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Eubacterium limosum is an acetogen that can produce butyrate along with acetate as the main fermentation end-product from methanol, a promising C1 feedstock. Although physiological characterization of E. limosum B2 during methylotrophy was previously performed, the strain was cultured in a semi-defined medium, limiting the scope for further metabolic insights. Here, we sequenced the complete genome of the native strain and performed adaptive laboratory evolution to sustain growth on methanol mineral medium. The evolved population significantly improved its maximal growth rate by 3.45-fold. Furthermore, three clones from the evolved population were isolated on methanol mineral medium without cysteine by the addition of sodium thiosulfate. To identify mutations related to growth improvement, the whole genomes of wild-type E. limosum B2, the 10th, 25th, 50th, and 75th generations, and the three clones were sequenced. We explored the total proteomes of the native and the best evolved clone (n°2) and noticed significant differences in proteins involved in gluconeogenesis, anaplerotic reactions, and sulphate metabolism. Furthermore, a homologous recombination was found in subunit S of the type I restriction-modification system between both strains, changing the structure of the subunit, its sequence recognition and the methylome of the evolved clone. Taken together, the genomic, proteomic and methylomic data suggest a possible epigenetic mechanism of metabolic regulation. [ABSTRACT FROM AUTHOR]

A library of Escherichia coli clones that express a particular chromosomal gene at a wide range of expression levels is produced developing a simple and highly efficient method. The aim was to replace all or part of the upstream of a coding sequence containing the elements involved in its expression with a PCR-generated library of expression cassettes.

The first isolation and preliminary biochemical characterization of a B12-independent glycerol dehydratase is presented. The crystal structure for the B12-independent GD is reported and proved that it is similar to PFL in both structure and the SAM-dependent activation mechanism.

The aldehyde/alcohol dehydrogenase (AADH) coding gene adhE2 of Clostridium acetobutylicum ATC 824 is the second gene identified in this bacterium besides adhE. The adhE2 is localized to the pSOL1 megaplasmid, is preferentially expressed in alcohologenic cultures, and is a NADH-dependent bifunctional AADH.

Results demonstrate that the genome sequence of Clostridium acetobutylicum accounts for majority of the genes involved in solvent-production. It consists of a 3.94-megabase chromosome and a 192-kilobase megaplasmid and exhibits both genome conservation and organization to that displayed by Bacillus subtilis.

Results show that Clostridium butyricum induces the formation of 1,3-propanediol and decreases the production of butyrate, acetate, lactate, and hydrogen when grown on glucose-glycerol mixtures. Data show that with glycerol alone, 1,3-propanediol will be the major product produced.

A study was conducted to describe a physical and genetic map of the Clostridium acetobutylicum ATCC 824 chromosome. Digestion of DNA was carried out in Eppendorf tubes at 37 degrees C while gels were stained in a fresh solution of ethidium bromide. Results indicated that the electron flow plays an important function in the regulation of C. acetobutylicum metabolism. They also showed that the chromosome backbone of C. acetobutylicum is similar to that of B. subtilis.

The role of genes for butanol and acetone formation in Clostridium acetobutylin ATCC 824 was analyzed to determine its effect on the degeneration of the strain. The study made use of a restriction map pf pSOL1 using ApaI, Sma1, Sst II and Nar I digestions. Results revealed that pSOL1 plasmid in C. acetobutylin ATCC 824 carries primary metabolic genes essential for the production of acetone and butanol.

The metabolic behaviour of Enterobacter agglomerans that grows anaerobically in a continous culture with glycerol or glucose as the only carbon source was investigated. Carbon flow distribution at branching metabolic points and the regeneration of reducing equivalents were monitored. The activities of major enzymes involved in the dissimilation of glycerol and nucleotide pools were quantified.

The partial inhibition of 1,3-propanediol dehydrogenase activity by NAD/NADH ratio causes the accumulation of 3-hydroxypropionaldehyde (3-HPA), during the fermentation of glycerol by Enterobacter agglomerans. With the decrease in NAD/NADH ratio at pH 8, further accumulation of 3-HPA is halted. 3-HPA accumulation suppresses the activity of glycerol dehydrogenase resulting in lower rates of glycerol dissimilation.

The putative hydrogenase gene (hydA) in Clostridium acetobutylicum encoding a 64,415-Da protein shows a high degree of similarity with the (Fe) hydrogenase genes of Desulfovibrio and Clostridium species. The hydA mRNA content is high in cells from an acidogenic or alcohologenic continuous culture and low in those from a solventogenic continuous culture. The regulation of hydA expression occurs at the transcriptional level.

The fermentation of glycerol to 1,3-propanediol by Enterobacter agglomerans produces 3-hydroxypropionaldehyde as an intermediate product that inhibits the formation of 1,3-propanediol. The synthesis of 1,3-propanediol stops after 430 mM of glycerol have been utilized by the enterobacterial species regardless of the initial glycerol concentration. 3-hydroxypropionaldehyde is also formed during the fermentation of glycerol by other enterobacterial species such as Klebsiella pneumoniae and Citrobacter freundii.

High NADH/NAD ratio of Clostridium acetobutylicum blocks the glyceraldehyde-3-phosphate dehydrogenase involved in glycolytic metabolism. High utilization of glycerol in steady-state continuous cultures is facilitated by partial substitution of glucose by pyruvate, with the glycerol levels maintained at constant. In these cultures, a high NADH/NAD ratio and a low intracellular ATP concentration accompany the activation of NADH-dependent butyraldehyde and ferrodoxin-NAD(P) reductases and high levels of alcohol dehydrogenase expression.

Mixtures of glucose and glycerol were used to study the metabolic flexibility of Clostridium acetobutylicum in chemostat culture by enhancing NAD(P)H pressure. The intracellular enzymatic distribution of glucose-grown-acid-producing cultures revealed the absence of butyraldehyde dehydrogenase activity, while cultures grown on mixtures generated alcohols and low level of hydrogen

The conditions promoting or inhibiting the lysis of Clostridium acetobutylicum, an acetone and butanol producer, were determined. The results showed that the optimum temperature for autolysis is 55 degress celsius, while the optimum pH is 6.3. Autolysis was more pronounced at the end of the exponential phase, and was enhanced by monovalent cations, while divalent cations were inhibitory. Characterization studies of the C. acetobutylicum autolysin showed that it is a muramidase which is inhibited by lipoteichoic acids and cardiolipin.

An extracellular enzyme capable oflyzing non-N-acetylated peptidoglycan has been recovered and purified from Clostridium acetobutylicum ATCC 824 cells. This enzyme has been characterized as a muramidase with a molecular weight of 41,000 and an acidic isoelectric point of 3.8. It is the first muramidase that cleaves only non-N-acetylated peptidoglycans. Its enzymatic activity is inhibited by N-acetylglucosamine and glucosamine and enhanced by choline and ethanolamine.

Examination of the metabolism of Clostridium acetobutylicum as a function of methyl viologen addition and iron limitation shows that both factors exhibit ability to induce metabolic shifts toward butanol formation. Additive physiological effects of these factors reveal different and complementary sites of action for these factors. Addition of methyl viologen increases the activity of C. acetobutylicum and generates an artifical electron transport chain.