Papers by Ralf-jörg Fischer
Small acid-soluble spore proteins of Clostridium acetobutylicum are able to protect DNA in vitro and are specifically cleaved by germination protease GPR and spore protease YyaC
Microbiology, 2015
Small acid-soluble proteins (SASP) play an important role in protection of DNA in dormant bacteri... more Small acid-soluble proteins (SASP) play an important role in protection of DNA in dormant bacterial endospores against damage by heat, UV-radiation or enzymatic degradation. In the genome of the strict anaerobe Clostridium acetobutylicum five genes encoding for SASP have been annotated and here a further sixth candidate is suggested. The ssp-genes are expressed in parallel dependent upon Spo0A, a master regulator of sporulation. Analysis of the transcription start points revealed a G or a F consensus promoter upstream of each ssp-gene confirming a forespore-specific gene expression. SASP were termed SspA (Cac2365), SspB (Cac1522), SspD (Cac1620), SspF (Cac2372), SspH (Cac1663), and Tlp (Cac1487). Here we show that with the exception of Tlp, every purified recombinant SASP is able to bind DNA in vitro thereby protecting it against enzymatic degradation by DNase I. Moreover, SspB and SspD were specifically cleaved by the two germination specific proteases GPR (Cac1275) and YyaC (Cac2857), which were overexpressed in Escherichia coli and activated by an autocleavage reaction. Thus, for the first time GPR-like activity and SASP specificity could be demonstrated for an YyaC-like protein. Collectively, our results assign SspA, SspB, SspD, SspF and SspH of C. acetobutylicum as members of α/β-type SASP, whereas Tlp seems to be a non DNA-binding spore protein of unknown function. In acidic acid extracted proteins of dormant spores of C. acetobutylicum, SspA was identified almost exclusively, indicating its dominant biological role as major α/β-type SASP in vivo.
Molecular Microbiology, 2008
Clostridia belong to those bacteria which are considered as obligate anaerobe, e.g. oxygen is har... more Clostridia belong to those bacteria which are considered as obligate anaerobe, e.g. oxygen is harmful or lethal to these bacteria. Nevertheless, it is known that they can survive limited exposure to air, and often eliminate oxygen or reactive derivatives via NAD(P)Hdependent reduction. This system does apparently contribute to survival after oxidative stress, but is insufficient to establish long-term tolerance of aerobic conditions. Here we show that manipulation of the regulatory mechanism of this defence mechanism can trigger aerotolerance in the obligate anaerobe Clostridium acetobutylicum. Deletion of a peroxide repressor (PerR)-homologous protein resulted in prolonged aerotolerance, limited growth under aerobic conditions and rapid consumption of oxygen from an aerobic environment. The mutant strain also revealed higher resistance to H 2O2 and activities of NADH-dependent scavenging of H2O2 and organic peroxides in cell-free extracts increased by at least one order of magnitude. Several genes encoding the putative enzymes were upregulated and identified as members of the clostridial PerR regulon, including the heat shock protein Hsp21, a reverse rubrerythrin which was massively produced and became the most abundant protein in the absence of PerR. This multifunctional protein is proposed to play the crucial role in the oxidative stress defence.
Identification of two genes encoding putative new members of the ECF subfamily of eubacterial RNA polymerase sigma factors in Clostridium acetobutylicum
Journal of molecular microbiology and biotechnology, 2000
Two genes from Clostridium acetobutylicum DSM 792 were identified which are predicted to encode n... more Two genes from Clostridium acetobutylicum DSM 792 were identified which are predicted to encode new members of the ECF subfamily of eubacterial RNA polymerase sigma factors. The sigX gene has the potential to encode a 184-amino acid protein with a molecular mass of 21,870 Da and with the highest overall similarity to Fecl of Escherichia coli (27 % identical residues). The second gene, which is predicted to encode an alternative sigma factor of the ECF subfamily, is the previously described orf2 gene (Gerischer and Dürre, 1990) located in the adc gene region of C. acetobutylicum. The deduced protein of orf2 has significant similarity to SigX of C. acetobutylicum (22 % identical residues) and shares structural features with other alternative sigma factors. Therefore, it is proposed to rename orf2 as sigY. Analysis of the phylogenetic relationship revealed that SigX from C. acetobutylicum, together with sigmaE from Streptomyces coelicolor and SigX from Bacillus subtilis, form a gram-po...
Identification of PTSFru as the major fructose uptake system of Clostridium acetobutylicum
Applied Microbiology and Biotechnology, 2014
As a member of the saccharolytic clostridia, a variety of different carbohydrates like glucose, f... more As a member of the saccharolytic clostridia, a variety of different carbohydrates like glucose, fructose, or mannose can be used as carbon and energy source by Clostridium acetobutylicum ATCC 824. Thirteen phosphoenolpyruvate-dependent phosphotransferase systems (PTS) have been identified in C. acetobutylicum, which are likely to be responsible for the uptake of hexoses, hexitols, or disaccharides. Here, we focus on three PTS which are expected to be involved in the uptake of fructose, PTS(Fru), PTS(ManI), and PTS(ManII). To analyze their individual functions, each PTS was inactivated via homologous recombination or insertional mutagenesis. Standardized comparative batch fermentations in a synthetic medium with glucose, fructose, or mannose as sole carbon source identified PTS(Fru) as primary uptake system for fructose, whereas growth with fructose was not impaired in PTS(ManI) and slightly altered in PTS(ManII)-deficient strains of C. acetobutylicum. The inactivation of PTS(ManI) resulted in slower growth on mannose whereas the loss of PTS(ManII) revealed no phenotype during growth on mannose. This is the first time that it has been shown that PTS(Fru) and PTS(ManI) of C. acetobutylicum are directly involved in fructose and mannose uptake, respectively. Moreover, comprehensive comparison of the fermentation products revealed that the loss of PTS(Fru) prevents the solvent shift as no butanol and only basic levels of acetone and ethanol could be determined.
Microbiology, 2009
An unusual non-haem diiron protein, reverse rubrerythrin (revRbr), is known to be massively upreg... more An unusual non-haem diiron protein, reverse rubrerythrin (revRbr), is known to be massively upregulated in response to oxidative stress in the strictly anaerobic bacterium Clostridium acetobutylicum. In the present study both in vivo and in vitro results demonstrate an H 2 O 2 and O 2 detoxification pathway in C. acetobutylicum involving revRbr, rubredoxin (Rd) and NADH : rubredoxin oxidoreductase (NROR). RevRbr exhibited both NADH peroxidase (NADH : H 2 O 2 oxidoreductase) and NADH oxidase (NADH : O 2 oxidoreductase) activities in in vitro assays using NROR as the electron-transfer intermediary from NADH to revRbr. Rd increased the NADH consumption rate by serving as an intermediary electron-transfer shuttle between NROR and revRbr. While H 2 O 2 was found to be the preferred substrate for revRbr, its relative oxidase activity was found to be significantly higher than that reported for other Rbrs. A revRbr-overexpressing strain of C. acetobutylicum showed significantly increased tolerance to H 2 O 2 and O 2 exposure. RevRbr thus appears to protect C. acetobutylicum against oxidative stress by functioning as the terminal component of an NADH peroxidase and NADH oxidase.
A modified pathway for the production of acetone in Escherichia coli
Metabolic Engineering, 2013
A modified synthetic acetone operon was constructed. It consists of two genes from Clostridium ac... more A modified synthetic acetone operon was constructed. It consists of two genes from Clostridium acetobutylicum (thlA coding for thiolase and adc coding for acetoacetate decarboxylase) and one from Bacillus subtilis or Haemophilus influenzae (teII(srf) or ybgC, respectively, for thioesterase). Expression of this operon in Escherichia coli resulted in the production of acetone starting from the common metabolite acetyl-CoA via acetoacetyl-CoA and acetoacetate. The thioesterases do not need a CoA acceptor for acetoacetyl-CoA hydrolysis. Thus, in contrast to the classic acetone pathway of Clostridium acetobutylicum and related microorganisms which employ a CoA transferase, the new pathway is acetate independent. The genetic background of the host strains was crucial. Only E. coli strains HB101 and WL3 were able to produce acetone via the modified plasmid based pathway, up to 64mM and 42mM in 5-ml cultures, respectively. Using glucose fed-batch cultures the concentration could be increased up to 122mM acetone with HB101 carrying the recombinant plasmid pUC19ayt (thioesterase from H. influenzae). The formation of acetone led to a decreased acetate production by E. coli.
Genome-Wide Gene Expression Analysis of the Switch between Acidogenesis and Solventogenesis in Continuous Cultures of Clostridium acetobutylicum
Journal of Molecular Microbiology and Biotechnology, 2011
Clostridium acetobutylicum is able to switch from acidogenic growth to solventogenic growth. We u... more Clostridium acetobutylicum is able to switch from acidogenic growth to solventogenic growth. We used phosphate-limited continuous cultures that established acidogenic growth at pH 5.8 and solventogenic growth at pH 4.5. These cultures allowed a detailed transcriptomic study of the switch from acidogenesis to solventogenesis that is not superimposed by sporulation and other growth phase-dependent parameters. These experiments led to new insights into the physiological role of several genes involved in solvent formation. The adc gene for acetone decarboxylase is upregulated well before the rest of the sol locus during the switch, and pyruvate decarboxylase is induced exclusively for the period of this switch. The aldehyde-alcohol dehydrogenase gene adhE1 located in the sol operon is regulated antagonistically to the paralog adhE2 that is expressed during acidogenic conditions. A similar antagonistic pattern can be seen with the two paralogs of thiolase genes, thlA and thlB. Interestingly, the genes coding for the putative cellulosome in C. acetobutylicum are exclusively transcribed throughout solventogenic growth. The genes for stress response are only induced during the shift but not in the course of solventogenesis when butanol is present in the culture. Finally, the data clearly indicate that solventogenesis is independent from sporulation.
Journal of Microbiological Methods, 2007
We report on the development of a Standard Operating Procedure (SOP) for extraction and handling ... more We report on the development of a Standard Operating Procedure (SOP) for extraction and handling of intra- and extracellular protein fractions of Clostridium acetobutylicum ATCC 824 for reproducible high quality two-dimensional gel electrophoresis (2-DE) analyses. Standardized cells from a phosphate-limited chemostat were used to evaluate different protein preparation methods. For the preparation of the secretome, a dialysis/ultrafiltration procedure resulted in higher protein yields and proved to be more reliable compared to different precipitation methods using TCA, DOC-TCA, acetone, and PEG 6000. Sonication was found to be the most efficient method among different tested techniques of cell disruption for the analysis of the intracellular proteome. Furthermore, the effect of protease inhibitors and sample storage conditions were tested for both intra- and extracellular protein samples. Significant changes in the protein pattern were observed depending on the addition of protease inhibitors. 2-DE gels with a pH gradient from 4 to 7 prepared according to the developed SOP contained at least 736 intracellular and 324 extracellular protein spots.
Journal of Bacteriology, 2006
The pst operon of Clostridium acetobutylicum ATCC 824 comprises five genes, pstS, pstC, pstA, pst... more The pst operon of Clostridium acetobutylicum ATCC 824 comprises five genes, pstS, pstC, pstA, pstB, and phoU, and shows a gene architecture identical to that of Escherichia coli. Deduced proteins are predicted to represent a high-affinity phosphate-specific ABC (ATP-binding cassette) transport system (Pst) and a protein homologous to PhoU, a negative phosphate regulon regulator. We analyzed the expression patterns of the pst operon in P i -limited chemostat cultures during acid production at pH 5.8 or solvent production at pH 4.5 and in response to P i pulses. Specific mRNA transcripts were found only when external P i concentrations had dropped below 0.2 mM. Two specific transcripts were detected, a 4.7-kb polycistronic mRNA spanning the whole operon and a quantitatively dominating 1.2-kb mRNA representing the first gene, pstS. The mRNA levels clearly differed depending on the external pH. The amounts of the full-length mRNA detected were about two times higher at pH 5.8 than at pH 4.5. The level of pstS mRNA increased by a factor of at least 8 at pH 5.8 compared to pH 4.5 results. Primer extension experiments revealed only one putative transcription start point 80 nucleotides upstream of pstS. Thus, additional regulatory sites are proposed in the promoter region, integrating two different extracellular signals, namely, depletion of inorganic phosphate and the pH of the environment. After phosphate pulses were applied to a phosphate-limited chemostat we observed faster phosphate consumption at pH 5.8 than at pH 4.5, although higher optical densities were recorded at pH 4.5.
FEMS Microbiology Reviews, 1995
The enzymes acetoacetate decarboxylase and coenzyme A transferase catalyse acetone production fro... more The enzymes acetoacetate decarboxylase and coenzyme A transferase catalyse acetone production from acetoacetyl-CoA in Clostridium acetobutylicum. The adc gene encoding the former enzyme is organized in a monocistronic operon, while the ctf genes form a common transcription unit with the gene (adhE) encoding a probable polyfunctional aldehyde/alcohol dehydrogenase. This genetic arrangement could reflect physiological requirements at the onset of solventogenesis. In addition to AdhE, two butanol dehydrogenase isozymes and a thiolase are involved in butanol synthesis. RNA analyses showed a sequential order of induction for the different butanol dehydrogenase genes, indicating an in vivo function of Bdhl in low level butanol formation. The physiological roles of AdhE and BdhlI most likely involve high level butanol formation, with AdhE being responsible for the onset of solventogenesis and BdhlI ensuring continued butanol production. Addition of methyl viologen results in artificially induced butanol synthesis which seems to be mediated by a still unknown set of enzymes. Although the signal that triggers the shift to solventogenesis has not yet been elucidated, recent investigations suggest a possible function of DNA supercoiling as a transcriptional sensor of the respective environmental stimuli.
FEMS Microbiology Letters, 2004
Comparison of the N-terminus of the heat shock protein Hsp21 of Clostridium acetobutylicum with p... more Comparison of the N-terminus of the heat shock protein Hsp21 of Clostridium acetobutylicum with proteins predicted to be encoded by the genome of this bacterium revealed that this stress protein is encoded by two almost identical open reading frames CAC3597 and CAC3598. These genes encode a rubrerythrin-like protein with the rubredoxin-like FeS 4 domain at the N-terminus and the ferritin-like diiron domain (rubrerythrin domain) at the C-terminus. Thus, the order of the two putative functional domains is reversed compared to ''normal'' rubrerythrins. This protein is proposed to be involved in the oxidative stress response of strict anaerobic bacteria. Northern blot analysis indicated that hsp21 is induced by heat and oxidative stress (air, H 2 O 2 ). Hsp21 of C. acetobutylicum can be considered as a ''reverse'' rubrerythrin and a role of this stress protein, which is conserved among clostridia and other strict anaerobic bacteria, in the heat and oxidative stress response is proposed.
Desulfoferrodoxin of Clostridium acetobutylicum functions as a superoxide reductase
FEBS Letters, 2007
Desulfoferrodoxin (cac2450) of Clostridium acetobutylicum was purified after overexpression in E.... more Desulfoferrodoxin (cac2450) of Clostridium acetobutylicum was purified after overexpression in E. coli. In an in vitro assay the enzyme exhibited superoxide reductase activity with rubredoxin (cac2778) of C. acetobutylicum as the proximal electron donor. Rubredoxin was reduced by ferredoxin:NADP(+) reductase from spinach and NADPH. The superoxide anions, generated from dissolved oxygen using Xanthine and Xanthine oxidase, were reduced to hydrogen peroxide. Thus, we assume that desulfoferrodoxin is the key factor in the superoxide reductase dependent part of an alternative pathway for detoxification of reactive oxygen species in this obligate anaerobic bacterium.
BMC Systems Biology, 2011
Background: Clostridium acetobutylicum is an anaerobic bacterium which is known for its solvent-p... more Background: Clostridium acetobutylicum is an anaerobic bacterium which is known for its solvent-producing capabilities, namely regarding the bulk chemicals acetone and butanol, the latter being a highly efficient biofuel. For butanol production by C. acetobutylicum to be optimized and exploited on an industrial scale, the effect of pH-induced gene regulation on solvent production by C. acetobutylicum in continuous culture must be understood as fully as possible. Results: We present an ordinary differential equation model combining the metabolic network governing solvent production with regulation at the genetic level of the enzymes required for this process. Parameterizing the model with experimental data from continuous culture, we demonstrate the influence of pH upon fermentation products: at high pH (pH 5.7) acids are the dominant product while at low pH (pH 4.5) this switches to solvents. Through steady-state analyses of the model we focus our investigations on how alteration in gene expression of C. acetobutylicum could be exploited to increase butanol yield in a continuous culture fermentation. Conclusions: Incorporating gene regulation into the model of solvent production by C. acetobutylicum enables an accurate representation of the pH-induced switch to solvent production to be obtained and theoretical investigations of possible synthetic-biology approaches to be pursued. Steady-state analyses suggest that, to increase butanol yield, alterations in the expression of single solvent-associated genes are insufficient; a more complex approach targeting two or more genes is required.
Archives of Microbiology, 2006
The small heat shock protein Hsp21 of Clostridium acetobutylicum was recently identified as a rub... more The small heat shock protein Hsp21 of Clostridium acetobutylicum was recently identified as a rubrerythrin-like protein with a rubredoxin-like FeS 4 domain at the N-terminus and a ferritin-like diiron domain at the C-terminus. Here, we report that the two identical tandem genes rbr3A and rbr3B, which encode the heat shock protein Hsp21, show the transcription pattern of general stress genes. Northern blot analysis indicated that the transcription of the rbr3AB operon is induced by various environmental stress conditions: in addition to heat and oxidative stress, an increase of the pH of the growth medium from 4.5 to 6.2, addition of the salt NaCl (400 mM) or of the solvent butanol (3.5% v/v), and lowering the incubation temperature from 37 to 25°C resulted in transiently increased transcript levels. The promoter region deduced from the 5¢ end of the mRNA has only limited similarity to the consensus promoter sequence of Gram-positive bacteria. A conserved inverted repeat between this promoter and the initiation codon is proposed to have a regulatory role. Although C. acetobutylicum is regarded as a strictly anaerobic bacterium, live/dead staining demonstrated that it can survive exposure to air or H 2 O 2 and other stressors to various extents.
Mutagenesis of conserved charged amino acids in SLH domains of Thermoanaerobacterium thermosulfurigenes EM1 affects attachment to cell wall sacculi
Archives of Microbiology, 2006
SLH domains (for surface layer homology) are involved in the attachment of proteins to bacterial ... more SLH domains (for surface layer homology) are involved in the attachment of proteins to bacterial cell walls. The data presented here assign the conserved TRAE motif within SLH domains a key role for the binding. The charged amino acids arginine (R) or/and glutamic acid (E) were replaced via site-directed mutagenesis by different amino acids. Effects were visualized in an in vitro binding assay using native cell wall sacculi of Thermoanaerobacterium thermosulfurigenes EM1 and different variants of an SLH protein which consisted of the triplicate SLH domain of xylanase XynA of this bacterium and which was purified after expression in Escherichia coli. The results indicated (1) that the TRAE motif is critical for the binding function of SLH domains, (2) that a functional TRAE motif is necessary in all three domains, (3) that a least one (preferentially positively) charged amino acid in the TRAE motif is required for the functionality of the SLH domain, and (4) that the position of the negatively and positively charged amino acids is important. The finding that the cell wall of T. thermosulfurigenes EM1 contains pyruvate (4 microg mg(-1)) is in agreement with the hypothesis that pyruvylated secondary cell wall polymers function as ligand for SLH domains.
Applied Microbiology and Biotechnology, 2010
The complex changes in the life cycle of Clostridium acetobutylicum, a promising biofuel producer... more The complex changes in the life cycle of Clostridium acetobutylicum, a promising biofuel producer, are not well understood. During exponential growth, sugars are fermented to acetate and butyrate, and in the transition phase, the metabolism switches to the production of the solvents acetone and butanol accompanied by the initiation of endospore formation. Using phosphate-limited chemostat cultures at pH 5.7, C. acetobutylicum was kept at a steady state of acidogenic metabolism, whereas at pH 4.5, the cells showed stable solvent production without sporulation. Novel proteome reference maps of cytosolic proteins from both acidogenesis and solventogenesis with a high degree of reproducibility were generated. Yielding a 21% coverage, 15 protein spots were specifically assigned to the acidogenic phase, and 29 protein spots exhibited a significantly higher abundance in the solventogenic phase. Besides well-known metabolic proteins, unexpected proteins were also identified. Among these, the two proteins CAP0036 and CAP0037 of unknown function were found as major striking indicator proteins in acidogenic cells. Proteome data were confirmed by genome-wide DNA microarray analyses of the identical cultures. Thus, a first systematic study of acidogenic and solventogenic chemostat cultures is presented, and similarities as well as differences to previous studies of batch cultures are discussed.
A transcriptional study of acidogenic chemostat cells of Clostridium acetobutylicum—Solvent stress caused by a transient n-butanol pulse
Journal of Biotechnology, 2012
The main product of the anaerobic fermentative bacterium Clostridium acetobutylicum is n-butanol,... more The main product of the anaerobic fermentative bacterium Clostridium acetobutylicum is n-butanol, an organic solvent with severe toxic effects on the cells. Therefore, the identification of the molecular factors related to n-butanol stress constitutes a major strategy for furthering the understanding of the biotechnological production of n-butanol, an important industrial biofuel. Previous reports concerning n-butanol stress in C. acetobutylicum dealt exclusively with batch cultures. In this study, for the first time a comprehensive transcriptional analysis of n-butanol-stressed C. acetobutylicum was conducted using stable steady state acidogenic chemostat cultures. A total of 358 differentially expressed genes were significantly affected by n-butanol stress. Similarities, such as the upregulation of general stress genes, and differences in gene expression were compared in detail with earlier DNA microarrays performed in batch cultivation experiments. The main result of this analysis was the observation that genes involved in amino acid and nucleotide biosynthesis, as well as genes for specific transport systems were upregulated by n-butanol. Our results exclude any transcriptional response triggered by exogenous pH changes or solventogenic n-butanol formation. Finally, our data suggest that metabolic flux through the glycerolipid biosynthetic pathway increases, confirming that C. acetobutylicum modifies the cytoplasmic membrane composition in response to n-butanol stress.
Uploads
Papers by Ralf-jörg Fischer