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ABSTRACT: 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.
Microbiology 05/2012; 158(Pt 7):1918-29. · 3.06 Impact Factor
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ABSTRACT: 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.
Journal of biotechnology 04/2012; 161(3):354-65. · 2.88 Impact Factor
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ABSTRACT: To gain more insight into the butanol stress response of Clostridium acetobutylicum the transcriptional response of a steady state acidogenic culture to different levels of n-butanol (0.25-1%) was investigated. No effect was observed on the fermentation pattern and expression of typical solvent genes (aad, ctfA/B, adc, bdhA/B, ptb, buk). Elevated levels of butanol mainly affected class I heat-shock genes (hrcA, grpE, dnaK, dnaJ, groES, groEL, hsp90), which were upregulated in a dose- and time-dependent manner, and genes encoding proteins involved in the membrane composition (fab and fad or glycerophospholipid related genes) and various ABC-transporters of unknown specificity. Interestingly, fab and fad genes were embedded in a large, entirely repressed cluster (CAC1988-CAC2019), which inter alia encoded an iron-specific ABC-transporter and molybdenum-cofactor synthesis proteins. Of the glycerophospholipid metabolism, the glycerol-3-phosphate dehydrogenase (glpA) gene was highly upregulated, whereas a glycerophosphodiester ABC-transporter (ugpAEBC) and a phosphodiesterase (ugpC) were repressed. On the megaplasmid, only a few genes showed differential expression, e.g. a rare lipoprotein (CAP0058, repressed) and a membrane protein (CAP0102, upregulated) gene. Observed transcriptional responses suggest that C. acetobutylicum reacts to butanol stress by induction of the general stress response and changing its cell envelope and transporter composition, but leaving the central catabolism unaffected.
Journal of biotechnology 03/2012; 161(3):366-77. · 2.88 Impact Factor
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ABSTRACT: Artificial electron carriers have been widely used to shift the solvent ratio toward butanol in acetone-butanol-ethanol (ABE) fermentation of solventogenic clostridia according to decreased hydrogen production. In this study, first insights on the molecular level were gained to explore the effect of methyl viologen addition to cultures of Clostridium acetobutylicum. Employing batch fermentation in mineral salts medium, the butanol:acetone ratio was successively increased from 2.3 to 12.4 on a 100-ml scale in serum bottles and from 1.4 to 16.5 on a 1300-ml scale in bioreactors, respectively. The latter cultures were used for DNA microarray analyses to provide new information on the transcriptional changes referring to methyl viologen exposure and thus, exhibit gene expression patterns according to the manipulation of the cellular redox balance. Methyl viologen-exposed cultures revealed lower expression levels of the sol operon (CAP0162-0164) and the adjacent adc gene (CAP0165) responsible for solvent formation as well as iron and sulfate transporters and the CAC0105-encoded ferredoxin. On the contrary, genes for riboflavin biosynthesis, for the butyrate/butanol metabolic pathway and genes coding for sugar transport systems were induced. Interestingly, the adhE2-encoded bifunctional NADH-dependent aldhehyde/alcohol-dehydrogenase (CAP0035) was upregulated up to more than 100-fold expression levels as compared to the control culture without methyl viologen addition. The data presented here indicate a transcriptional regulation for decreased acetone biosynthesis and the redox-dependent substitution of adhE1 (CAP0162) by adhE2.
New Biotechnology 01/2012; 29(4):485-93. · 2.76 Impact Factor
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ABSTRACT: 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 Molecular Microbiology and Biotechnology 01/2011; 20(1):1-15. · 1.95 Impact Factor
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ABSTRACT: By taking advantage of the available genome sequence of Ralstonia eutropha H16, glucose uptake in the UV-generated glucose-utilizing mutant R. eutropha G(+)1 was investigated by transcriptomic and proteomic analyses. Data revealed clear evidence that glucose is transported by a usually N-acetylglucosamine-specific phosphotransferase system (PTS)-type transport system, which in this mutant is probably overexpressed due to a derepression of the encoding nag operon by an identified insertion mutation in gene H16_A0310 (nagR). Furthermore, a missense mutation in nagE (membrane component EIICB), which yields a substitution of an alanine by threonine in NagE and may additionally increase glucose uptake, was identified. Phosphorylation of glucose is subsequently mediated by NagF (cytosolic PTS component EIIA-HPr-EI) or glucokinase (GlK), respectively. The inability of the defined deletion mutant R. eutropha G(+)1 ΔnagFEC to utilize glucose strongly confirms this finding. In addition, secondary effects of glucose, which is now intracellularly available as a carbon source, on the metabolism of the mutant cells in the stationary growth phase occurred: intracellular glucose degradation is stimulated by the stronger expression of enzymes involved in the 2-keto-3-deoxygluconate 6-phosphate (KDPG) pathway and in subsequent reactions yielding pyruvate. The intermediate phosphoenolpyruvate (PEP) in turn supports further glucose uptake by the Nag PTS. Pyruvate is then decarboxylated by the pyruvate dehydrogenase multienzyme complex to acetyl coenzyme A (acetyl-CoA), which is directed to poly(3-hydroxybutyrate). The polyester is then synthesized to a greater extent, as also indicated by the upregulation of various enzymes of poly-β-hydroxybutyrate (PHB) metabolism. The larger amounts of NADPH required for PHB synthesis are delivered by significantly increased quantities of proton-translocating NAD(P) transhydrogenases. The current study successfully combined transcriptomic and proteomic investigations to unravel the phenotype of this hitherto-undefined glucose-utilizing mutant.
Applied and environmental microbiology 01/2011; 77(6):2058-70. · 3.69 Impact Factor
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ABSTRACT: In this study, we have investigated the transcriptome of Ralstonia eutropha H16 during cultivation with gluconate in presence of 3,3'-thiodipropionic acid (TDP) or 3,3'-dithiodipropionic acid (DTDP) during biosynthesis of poly(3-hydroxybutyrate-co-3-mercaptopropionate). Genome-wide transcriptome analyses revealed several genes which were upregulated during cultivation in presence of the above-mentioned compounds. Obtained data strongly suggest that two ABC-type transport system and three probable extracytoplasmic solute receptors mediate the uptake of TDP and DTDP, respectively. In addition, genes encoding the hydrolase S-adenosylhomocysteinase AhcY and the thiol-disulfide interchange proteins DsbA, DsbD, and FrnE were upregulated during cultivation on DTDP and, in case of AhcY and FrnE, on TDP as well. It is assumed that the corresponding enzymes are involved in the cleavage of TDP and DTDP. Several genes of the fatty acid metabolism exhibited increased expression levels: genes encoding two acetyltransferases, a predicted acyltransferase, the acetoacetyl-CoA reductase phaB3, an enoyl-CoA hydratase as well as an acyl dehydratase, an acetyl-CoA synthetase, two acyl-CoA dehydrogenases, the methylmalonyl-CoA mutase encoded by sbm1 and sbm2 and phaY1 were detected. Furthermore, ORF H16_A0217 encoding a hypothetical protein and exhibiting 54% amino acids identical to an acyl-CoA thioesterase from Saccharomonospora viridis was found to be highly upregulated. As the 2-methylcitrate synthase PrpC exhibited a three- to fourfold increased activity in cells grown in presence of TDP or DTDP as compared to gluconate, metabolization of the cleavage products 3MP and 3-hydroxypropionate to propionyl-CoA is proposed.
Applied Microbiology and Biotechnology 10/2010; 88(5):1145-59. · 3.42 Impact Factor
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ABSTRACT: Clostridium acetobutylicum is a strict anaerobic organism that is used for biotechnological butanol fermentation. It ferments various hexoses and pentoses to solvents but prefers glucose presumably using a catabolite repression mechanism. Accordingly during growth on a mixture of D-glucose and D-xylose a typical diauxic growth pattern was observed. We used DNA microarrays and real-time RT-PCR to study gene expression during growth on D-glucose, D-xylose mixtures on a defined minimal medium together with monitoring substrate consumption and product formation. We identified two putative operons involved in D-xylose degradation. The first operon (CAC1344-CAC1349) includes a transporter, a xylulose-kinase, a transaldolase, a transketolase, an aldose-1-epimerase and a putative xylose isomerase that has been annotated as an arabinose isomerase. This operon is induced by D-xylose but was catabolite repressed by D-glucose. A second operon (CAC2610-CAC2612) consists of a xylulose-kinase, a hypothetical protein and a gene that has been annotated as a L-fucose isomerase that might in fact code for a xylose isomerase. This operon was induced by D-xylose but was not subject to catabolite repression. In accordance with these results we identified a CRE site in the catabolite repressed operon but not in the operon that was not subject to catabolite repression.
Journal of biotechnology 09/2010; 150(3):315-23. · 2.88 Impact Factor
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Trung Nguyen Thanh,
Britta Jürgen,
Melanie Bauch,
Manuel Liebeke,
Michael Lalk, Armin Ehrenreich,
Stefan Evers,
Karl-Heinz Maurer,
Haike Antelmann,
Florian Ernst,
Georg Homuth,
Michael Hecker,
Thomas Schweder
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ABSTRACT: The acoABCL and acuABC operons of Bacillus licheniformis DSM13 are strongly induced at the transcriptional level during glucose starvation conditions. Primer extension analyses of this study indicate that the acoABCL operon is controlled by a sigmaL-dependent promoter and the acuABC operon by a sigmaA-dependent promoter. Transcription at the acoA promoter is repressed by glucose but induced by acetoin as soon as the preferred carbon source glucose is exhausted. The acuA promoter shows a similar induction pattern, but its activity is independent from the presence of acetoin. It is demonstrated that the acoABCL operon is mainly responsible for acetoin and 2,3-butanediol degradation in B. licheniformis.
Applied Microbiology and Biotechnology 08/2010; 87(6):2227-35. · 3.42 Impact Factor
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ABSTRACT: The genetic manageability of the biotechnologically important Bacillus licheniformis is hampered due to its poor transformability, whereas Bacillus subtilis efficiently takes up DNA during genetic competence, a quorum-sensing-dependent process. Since the sensor histidine kinase ComP, encoded by a gene of the quorum-sensing module comQXPA of B. licheniformis DSM13, was found to be inactive due to an insertion element within comP, the coding region was exchanged with a functional copy. Quorum sensing was restored, but the already-poor genetic competence dropped further. The inducible expression of the key regulator for the transcription of competence genes, ComK, in trans resulted in highly competent strains and facilitated the direct disruption of genes, as well as the conditional knockout of an essential operon. As ComK is inhibited at low cell densities by a proteolytic complex in which MecA binds ComK and such inhibition is antagonized by the interaction of MecA with ComS (the expression of the latter is controlled by cell density in B. subtilis), we performed an in silico analysis of MecA and the hitherto unidentified ComS, which revealed differences for competent and noncompetent strains, indicating that the reduced competence possibly is due to a nonfunctional coupling of the comQXPA-encoded quorum module and ComK. The obtained increased genetic tractability of this industrial workhorse should improve a wide array of scientific investigations.
Applied and environmental microbiology 08/2010; 76(15):5046-57. · 3.69 Impact Factor
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ABSTRACT: beta-Ketothiolases catalyze the first step of poly(3-hydroxybutyrate) [poly(3HB)] synthesis in bacteria by condensing two molecules of acetyl coenzyme A (acetyl-CoA) to acetoacetyl-CoA. Analyses of the genome sequence of Ralstonia eutropha H16 revealed 15 isoenzymes of PhaA in this bacterium. In this study, we generated knockout mutants of various phaA homologues to investigate their role in and contributions to poly(3HB) metabolism and to suppress biosynthesis of 3HB-CoA for obtaining enhanced molar 3-mercaptopriopionate (3MP) contents in poly(3HB-co-3MP) copolymers when cells were grown on gluconate plus 3-mercaptopropionate or 3,3'-dithiodipropionate. In silico sequence analysis of PhaA homologues, transcriptome data, and other aspects recommended the homologues phaA, bktB, H16_A1713/H16_B1771, H16_A1528, H16_B1369, H16_B0381, and H16_A0170 for further analysis. Single- and multiple-deletion mutants were generated to investigate the influence of these beta-ketothiolases on growth and polymer accumulation. The deletion of single genes resulted in no significant differences from the wild type regarding growth and polymer accumulation during cultivation on gluconate or gluconate plus 3MP. Deletion of phaA plus bktB (H16Delta2 mutant) resulted in approximately 30% less polymer accumulation than in the wild type. Deletion of H16_A1713/H16_B1771, H16_A1528, H16_B0381, and H16_B1369 in addition to phaA and bktB gave no differences in comparison to the H16Delta2 mutant. In contrast, deletion of H16_A0170 additionally to phaA and bktB yielded a mutant which accumulated about 30% poly(3HB) (wt/wt of the cell dry weight [CDW]). Although we were not able to suppress poly(3HB) biosynthesis completely, the copolymer compositions could be altered significantly with a lowered percentage ratio of 3HB constituents (from 85 to 52 mol%) and an increased percentage ratio of 3MP constituents (from 15 to 48 mol%), respectively. In this study, we demonstrated that PhaA, BktB, and H16_A0170 are majorly involved in poly(3HB) synthesis in R. eutropha H16. A fourth beta-ketothiolase or a combination of several of the other beta-ketothiolases contributed to a maximum of only 30% (wt/wt of CDW) of the remaining (co)polymer.
Applied and environmental microbiology 08/2010; 76(16):5373-82. · 3.69 Impact Factor
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ABSTRACT: 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.
Applied Microbiology and Biotechnology 08/2010; 87(6):2209-26. · 3.42 Impact Factor
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ABSTRACT: Clostridium ljungdahlii is an anaerobic homoacetogen, able to ferment sugars, other organic compounds, or CO(2)/H(2) and synthesis gas (CO/H(2)). The latter feature makes it an interesting microbe for the biotech industry, as important bulk chemicals and proteins can be produced at the expense of CO(2), thus combining industrial needs with sustained reduction of CO and CO(2) in the atmosphere. Sequencing the complete genome of C. ljungdahlii revealed that it comprises 4,630,065 bp and is one of the largest clostridial genomes known to date. Experimental data and in silico comparisons revealed a third mode of anaerobic homoacetogenic metabolism. Unlike other organisms such as Moorella thermoacetica or Acetobacterium woodii, neither cytochromes nor sodium ions are involved in energy generation. Instead, an Rnf system is present, by which proton translocation can be performed. An electroporation procedure has been developed to transform the organism with plasmids bearing heterologous genes for butanol production. Successful expression of these genes could be demonstrated, leading to formation of the biofuel. Thus, C. ljungdahlii can be used as a unique microbial production platform based on synthesis gas and carbon dioxide/hydrogen mixtures.
Proceedings of the National Academy of Sciences 07/2010; 107(29):13087-92. · 9.68 Impact Factor
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ABSTRACT: Ralstonia eutropha H16 is probably the best-studied 'Knallgas' bacterium and producer of poly(3-hydroxybutyrate) (PHB). Genome-wide transcriptome analyses were employed to detect genes that are differentially transcribed during PHB biosynthesis. For this purpose, four transcriptomes from different growth phases of the wild-type H16 and of the two PHB-negative mutants PHB(-)4 and Delta phaC1 were compared: (i) cells from the exponential growth phase with cells that were in transition to stationary growth phase, and (ii) cells from the transition phase with cells from the stationary growth phase of R. eutropha H16, as well as (iii) cells from the transition phase of R. eutropha H16 with those from the transition phase of R. eutropha PHB(-)4 and (iv) cells from the transition phase of R. eutropha Delta phaC1 with those from the transition phase of R. eutropha PHB(-)4. Among a large number of genes exhibiting significant changes in transcription level, several genes within the functional class of lipid metabolism were detected. In strain H16, phaP3, accC2, fabZ, fabG and H16_A3307 exhibited a decreased transcription level in the stationary growth phase compared with the transition phase, whereas phaP1, H16_A3311, phaZ2 and phaZ6 were found to be induced in the stationary growth phase. Compared with PHB(-)4, we found that phaA, phaB1, paaH1, H16_A3307, phaP3, accC2 and fabG were induced in the wild-type, and phaP1, phaP4, phaZ2 and phaZ6 exhibited an elevated transcription level in PHB(-)4. In strain Delta phaC1, phaA and phaB1 were highly induced compared with PHB(-)4. Additionally, the results of this study suggest that mutant strain PHB(-)4 is defective in PHB biosynthesis and fatty acid metabolism. A significant downregulation of the two cbb operons in mutant strain PHB(-)4 was observed. The putative polyhydroxyalkanoate (PHA) synthase phaC2 identified in strain H16 was further investigated by several functional analyses. Mutant PHB(-)4 could be phenotypically complemented by expression of phaC2 from a plasmid; on the other hand, in the mutant H16Delta phaC1, no PHA production was observed. PhaC2 activity could not be detected in any experiment.
Microbiology 07/2010; 156(Pt 7):2136-52. · 3.06 Impact Factor
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ABSTRACT: The growth of Gluconobacter oxydans DSM 7145 on meso-erythritol is characterized by two stages: in the first stage, meso-erythritol is oxidized almost stoichiometrically to L-erythrulose according to the Bertrand-Hudson rule. The second phase is distinguished from the first phase by a global metabolic change from membrane-bound meso-erythritol oxidation to L-erythrulose assimilation with concomitant accumulation of acetic acid. The membrane-associated erythritol-oxidizing enzyme was found to be encoded by a gene homologous to sldA known from other species of acetic acid bacteria. Disruption of this gene in the genome of G. oxydans DSM 7145 revealed that the membrane-bound polyol dehydrogenase not only oxidizes meso-erythritol but also has a broader substrate spectrum which includes C3-C6 polyols and D-gluconate and supports growth on these substrates. Cultivation of G. oxydans DSM 7145 on different substrates indicated that expression of the polyol dehydrogenase was not regulated, implying that the production of biomass of G. oxydans to be used as whole-cell biocatalysts in the biotechnological conversion of meso-erythritol to L-erythrulose, which is used as a tanning agent in the cosmetics industry, can be conveniently carried out with glucose as the growth substrate.
Microbiology 03/2010; 156(Pt 6):1890-9. · 3.06 Impact Factor
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ABSTRACT: 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.
Journal of bacteriology 08/2009; 191(19):6082-93. · 3.94 Impact Factor
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ABSTRACT: The genome sequence of Bacillus subtilis was published in 1997 and since then many other bacterial genomes have been sequenced, among them Bacillus licheniformis in 2004. B. subtilis and B. licheniformis are closely related and feature similar saprophytic lifestyles in the soil. Both species can secrete numerous proteins into the surrounding medium enabling them to use high-molecular-weight substances, which are abundant in soils, as nutrient sources. The availability of complete genome sequences allows for the prediction of the proteins containing signals for secretion into the extracellular milieu and also of the proteins which form the secretion machinery needed for protein translocation through the cytoplasmic membrane. To confirm the predicted subcellular localization of proteins, proteomics is the best choice. The extracellular proteomes of B. subtilis and B. licheniformis have been analyzed under different growth conditions allowing comparisons of the extracellular proteomes and conclusions regarding similarities and differences of the protein secretion mechanisms between the two species.
Journal of Molecular Microbiology and Biotechnology 02/2009; 16(1-2):53-68. · 1.95 Impact Factor
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ABSTRACT: Acetic acid bacteria are a distinct group of microorganisms within the family Acetobacteriaceae. They are characterized by their ability to incompletely oxidize a wide range of carbohydrates and alcohols. The great advantage of these reactions is that many substrates are regio- and stereoselectively oxidized. This feature is already exploited in several combined biotechnological-chemical procedures for the synthesis of sugar derivatives. Therefore, it is important to understand the basic concepts of this type of physiology to construct strains for improved or new oxidative fermentations. Based on the genome sequence of Gluconobacteroxydans, we will shed light on the central carbon metabolism, the composition of the respiratory chain and the analysis of uncharacterized oxidoreductases. In this context, the role of membrane-bound and -soluble dehydrogenases are of major importance in the process of incomplete oxidation. Other topics deal with the question of how these organisms generate energy and assimilate carbon. Furthermore, we will discuss how acetic acid bacteria thrive in their nutrient-rich environment and how they outcompete other microorganisms.
Journal of Molecular Microbiology and Biotechnology 02/2009; 16(1-2):69-80. · 1.95 Impact Factor
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ABSTRACT: The methanogenic archaeon Methanosarcina mazei strain Gö1 contains two homologues of NrpR, the transcriptional repressor of nitrogen assimilation genes recently discovered and characterized in Methanococcus maripaludis. Insertion of a puromycin-resistance conferring cassette into MM1085 encoding a single NrpR domain with an N-terminal helix-turn-helix domain (NrpRI) lead to a significant reduction of the lag-phase after a shift from nitrogen sufficiency to nitrogen limitation. Consistent with this finding, loss of NrpRI resulted in significantly increased transcript levels of genes involved in nitrogen fixation or nitrogen assimilation though growing under nitrogen sufficiency as demonstrated by quantitative reverse transcriptional PCR analysis. Genome-wide analysis using DNA-microarrays confirmed that transcript levels of 27 ORFs were significantly elevated in the M. mazei MM1085::pac mutant under nitrogen sufficiency, including genes known to be up-regulated under nitrogen limitation (e.g., nifH, glnA(1), glnK(1)), and 17 additional genes involved in metabolism (4), encoding a flagella related protein (1) and genes encoding hypothetical proteins (12). Using cell extracts of Escherichia coli expressing MM1085 fused to the maltose binding protein (MBP-NrpRI) and employing promoter binding studies by DNA-affinity chromatography demonstrated that MBP-NrpRI binds specifically to the nifH-promoter. Deletion of various bases in the promoter region of nifH confirmed that the regulatory element ACC-N(7)-GGT is required for specific binding of NrpRI to the promoter.
Archives of Microbiology 05/2008; 190(3):319-32. · 1.43 Impact Factor
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ABSTRACT: Methanosarcina mazei is a nonhalophilic methanogen that can adapt to 800 mM NaCl. Microarray studies have been used to examine the effect of elevated salinities on the regulation of gene expression in M. mazei. Eighty-four genes of different functional categories, such as solute transport and biosynthesis, Na(+) export, stress response, ion, protein and phosphate transport, metabolic enzymes, regulatory proteins, DNA-modification systems, and cell-surface modulators, were found to be stronger expressed at high salinities. Moreover, 10 genes encoding different metabolic functions including potassium uptake and ATP synthesis were reduced in expression under high salt. The overall expression profiles suggest that M. mazei is able to adapt to high salinities by multiple upregulation of many different cellular functions including protective pathways such as solute transport and biosynthesis, import of phosphate, export of Na(+), and upregulation of pathways for modification of DNA and cell surface architecture.
FEMS Microbiology Letters 01/2008; 277(1):79-89. · 2.04 Impact Factor
