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Relationship between Glycosyl Hydrolase Inventory and Growth Physiology of the Hyperthermophile Pyrococcus furiosus on Carbohydrate-Based Media

Driskill, Lance E.; Kusy, Kevin; Bauer, Michael W.; Kelly, Robert M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /03/1999 EN
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Utilization of a range of carbohydrates for growth by the hyperthermophile Pyrococcus furiosus was investigated by examining the spectrum of glycosyl hydrolases produced by this microorganism and the thermal labilities of various saccharides. Previously, P. furiosus had been found to grow in batch cultures on several α-linked carbohydrates and cellobiose but not on glucose or other β-linked sugars. Although P. furiosus was not able to grow on any nonglucan carbohydrate or any form of cellulose in this study (growth on oat spelt arabinoxylan was attributed to glucan contamination of this substrate), significant growth at 98°C occurred on β-1,3- and β-1,3–β-1,4-linked glucans. Oligosaccharides generated by digestion with a recombinant laminarinase derived from P. furiosus were the compounds that were most effective in stimulating growth of the microorganism. In several cases, periodic addition of β-glucan substrates to fed-batch cultures limited adverse thermochemical modifications of the carbohydrates (i.e., Maillard reactions and caramelization) and led to significant increases (as much as two- to threefold) in the cell yields. While glucose had only a marginally positive effect on growth in batch culture, the final cell densities nearly tripled when glucose was added by the fed-batch procedure. Nonenzymatic browning reactions were found to be significant at 98°C for saccharides with degrees of polymerization (DP) ranging from 1 to 6; glucose was the most labile compound on a mass basis and the least labile compound on a molar basis. This suggests that for DP of 2 or greater protection of the nonreducing monosaccharide component may be a factor in substrate availability. For P. furiosus...

Overexpression of the Saccharomyces cerevisiae Mannosylphosphodolichol Synthase-Encoding Gene in Trichoderma reesei Results in an Increased Level of Protein Secretion and Abnormal Cell Ultrastructure

Kruszewska, Joanna S.; Butterweck, Arno H.; Kurzątkowski, Wiesław; Migdalski, Andrzej; Kubicek, Christian P.; Palamarczyk, Grażyna
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /06/1999 EN
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Production of extracellular proteins plays an important role in the physiology of Trichoderma reesei and has potential industrial application. To improve the efficiency of protein secretion, we overexpressed in T. reesei the DPM1 gene of Saccharomyces cerevisiae, encoding mannosylphosphodolichol (MPD) synthase, under homologous, constitutively acting expression signals. Four stable transformants, each with different copy numbers of tandemly integrated DPM1, exhibited roughly double the activity of MPD synthase in the respective endoplasmic reticulum membrane fraction. On a dry-weight basis, they secreted up to sevenfold-higher concentrations of extracellular proteins during growth on lactose, a carbon source promoting formation of cellulases. Northern blot analysis showed that the relative level of the transcript of cbh1, which encodes the major cellulase (cellobiohydrolase I [CBH I]), did not increase in the transformants. On the other hand, the amount of secreted CBH I and, in all but one of the transformants, intracellular CBH I was elevated. Our results suggest that posttranscriptional processes are responsible for the increase in CBH I production. The carbohydrate contents of the extracellular proteins were comparable in the wild type and in the transformants...

Key Physiology of Anaerobic Ammonium Oxidation

Strous, Marc; Kuenen, J. Gijs; Jetten, Mike S. M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/1999 EN
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The physiology of anaerobic ammonium oxidizing (anammox) aggregates grown in a sequencing batch reactor was investigated quantitatively. The physiological pH and temperature ranges were 6.7 to 8.3 and 20 to 43°C, respectively. The affinity constants for the substrates ammonium and nitrite were each less than 0.1 mg of nitrogen per liter. The anammox process was completely inhibited by nitrite concentrations higher than 0.1 g of nitrogen per liter. Addition of trace amounts of either of the anammox intermediates (1.4 mg of nitrogen per liter of hydrazine or 0.7 mg of nitrogen per liter of hydroxylamine) restored activity completely.

Effect of Organic Complex Compounds on Bacillus thermoamylovorans Growth and Glucose Fermentation

Combet-Blanc, Y.; Dieng, M. C.; Kergoat, P. Y.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/1999 EN
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The effect of the concentration of a mixture (1/1 [wt/wt]) of yeast extract and bioTrypcase (YE+bT) on the growth and physiology of a new species, Bacillus thermoamylovorans, a moderately thermophilic, non-spore-forming, lactic acid-producing bacterium isolated from palm wine, was studied. At an initial glucose concentration of 100 mM, B. thermoamylovorans growth was limited when the concentration of YE+bT was lower than 5.0 g liter−1; under these conditions, cellular yield reached a maximum value of 0.4 g of cells per g of YE+bT. Growth limitation due to deficiency in growth factors led to a significant shift in glucose metabolism towards lactate production. Lactate constituted 27.5 and 76% of the end products of glucose fermentation in media containing YE+bT at 20.0 and 1.0 g liter−1, respectively. This result markedly differed from published data for lactic bacteria, which indicated that fermentative metabolism remained homolactic regardless of the concentration of YE. Our results showed that the ratio between cellular synthesis and energy production increased with the concentration of YE+bT in the culture medium. They indicate that the industrial production of lactic acid through glucose fermentation by B. thermoamylovorans can be optimized by using a medium where glucose is present in excess and the organic additives are limiting.

The H+-ATPase in the Plasma Membrane of Saccharomyces cerevisiae Is Activated during Growth Latency in Octanoic Acid-Supplemented Medium Accompanying the Decrease in Intracellular pH and Cell Viability

Viegas, Cristina A.; Almeida, Paulo F.; Cavaco, Miguel; Sá-Correia, Isabel
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/1998 EN
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Saccharomyces cerevisiae plasma membrane H+-ATPase activity was stimulated during octanoic acid-induced latency, reaching maximal values at the early stages of exponential growth. The time-dependent pattern of ATPase activation correlated with the decrease of cytosolic pH (pHi). The cell population used as inoculum exhibited a significant heterogeneity of pHi, and the fall of pHi correlated with the loss of cell viability as determined by plate counts. When exponential growth started, only a fraction of the initial population was still viable, consistent with the role of the physiology and number of viable cells in the inoculum in the duration of latency under acid stress.

Effect of Pyruvate Carboxylase Overexpression on the Physiology of Corynebacterium glutamicum

Koffas, Mattheos A. G.; Jung, Gyoo Yeol; Aon, Juan C.; Stephanopoulos, Gregory
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/2002 EN
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Pyruvate carboxylase was recently sequenced in Corynebacterium glutamicum and shown to play an important role of anaplerosis in the central carbon metabolism and amino acid synthesis of these bacteria. In this study we investigate the effect of the overexpression of the gene for pyruvate carboxylase (pyc) on the physiology of C. glutamicum ATCC 21253 and ATCC 21799 grown on defined media with two different carbon sources, glucose and lactate. In general, the physiological effects of pyc overexpression in Corynebacteria depend on the genetic background of the particular strain studied and are determined to a large extent by the interplay between pyruvate carboxylase and aspartate kinase activities. If the pyruvate carboxylase activity is not properly matched by the aspartate kinase activity, pyc overexpression results in growth enhancement instead of greater lysine production, despite its central role in anaplerosis and aspartic acid biosynthesis. Aspartate kinase regulation by lysine and threonine, pyruvate carboxylase inhibition by aspartate (shown in this study using permeabilized cells), as well as well-established activation of pyruvate carboxylase by lactate and acetyl coenzyme A are the key factors in determining the effect of pyc overexpression on Corynebacteria physiology.

Ethanol Production by Thermophilic Bacteria: Physiological Comparison of Solvent Effects on Parent and Alcohol-Tolerant Strains of Clostridium thermohydrosulfuricum

Lovitt, R. W.; Longin, R.; Zeikus, J. G.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1984 EN
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The effects of temperature, solvents, and cultural conditions on the fermentative physiology of an ethanol-tolerant (56 g/liter at 60°C) and parent strain of Clostridium thermohydrosulfuricum were compared. An ethanol-tolerant mutant was selected by successive transfer of the parent strain into media with progressively higher ethanol concentrations. Physiological differences noted in the mutant included enhanced growth, tolerance to various solvents, and alterations in the substrate range and the fermentation end product ratio. Ethanol tolerance was temperature dependent in the mutant but not in the parent strain. The mutant grew with ethanol concentrations up to 8.0% (wt/vol) at 45°C, but only up to 3.3% (wt/vol) at 68°C. Low ethanol concentration (0.2 to 1.6% [wt/vol]) progressively inhibited the parent strain to where glucose was not fermented at 2.0% (wt/vol) ethanol. Both strains grew and produced alcohols on glucose complex medium at 60°C in the presence of either 5% methanol or acetone, and these solvents when added at low concentration stimulated fermentative metabolism. The mutant produced ethanol at high concentrations and displayed an ethanol/glucose ratio (mole/mole) of 1.0 in media where initial ethanol concentrations were ≤4.0% (wt/vol)...

Overproduction of Trehalose: Heterologous Expression of Escherichia coli Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in Corynebacterium glutamicum

Padilla, Leandro; Krämer, Reinhard; Stephanopoulos, Gregory; Agosin, Eduardo
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/2004 EN
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Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum.

Growth of Polychlorinated-Biphenyl-Degrading Bacteria in the Presence of Biphenyl and Chlorobiphenyls Generates Oxidative Stress and Massive Accumulation of Inorganic Polyphosphate

Chávez, Francisco P.; Lünsdorf, Heinrich; Jerez, Carlos A.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2004 EN
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Inorganic polyphosphate (polyP) plays a significant role in increasing bacterial cell resistance to unfavorable environmental conditions and in regulating different biochemical processes. Using transmission electron microscopy of the polychlorinated biphenyl (PCB)-degrading bacterium Pseudomonas sp. strain B4 grown in defined medium with biphenyl as the sole carbon source, we observed large and abundant electron-dense granules at all stages of growth and following a shift from glucose to biphenyl or chlorobiphenyls. Using energy dispersive X-ray analysis and electron energy loss spectroscopy with an integrated energy-filtered transmission electron microscope, we demonstrated that these granules were mainly composed of phosphate. Using sensitive enzymatic methods to quantify cellular polyP, we confirmed that this polymer accumulates in PCB-degrading bacteria when they grow in the presence of biphenyl and chlorobiphenyls. Concomitant increases in the levels of the general stress protein GroEl and reactive oxygen species were also observed in chlorobiphenyl-grown cells, indicating that these bacteria adjust their physiology with a stress response when they are confronted with compounds that serve as carbon and energy sources and at the same time are chemical stressors.

CO2- and Anaerobiosis-Induced Changes in Physiology and Gene Expression of Different Listeria monocytogenes Strains

Jydegaard-Axelsen, Anne-Marie; Høiby, Poul Erik; Holmstrøm, Kim; Russell, Nicholas; Knøchel, Susanne
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/2004 EN
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67.15914%
Although carbon dioxide (CO2) is known to inhibit growth of most bacteria, very little is known about the cellular response. The food-borne pathogen Listeria monocytogenes is characterized by its ability to grow in high CO2 concentrations at refrigeration temperatures. We examined the listerial responses of different strains to growth in air, 100% N2, and 100% CO2. The CO2-induced changes in membrane lipid fatty acid composition and expression of selected genes were strain dependent. The acid-tolerant L. monocytogenes LO28 responded in the same manner to CO2 as to other anaerobic, slightly acidic environments (100% N2, pH 5.7). An increase in the expression of the genes encoding glutamate decarboxylase (essential for survival in strong acid) as well as an increased amount of branched-chain fatty acids in the membrane was observed in both atmospheres. In contrast, the acid-sensitive L. monocytogenes strain EGD responded differently to CO2 and N2 at the same pH. In a separate experiment with L. monocytogenes 412, an increased isocitrate dehydrogenase activity level was observed for cells grown in CO2-containing atmospheres. Together, our findings demonstrate that the CO2-response is a partly strain-dependent complex mechanism. The possible links between the CO2-dependent changes in isocitrate dehydrogenase activity...

Effects of Growth in the Presence of Subinhibitory Concentrations of Dicloxacillin on Staphylococcus epidermidis and Staphylococcus haemolyticus Biofilms

Cerca, Nuno; Martins, Silvia; Sillankorva, Sanna; Jefferson, Kimberly K.; Pier, Gerald B.; Oliveira, Rosario; Azeredo, Joana
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2005 EN
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Low concentrations of antibiotics can inhibit microbial adherence to medical device surfaces. However, little is known about the changes that occur in the physiology of bacteria within biofilms formed in the presence of subinhibitory (sub-MIC) concentrations of antibiotics. In this study, the densities and matrix compositions ofbiofilms formed by two coagulase-negative Staphylococcus species in the absence and in the presence of sub-MIC concentrations of dicloxacillin were evaluated. Biofilms formed in the presence of sub-MIC concentrations of dicloxacillin contained less biomass, and there were notable changes in the composition of the biofilm matrix. Changes in the spatial structure were also verified by confocal scanning laser microscopy, indicating that biofilms grown in the presence of sub-MIC concentrations of dicloxicilln had a lower cell density. Physiological alterations in the bacteria within biofilms grown in the presence of subinhibitory concentrations of the antibiotic were also evaluated. The results showed that there were differences in bacterial surface characteristics when cultures were grown in the presence of sub-MIC concentrations of dicloxacillin, including decreased hydrophobicity and decreased expression of the exopolysaccharide poly-N-acetylglucosamine. The elemental composition of the cell surface was also analyzed...

Listeria monocytogenes PerR Mutants Display a Small-Colony Phenotype, Increased Sensitivity to Hydrogen Peroxide, and Significantly Reduced Murine Virulence

Rea, Rosemarie; Hill, Colin; Gahan, Cormac G. M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2005 EN
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Deletion of perR in Listeria monocytogenes results in a small-colony phenotype (ΔperRsm) that is slow growing and exhibits increased sensitivity to H2O2. At a relatively high frequency, large-colony variants (ΔperRlg) arise, which are more resistant to H2O2 than the wild-type and ultimately dominate the culture. Transcriptional analysis revealed that the kat gene (catalase) is up-regulated in both types of mutants and that the highest level is apparent in ΔperRsm mutants, demonstrating PerR regulation of this gene. Overexpression of the catalase gene in the wild-type background resulted in a slower-growing strain with a smaller colony size similar to that of ΔperRsm. By combining a bioinformatic approach with experimental evidence, other PerR-regulated genes were identified, including fur, lmo0641, fri, lmo1604, hemA, and trxB. The transcriptional profile of these genes in both mutant backgrounds was similar to that of catalase in that a higher level of expression was observed in ΔperRsm than in the wild type or ΔperRlg. Murine studies revealed that the virulence potential of the ΔperRsm mutant is substantially reduced compared to that of the wild-type and ΔperRlg strains. Collectively, the data demonstrate that the ΔperRsm mutant represents the true phenotype associated with the absence of PerR...

Long Serial Analysis of Gene Expression for Gene Discovery and Transcriptome Profiling in the Widespread Marine Coccolithophore Emiliania huxleyi†

Dyhrman, Sonya T.; Haley, Sheean T.; Birkeland, Shanda R.; Wurch, Louie L.; Cipriano, Michael J.; McArthur, Andrew G.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/2006 EN
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The abundant and widespread coccolithophore Emiliania huxleyi plays an important role in mediating CO2 exchange between the ocean and the atmosphere through its impact on marine photosynthesis and calcification. Here, we use long serial analysis of gene expression (SAGE) to identify E. huxleyi genes responsive to nitrogen (N) or phosphorus (P) starvation. Long SAGE is an elegant approach for examining quantitative and comprehensive gene expression patterns without a priori knowledge of gene sequences via the detection of 21-bp nucleotide sequence tags. E. huxleyi appears to have a robust transcriptional-level response to macronutrient deficiency, with 42 tags uniquely present or up-regulated twofold or greater in the N-starved library and 128 tags uniquely present or up-regulated twofold or greater in the P-starved library. The expression patterns of several tags were validated with reverse transcriptase PCR. Roughly 48% of these differentially expressed tags could be mapped to publicly available genomic or expressed sequence tag (EST) sequence data. For example, in the P-starved library a number of the tags mapped to genes with a role in P scavenging, including a putative phosphate-repressible permease and a putative polyphosphate synthetase. In short...

The Thin Pili of Acinetobacter sp. Strain BD413 Mediate Adhesion to Biotic and Abiotic Surfaces

Gohl, Olivia; Friedrich, Alexandra; Hoppert, Michael; Averhoff, Beate
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/2006 EN
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57.165654%
Two structurally different appendages, thin and thick pili, are found in members of the genus Acinetobacter. The presence of pilus structures correlates with different phenotypes, such as adherence to surfaces, a trait not only observed in pathogenic Acinetobacter species, as well as motility. However, their distinct individual roles were unknown. To characterize the role of different pili in the physiology of Acinetobacter, we isolated the thin pili from the cell surface of Acinetobacter sp. strain BD413 (recently recognized as representative of Acinetobacter baylyi), a soil bacterium that rapidly takes up naked DNA from its environment. Electron microcopy revealed that the pilus has an external diameter of 2 to 3 nm for single filaments. The filaments are packed into right-handed bundles. The major protein constituting the pilus was purified, and the encoding gene, acuA, was cloned. AcuA was found to be weakly related to the structural subunit of F17 pili of Escherichia coli. Analyses of the acuA flanking DNA region led to the identification of three closely associated genes, acuD, acuC, and acuG, whose deduced proteins are similar to chaperone, usher, and adhesin of F17-related pili, respectively. Transcriptional analyses revealed that acuA expression is maximal in the late-stationary-growth phase. Mutation of acuA led to a loss of thin pili and concomitantly loss of adhesion to polystyrene and erythrocytes but not loss of competence. Therefore...

Global Transcriptional and Physiological Responses of Saccharomyces cerevisiae to Ammonium, l-Alanine, or l-Glutamine Limitation†

Usaite, Renata; Patil, Kiran R.; Grotkjær, Thomas; Nielsen, Jens; Regenberg, Birgitte
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/2006 EN
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The yeast Saccharomyces cerevisiae encounters a range of nitrogen sources at various concentrations in its environment. The impact of these two parameters on transcription and metabolism was studied by growing S. cerevisiae in chemostat cultures with l-glutamine, l-alanine, or l-ammonium in limitation and by growing cells in an excess of ammonium. Cells grown in l-alanine-limited cultures had higher biomass yield per nitrogen mole (19%) than those from ammonium-limited cultures. Whole-genome transcript profiles were analyzed with a genome-scale metabolic model that suggested increased anabolic activity in l-alanine-limited cells. The changes in these cells were found to be focused around pyruvate, acetyl coenzyme A, glyoxylate, and α-ketoglutarate via increased levels of ALT1, DAL7, PYC1, GDH2, and ADH5 and decreased levels of GDH3, CIT2, and ACS1 transcripts. The transcript profiles were then clustered. Approximately 1,400 transcripts showed altered levels when amino acid-grown cells were compared to those from ammonium. Another 400 genes had low transcript levels when ammonium was in excess. Overrepresentation of the GATAAG element in their promoters suggests that nitrogen catabolite repression (NCR) may be responsible for this regulation. Ninety-one genes had transcript levels on both l-glutamine and ammonium that were decreased compared to those on l-alanine...

Quantifying Genes and Transcripts To Assess the In Situ Physiology of “Dehalococcoides” spp. in a Trichloroethene-Contaminated Groundwater Site▿ †

Lee, Patrick K. H.; Macbeth, Tamzen W.; Sorenson, Kent S.; Deeb, Rula A.; Alvarez-Cohen, Lisa
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
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Quantitative PCR (qPCR) was coupled with reverse transcription (RT) to analyze both gene copy numbers and transcripts of the 16S rRNA gene and three reductive dehalogenase (RDase) genes (tceA, vcrA, and bvcA) as biomarkers of “Dehalococcoides” spp. in the groundwater of a trichloroethene-dense nonaqueous-phase liquid site at Fort Lewis, WA, that was sequentially subjected to biostimulation and bioaugmentation. Dehalococcoides cells carrying the tceA, vcrA, and bvcA genes were indigenous to the site. The sum of the three identified RDase gene copy numbers closely correlated to 16S rRNA gene copy numbers throughout the biostimulation and bioaugmentation activity, suggesting that these RDase genes represented the major Dehalococcoides metabolic functions at this site. Biomarker quantification revealed an overall increase of more than 3 orders of magnitude in the total Dehalococcoides population through the 1-year monitoring period (spanning biostimulation and bioaugmentation), and measurement of the respective RDase gene concentrations indicated different growth dynamics among Dehalococcoides cells. The Dehalococcoides cells containing the tceA gene consistently lagged behind other Dehalococcoides cells in population numbers and made up less than 5% of the total Dehalococcoides population...

Synthesis and Accumulation of Cyanophycin in Transgenic Strains of Saccharomyces cerevisiae▿

Steinle, Anna; Oppermann-Sanio, Fred Bernd; Reichelt, Rudolf; Steinbüchel, Alexander
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
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Cyanophycin [multi-l-arginyl-poly(l-aspartic acid) (CGP)] was, for the first time, produced in yeast. As yeasts are very important production organisms in biotechnology, it was determined if CGP can be produced in two different strains of Saccharomyces cerevisiae. The episomal vector systems pESC (with the galactose-inducible promoter GAL1) and pYEX-BX (with the copper ion-inducible promoter CUP1) were chosen to express the cyanophycin synthetase gene from the cyanobacterium Synechocystis sp. strain PCC 6308 (cphA6308) in yeast. Expression experiments with transgenic yeasts revealed that the use of the CUP1 promoter is much more efficient for CGP production than the GAL1 promoter. As observed by electrophoresis of isolated CGP in sodium dodecyl sulfate-polyacrylamide gels, the yeast strains produced two different types of polymer: the water-soluble and the water-insoluble CGP were observed as major and minor forms of the polymer, respectively. A maximum CGP content of 6.9% (wt/wt) was detected in the cells. High-performance liquid chromatography analysis showed that the isolated polymers consisted mainly of the two amino acids aspartic acid and arginine and that, in addition, a minor amount (2 mol%) of lysine was present. Growth of transgenic yeasts in the presence of 15 mM lysine resulted in an incorporation of up to 10 mol% of lysine into CGP. Anti-CGP antibodies generated against CGP isolated from Escherichia coli TOP10 harboring cphA6308 reacted with insoluble CGP but not with soluble CGP...

Synthesis and Uptake of the Compatible Solutes Ectoine and 5-Hydroxyectoine by Streptomyces coelicolor A3(2) in Response to Salt and Heat Stresses▿

Bursy, Jan; Kuhlmann, Anne U.; Pittelkow, Marco; Hartmann, Holger; Jebbar, Mohamed; Pierik, Antonio J.; Bremer, Erhard
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
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Streptomyces coelicolor A3(2) synthesizes ectoine and 5-hydroxyectoine upon the imposition of either salt (0.5 M NaCl) or heat stress (39°C). The cells produced the highest cellular levels of these compatible solutes when both stress conditions were simultaneously imposed. Protection against either severe salt (1.2 M NaCl) or heat stress (39°C) or a combination of both environmental cues could be accomplished by adding low concentrations (1 mM) of either ectoine or 5-hydroxyectoine to S. coelicolor A3(2) cultures. The best salt and heat stress protection was observed when a mixture of ectoine and 5-hydroxyectoine (0.5 mM each) was provided to the growth medium. Transport assays with radiolabeled ectoine demonstrated that uptake was triggered by either salt or heat stress. The most effective transport and accumulation of [14C]ectoine by S. coelicolor A3(2) were achieved when both environmental cues were simultaneously applied. Our results demonstrate that the accumulation of the compatible solutes ectoine and 5-hydroxyectoine allows S. coelicolor A3(2) to fend off the detrimental effects of both high salinity and high temperature on cell physiology. We also characterized the enzyme (EctD) required for the synthesis of 5-hydroxyectoine from ectoine...

Transcriptional Analysis of Clostridium beijerinckii NCIMB 8052 and the Hyper-Butanol-Producing Mutant BA101 during the Shift from Acidogenesis to Solventogenesis▿ †

Shi, Zhen; Blaschek, Hans P.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
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Clostridium beijerinckii is an anaerobic bacterium used for the fermentative production of acetone and butanol. The recent availability of genomic sequence information for C. beijerinckii NCIMB 8052 has allowed for an examination of gene expression during the shift from acidogenesis to solventogenesis over the time course of a batch fermentation using a ca. 500-gene set DNA microarray. The microarray was constructed using a collection of genes which are orthologs of members of gene families previously found to be important to the physiology of C. acetobutylicum ATCC 824. Similar to the onset of solventogenesis in C. acetobutylicum 824, the onset of solventogenesis in C. beijerinckii 8052 was concurrent with the initiation of sporulation. However, forespores and endospores developed more rapidly in C. beijerinckii 8052 than in C. acetobutylicum 824, consistent with the accelerated expression of the sigE- and sigG-regulated genes in C. beijerinckii 8052. The comparison of gene expression patterns and morphological changes in C. beijerinckii 8052 and the hyper-butanol-producing C. beijerinckii strain BA101 indicated that BA101 was less efficient in sporulation and phosphotransferase system-mediated sugar transport than 8052 but that it exhibited elevated expression of several primary metabolic genes and chemotaxis/motility genes.

Development of Chemically Defined Media Supporting High-Cell-Density Growth of Lactococci, Enterococci, and Streptococci▿ †

Zhang, Guiying; Mills, David A.; Block, David E.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
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Lactococcus lactis IL1403 was used as an experimental strain to develop a chemically defined medium for study of the physiology and metabolic pathways of lactococci. An experimental leave-one-out technique was employed to determine the necessity of each of the 57 chemical components used in medium development. A statistical experimental design approach including three fractional factorial designs and a central composite design was used to optimize the fermentation process with 21 variables composed of 19 nutritional factors grouped from the 57 components and two environmental factors (initial pH and temperature). For L. lactis IL1403, the maximum biomass concentrations obtained with the two optimal chemically defined media developed in this study (ZMB1 and ZMB2) were generally 3.5- to 4-fold higher than the maximum biomass concentrations obtained with the previously described best synthetic media (SA) and 50% to 68% higher than the maximum biomass concentrations obtained with M17, a complex medium commonly used for lactococci. The new chemically defined media support high-cell-density growth of numerous strains of L. lactis, Enterococcus faecalis, and Streptococcus thermophilus.