Ethanol synthesis from glycerol by Escherichia coli redox mutants expressing adhE from Leuconostoc mesenteroides.

AIMS: Analysis of the physiology and metabolism of Escherichia coli arcA and creC mutants expressing a bifunctional alcohol-acetaldehyde dehydrogenase from Leuconostoc mesenteroides growing on glycerol under oxygen-restricted conditions. The effect of an ldhA mutation and different growth medium modifications was also assessed. METHODS AND RESULTS: Expression of adhE in E. coli CT1061 [arcA creC(Con)] resulted in a 1.4-fold enhancement in ethanol synthesis. Significant amounts of lactate were produced during micro-oxic cultures and strain CT1061LE, in which fermentative lactate dehydrogenase was deleted, produced up to 6.5 +/- 0.3 g l(-1) ethanol in 48 h. Escherichia coli CT1061LE derivatives resistant to >25 g l(-1) ethanol were obtained by metabolic evolution. Pyruvate and acetaldehyde addition significantly increased both biomass and ethanol concentrations, probably by overcoming acetyl-coenzyme A (CoA) shortage. Yeast extract also promoted growth and ethanol synthesis, and this positive effect was mainly attributable to its vitamin content. Two-stage bioreactor cultures were conducted in a minimal medium containing 100 microg l(-1) calcium d-pantothenate to evaluate oxic acetyl-CoA synthesis followed by a switch into fermentative conditions. Ethanol reached 15.4 +/- 0.9 g l(-1) with a volumetric productivity of 0.34 +/- 0.02 g l(-1) h(-1). CONCLUSIONS: Escherichia coli responded to adhE over-expression by funnelling carbon and reducing equivalents into a highly reduced metabolite, ethanol. Acetyl-CoA played a key role in micro-oxic ethanol synthesis and growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Insight into the micro-oxic metabolism of E. coli growing on glycerol is essential for the development of efficient industrial processes for reduced biochemicals production from this substrate, with special relevance to biofuels synthesis.

AAV2-mediated CLN2 gene transfer to rodent and non-human primate brain results in long-term TPP-I expression compatible with therapy for LINCL.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal, autosomal recessive disease resulting from mutations in the CLN2 gene with consequent deficiency in its product tripeptidyl peptidase I (TPP-I). In the central nervous system (CNS), the deficiency of TPP-I results in the accumulation of proteins in lysosomes leading to a loss of neurons causing progressive neurological decline, and death by ages 10-12 years. To establish the feasibility of treating the CNS manifestations of LINCL by gene transfer, an adeno-associated virus 2 (AAV2) vector encoding the human CLN2 cDNA (AAV2CUhCLN2) was assessed for its ability to establish therapeutic levels of TPP-I in the brain. In vitro studies demonstrated that AAV2CUhCLN2 expressed CLN2 and produced biologically active TPP-I protein of which a fraction was secreted as the pro-TPP-I precursor and was taken up by nontransduced cells (ie, cross-correction). Following AAV2-mediated CLN2 delivery to the rat striatum, enzymatically active TPP-I protein was detected. By immunohistochemistry TPP-I protein was detected in striatal neurons (encompassing nearly half of the target structure) for up to 18 months. At the longer time points following striatal administration, TPP-I-positive cell bodies were also observed in the substantia nigra, frontal cerebral cortex and thalamus of the injected hemisphere, and the frontal cerebral cortex of the noninjected hemisphere. These areas of the brain contain neurons that extend axons into the striatum, suggesting that CNS circuitry may aid the distribution of the gene product. To assess the feasibility of human CNS delivery, a total of 3.6 x 10(11) particle units of AAV2CUhCLN2 was administered to the CNS of African green monkeys in 12 distributed doses. Assessment at 5 and 13 weeks demonstrated widespread detection of TPP-I in neurons, but not glial cells, at all regions of injection. The distribution of TPP-I-positive cells was similar between the two time points at all injection sites. Together, these data support the development of direct CNS gene transfer using an AAV2 vector expressing the CLN2 cDNA for the CNS manifestations of LINCL.

Poly(3-hydroxybutyrate) synthesis genes in Azotobacter sp. strain FA8.

Genes responsible for the synthesis of poly(3-hydroxybutyrate) (PHB) in Azotobacter sp. FA8 were cloned and analyzed. A PHB polymerase gene (phbC) was found downstream from genes coding for beta-ketothiolase (phbA) and acetoacetyl-coenzyme A reductase (phbB). A PHB synthase mutant was obtained by gene inactivation and used for genetic studies. The phbC gene from this strain was introduced into Ralstonia eutropha PHB-4 (phbC-negative mutant), and the recombinant accumulated PHB when either glucose or octanoate was used as a source of carbon, indicating that this PHB synthase cannot incorporate medium-chain-length hydroxyalkanoates into PHB.

Phosphotransbutyrylase expression in Bacillus megaterium.

The molecular analysis of a genomic region of B. megaterium revealed the presence of a gene coding for the enzyme phosphotransbutyrylase (Ptb). The enzyme activity was measured throughout the different phases of growth in B. megaterium, and its activity was found to be maximal in the late exponential growth phase. The branched amino acids isoleucine and valine activated Ptb expression. PtbBm was capable of using butyryl-CoA and 2-methyl-propionyl CoA as substrates. ActBm, a final sigma54 regulator from B. megaterium whose gene is situated upstream from the ptb gene, activated its expression.

Polyhydroxyalkanoate degradation is associated with nucleotide accumulation and enhances stress resistance and survival of Pseudomonas oleovorans in natural water microcosms.

Pseudomonas oleovorans GPo1 and its polyhydroxyalkanoic acid (PHA) depolymerization-minus mutant, GPo500 phaZ, residing in natural water microcosms, were utilized to asses the effect of PHA availability on survival and resistance to stress agents. The wild-type strain showed increased survival compared to the PHA depolymerase-minus strain. The appearance of a round cellular shape, characteristic of bacteria growing under starvation conditions, was delayed in the wild type in comparison to the mutant strain. Percent survival at the end of ethanol and heat challenges was always higher in GPo1 than in GPo500. Based on these results and on early experiments (H. Hippe, Arch. Mikrobiol. 56:248-277, 1967) that suggested an association of PHA utilization with respiration and oxidative phosphorylation, we investigated the association between PHA degradation and nucleotide accumulation. ATP and guanosine tetraphosphate (ppGpp) production was analyzed under culture conditions leading to PHA depolymerization. A rise in the ATP and ppGpp levels appeared concomitant with PHA degradation, while this phenomenon was not observed in the mutant strain unable to degrade the polymer. Complementation of the phaZ mutation restored the wild-type phenotype.

Polyhydroxyalkanoates degradation affects survival of Pseudomonas oleovorans in river water microcosms

Bacterial survival in natural environments involves the ability of scavenging nutrients and energy sources. Polyhydroxyalkanoates (PHAs) are intracellular polymers that endow bacteria with enhanced survival capabilities in adverse environmental conditions. In this paper we compared survival of Pseudomonas oleovorans wild type and PHA depolymerase mutant strains in natural river waters by using microcosms. Experiments were performed with water samples collected from the Rio de la Plata. The survival of the P. oleovorans strain capable of degrading PHA was higher in raw river water compared to the depolymerase negative mutant strain. Bacterial numbers decreased during the experiment. At the end of the experiment, the difference in the number of cells between wild type and mutant strains was of 3 orders of magnitude. Mutants deficient in PHA degradation are useful to study the importance of reserve polymers in the survival of bacterial species in natural environments. They could also provide an adequate system for the analysis of the role of PHA in the tolerance to physical or chemical stress agents.

Polyhydroxyalkanoates degradation affects survival of Pseudomonas oleovorans in river water microcosms

Bacterial survival in natural environments involves the ability of scavenging nutrients and energy sources. Polyhydroxyalkanoates (PHAs) are intracellular polymers that endow bacteria with enhanced survival capabilities in adverse environmental conditions. In this paper we compared survival of Pseudomonas oleovorans wild type and PHA depolymerase mutant strains in natural river waters by using microcosms. Experiments were performed with water samples collected from the Rio de la Plata. The survival of the P. oleovorans strain capable of degrading PHA was higher in raw river water compared to the depolymerase negative mutant strain. Bacterial numbers decreased during the experiment. At the end of the experiment, the difference in the number of cells between wild type and mutant strains was of 3 orders of magnitude. Mutants deficient in PHA degradation are useful to study the importance of reserve polymers in the survival of bacterial species in natural environments. They could also provide an adequate system for the analysis of the role of PHA in the tolerance to physical or chemical stress agents.(AU)

Evaluation of Xanthomonas campestris survival in a soil microcosm system.

Xanthomonas campestris pv. campestris is a pathogen of cruciferous plants. We studied the survival of the wild type strain and mutant derivatives which are deficient in exopolysaccharide (EPS) or in extracellular protease synthesis in soil microcosms in order to test the hypothesis that, in this environment, adherence to soil particles and scavenging of nutrients are very important strategies for bacterial survival. In sterile soil microcosms, differences in survival were only observed between the EPS producer and its mutant. In non-sterile soil experiments, survival of Prt- mutant was similar to EPS- mutant, suggesting that both characteristics have a strong influence in survival in the presence of the natural bacterial community. Bacterial decrease represented by the slope of regression lines was higher in non-sterile soil microcosms due to the influence of biotic interactions.

Polyhydroxyalkanoates degradation affects survival of Pseudomonas oleovorans in river water microcosms.

Bacterial survival in natural environments involves the ability of scavenging nutrients and energy sources. Polyhydroxyalkanoates (PHAs) are intracellular polymers that endow bacteria with enhanced survival capabilities in adverse environmental conditions. In this paper we compared survival of Pseudomonas oleovorans wild type and PHA depolymerase mutant strains in natural river waters by using microcosms. Experiments were performed with water samples collected from the Rio de la Plata. The survival of the P. oleovorans strain capable of degrading PHA was higher in raw river water compared to the depolymerase negative mutant strain. Bacterial numbers decreased during the experiment. At the end of the experiment, the difference in the number of cells between wild type and mutant strains was of 3 orders of magnitude. Mutants deficient in PHA degradation are useful to study the importance of reserve polymers in the survival of bacterial species in natural environments. They could also provide an adequate system for the analysis of the role of PHA in the tolerance to physical or chemical stress agents.

Trans activation of the Escherichia coli ato structural genes by a regulatory protein from Bacillus megaterium: potential use in polyhydroxyalkanoate production.

A Bacillus megaterium genomic fragment, which encoded an activator homologous to sigma 54 regulators and which was capable of activating Escherichia coli ato genes in trans, was detected in a gene library of B. megaterium screened for beta-ketothiolase activity. The fragment presented only one complete open reading frame (ORF1), which encoded a protein of 398 amino acids. The recombinant plasmid complemented mutations in the Escherichia coli atoC regulatory gene. The constitutive expression of the E. coli ato operon mediated by ORF1 could be useful for the synthesis of polyhydroxyalkanoates with different flexibility properties by recombinant E. coli strains.

Monitoring by PCR amplification of the polyphosphate kinase gene added to natural water samples.

A fast, simple method for the detection of the Escherichia coli polyphosphate kinase (ppk) gene by means of PCR amplification is described. The method uses filters to recover cells from the samples, which makes it suitable for environmental studies. The detection of the ppk gene was achieved from samples containing 10(2) E. coli cells, either in saline solution or in river water.

[Poly-(3-hydroxybutyrate) granules in Bacillus megaterium. Isolation and analysis of associated proteins]. / Gránulos de poli(3-hidroxibutirato) en Bacillus megaterium. Aislamiento y análisis de las proteínas asociadas.

Bacillus megaterium accumulates poly-(3-hydroxybutyrate) (PHB) as a reserve material in intracellular granules. In this work we described a method for the preparation of PHB granules from B. megaterium PV447 and the analysis by polyacrylamide gel electrophoresis of the associated proteins. By comparison with another species a function is proposed for some of these proteins.

Conjugal transfer of enterococcal transposons in Bacillus megaterium.

The conjugative enterococcal transposons Tn916 and Tn919 were introduced into Bacillus megaterium by a filtermating technique. The transfer frequencies obtained ranged from 1.3×10(-6) to 6.6×10(-7). The transposons integrated stably into the B. megaterium chromosome. Tn916 could generate auxotrophs and was transferred from B. megaterium Tn916 transconjugants to other species.

[Enrichment for mutants of Bacillus megaterium deficient in the synthesis of poly-beta-hydroxybutyrate (PHB)]. / Enriquecimiento en mutantes de Bacillus megaterium deficientes en la síntesis de poli-beta-hidroxibutirato (PHB).

Centrifugation through sucrose gradients was adapted to separate spore-forming cells of B. megaterium deficient in poly-beta-hydroxybutyrate synthesis from wild type cells. The conditions described allowed the detection of mutant clones screening a low percentage of the mutagenized population.

Enriquecimiento en mutantes de Bacillus megaterium deficientes en la síntesis de poli-beta-hidroxibutirato (PHB). / [Enrichment for mutants of Bacillus megaterium deficient in the synthesis of poly-beta-hydroxybutyrate (PHB)]

Centrifugation through sucrose gradients was adapted to separate spore-forming cells of B. megaterium deficient in poly-beta-hydroxybutyrate synthesis from wild type cells. The conditions described allowed the detection of mutant clones screening a low percentage of the mutagenized population.

[Mutants with high butanol production from a strain of solventogenic Clostridium isolated from olive black-water]. / Mutantes con alta producción de butanol de una cepa de Clostridium solventogénica aislada de alpechín.

Butanol high producing mutants of a solventogenic Clostridium sp. capable of degrading olive black water, were selected according to ethanol or butanol resistance after treatment with N-methyl-N'-nitro-N-nitrosoguanidine. Mutants were quickly screened from isolated colonies and then characterized in standard culture conditions.

Direct Selection of Clostridium acetobutylicum Fermentation Mutants by a Proton Suicide Method.

Clostridium acetobutylicum ATCC 10132 mutants altered in acetic acid synthesis or in the shift to solventogenesis were directly selected by a proton suicide method after mutagenic treatment, by using bromide and bromate as selective agents. The mutants were characterized according to their solvent and acid production. On the selection plates they differed in colony phenotype from the parent strain.

Mutantes con alta producción de butanol de una cepa de Clostridium solventogénica aislada de alpechín. / [Mutants with high butanol production from a strain of solventogenic Clostridium isolated from olive black-water]

Butanol high producing mutants of a solventogenic Clostridium sp. capable of degrading olive black water, were selected according to ethanol or butanol resistance after treatment with N-methyl-N-nitro-N-nitrosoguanidine. Mutants were quickly screened from isolated colonies and then characterized in standard culture conditions.

Mutantes con alta producción de butanol de una cepa de Clostridium solventogénica aislada de alpechín / [Mutants with high butanol production from a strain of solventogenic Clostridium isolated from olive black-water]

Butanol high producing mutants of a solventogenic Clostridium sp. capable of degrading olive black water, were selected according to ethanol or butanol resistance after treatment with N-methyl-N’-nitro-N-nitrosoguanidine. Mutants were quickly screened from isolated colonies and then characterized in standard culture conditions.