionMolecular hydrogen is a key intermediate in the metabolic interactions of a wide range of micro-organisms. The main routes for biohydrogen production are photoproduction and dark fermentation with the latter providing higher rates of gas evolution without external energy requirements and the possibility of converting a wide range of biomassbased substrates into hydrogen. Among a large variety of micro-organisms capable of fermentative hydrogen production, strict anaerobes such as members of the genus Clostridium are the most widely studied (Levin et al., 2004). Clostridia are the dominant micro-organisms in mixed microaerophilic communities capable of hydrogen production from biomass waste treatment. They can produce hydrogen by butyric and mixed acid fermentations at optimal pH values ranging from 4.5 to 5.5 (Fang & Liu, 2002). While fermentative conditions, such as substrate type, pH, hydraulic and solid retention time, H 2 partial pressure and the concentration of acids produced, have been extensively studied and optimized (Li & Fang, 2007;Van Ginkel et al., 2005;Khanal et al., 2004), relatively little is known about the different forms of hydrogenases present in clostridia.Three classes of enzymes are capable of hydrogen production: nitrogenases (Masukawa et al., 2002), alkaline phosphatases (Yang & Metcalf, 2004) and hydrogenases (Heinekey, 2009;Meyer, 2007;Vignais & Colbeau, 2004;Vignais et al., 2001). However, owing to their highly reactive and complex metallocenters, hydrogenases are regarded as the most efficient with turnover rates 1000 times hig...

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Complete activity profile ofClostridium acetobutylicum[FeFe]-hydrogenase and kinetic parameters for endogenous redox partners

Cited by 74 publications

References 42 publications

Effect of iron limitation and fur gene inactivation on the transcriptional profile of the strict anaerobe Clostridium acetobutylicum

Effect of iron limitation and fur gene inactivation on the transcriptional profile of the strict anaerobe Clostridium acetobutylicum

The Role of PerR in O₂-Affected Gene Expression ofClostridium acetobutylicum

The surprising diversity of clostridial hydrogenases: a comparative genomic perspective

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Complete activity profile ofClostridium acetobutylicum[FeFe]-hydrogenase and kinetic parameters for endogenous redox partners

Cited by 74 publications

References 42 publications

Effect of iron limitation and fur gene inactivation on the transcriptional profile of the strict anaerobe Clostridium acetobutylicum

Effect of iron limitation and fur gene inactivation on the transcriptional profile of the strict anaerobe Clostridium acetobutylicum

The Role of PerR in O2-Affected Gene Expression ofClostridium acetobutylicum

The surprising diversity of clostridial hydrogenases: a comparative genomic perspective

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The Role of PerR in O₂-Affected Gene Expression ofClostridium acetobutylicum