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Effects of carbon sources on chemical composition of cell envelopes of Pseudomonas aeruginosa in association with polymyxin resistance.

Cells of Pseudomonas aeruginosa 015 were grown in basal medium with isobutyrate, DL-2-methylbutyrate, isovalerate, L-valine, L-isoleucine, L-leucine, D-glucose, or L-glutamate as the carbon source. Their resultant susceptibility to polymyxin B varied from a minimal inhibitory concentration of 2 U of polymyxin per ml for isobutyrate-grown cells to 975 U/ml for L-glutamate-grown cells. Cell envelopes from cells grown with each carbon source were compared with cell envelopes from cells grown in Mueller-Hinton broth as to their content of total protein, carbohydrate, and 2-keto-3-deoxyoctonate and as to their protein composition as determined by slab polyacrylamide gel electrophoresis. No pattern of cell envelope content of total protein, carbohydrate, 2-keto-3-deoxyoctonate, or outer membrane protein concentrations could be correlated with the degree of resistance to polymyxin. In these cells increased resistance to polymyxin was not associated with the loss of outer membrane proteins and lipopolysaccharide by the cell envelope.

Bacterial Proteins↗

[Production of polygalacturonase by Bacillus subtilis cultured with waste and residues as carbon sources].

In the recent study, it was observed that if when the initial pH was 7.0, incubation temperature was 37 degrees C and aeration (150 rpm) was supplied polygalacturonase production by Bacillus subtilis reached to its maximum. Molasses, Vinase and Peach or Apricot pomace were used as carbon sources at differing concentrations and the highest differential rate of enzyme synthesis was detected in the medium prepared with Vinase having a 5% of total sugar content (6.74 delta U/delta O.D.). It also was detected that the waste or residues mentioned above could be used as carbon sources for the polygalacturonase enzyme production by Bacillus subtilis.

Bacillus subtilis↗

[Utilization of paraffins and other noncarbohydrate carbon sources for microbial citric acid synthesis].

This article reviews the developments achieved in citrate and isocitrate accumulation with non-carbohydrate substrates by microorganisms presented as well in academic publications as in patients. The efficiency of citrate and isocitrate overproducing microorganisms and of mutants obtained thereof with respect to different carbon sources (n-alkanes, triglycerides, organic acids, etc.) is discussed. The influence of environmental conditions (media, pH etc.) and biochemical mechanisms which lead to metabolic overflow are emphasized. The kinetics of fermentation processes are described, calculations concerning carbon balances are involved. The production of by-products and the conversion of isocitrate to citrate is considered. The production of citric acid by yeasts which utilize different carbon sources may be economically feasible and an accession to the practized molasse-Aspergillus-process.

Aconitate Hydratase↗

The protein disulphide isomerase gene of the fungus Trichoderma reesei is induced by endoplasmic reticulum stress and regulated by the carbon source.

The gene pdi1 encoding protein disulphide isomerase was isolated from the filamentous fungus Trichoderma reesei by degenerate PCR based on a consensus PDI active-site sequence. It was shown that the Trichoderma pdi1 cDNA is able to complement a yeast mutant with a disrupted PDI1 gene. The putative T. reesei PD1I protein has a predicted 20-amino acid N-terminal signal sequence and the C-terminal fungal consensus ER retention signal HDEL. The mature protein shows strong conservation relative to other fungal protein disulphide isomerases. The T. reesei pdi1 promoter has two possible unfolded protein response (UPR) elements and it was shown by treatments with dithiothreitol and tunicamycin that the gene is under the control of the UPR pathway. Expression of a heterologous protein, an IgG antibody Fab fragment, in Trichoderma increases pdi1 expression, probably by inducing the UPR. The level of T. reesei pdi1 mRNA is also regulated by the carbon source, being lowest in glucose-containing media and highest on carbon sources that induce the genes encoding extracellular enzymes. The mechanism of this regulation was studied by examining pdi1 mRNA levels under conditions where the extracellular enzymes are induced by sophorose, as well as in the strain RutC-30, which is mutant for the glucose repressor gene cre1. The results suggest that neither sophorose induction nor glucose repression by the CREI protein affect the pdi1 promoter directly.

Amino Acid Sequence↗

Improvement of biomass yield and recombinant gene expression in Escherichia coli by using fructose as the primary carbon source.

The feasibility of substituting glucose with fructose as a carbon source in Escherichia coli fermentations was investigated. Glucose, the most commonly used sugar in bacterial cultivations, is well-known to pose a number of drawbacks; the most important of which is the Crabtree effect, which results in acidogenesis. Fructose, a glucose structural isomer, offers a reasonable alternative for glucose, since its uptake and utilization are more tightly regulated. Comparative fermentation studies indicate that lower acetate excretion and higher biomass yields were attained in fructose-supplemented growth media compared with those of glucose media. More specifically, cells grown in defined media supplemented with fructose do not excrete detectable amounts of acetate, while about 40 mM of acetate was detected extracellularly in similar glucose cultures. A reduction in the initial growth rate of about 20% was observed with fructose, but final cell densities were about 70% higher compared with glucose supplements. Growth in complex LB media supplemented with fructose again resulted in higher biomass yields (up to 40%) and lower acetate excretion (30-40%) than the comparable glucose media. In bioreactor studies using LB media, acetate levels were reduced from 90 to less than 6 mM, while achieving a 25% improvement in biomass yield. When using richer media, cell densities of more than 40 g L-1 dry cell weight were attained in batch cultivation using fructose compared with 30 g L-1 for glucose. These results have immense applicability in the area of recombinant protein processes. Recombinant E. coli, overexpressing beta-galactosidase under the control of the strong pH-inducible promoter, achieved a volumetric recombinant protein yield of 2.2 million U mL-1 (corresponding to approximately 1.5 g L-1) in batch fructose cultures. This represents a 65% recombinant protein yield enhancement when compared to similar glucose cultivations.

Biomass↗

Enrichment from activated sludges of aerobic mixed cultures capable to degrade vinyl chloride (VC) as the sole carbon source.

Two microbial cultures able to degrade high concentrations of VC as the sole carbon source have been obtained by enrichment from activated sludge. The cultures began consuming VC (0.02 mmol l(-1)) only after a long initial acclimation period (1-2 months). After then the concentration of VC was gradually increased (from 0.02 to 0.8 mmol l(-1)) and the cultures were able to maintain VC degrading ability for long time (over 500 days). VC-degrading biomass in the two cultures was characterized by low specific maximum growth rates (0.19-0.21 d(-1)) compared to heterotrophic organisms typically present in activated sludge processes. Monod half-saturation constant was rather low (0.7-1.6 mg VC l(-1)) indicating that it is possible to effectively remove VC to low residual concentrations. The cultures were highly sensitive to even short periods of VC lack (with quick decrease of VC degradation rates) whereas they were not to sudden load increases (up to 3.4 mmol l(-1)). After being cultured with only ethene as the sole carbon and energy source, the cultures kept the ability of degrading VC. Possibility of maintaining the mixed cultures on non-toxic ethene, without loosing VC degradation ability, is very promising for bioaugmentation treatments.

Bacteria, Aerobic↗

Denitrification rate and carbon source consumption in full-scale wastewater filtration.

In response to new demands for increased removal of nitrogen and phosphorus, the Henriksdal and Bromma treatment plants, with hydraulic loads of 283,000 and 148,000 m3/d, respectively, built filtration steps as a final process step in the plants. The denitrification rates in a full-scale and in a pilot plant filter are calculated to 13.1 and 21.3 g (NO3+NO2)-N/(m3 x h), respectively, in the total filter bed after 2.5-24.2 and 16.0-28.0 h of operational time, and 6.4 and 18.7 g (NO3+NO2)-N/(m3 x h), respectively, after 1.0 and 0.1-0.9 h of operational time. In composite samples, the denitrification rate in the total filter bed is 10-20 g (NO3+NO2)-N/(m3 x h) in the full-scale filter. The average values for k = deltaCODf/deltaC(T) are 1.6 and around 3 in the total filter bed in steady state and in the beginning of the experiments, respectively, both in the full-scale and in the pilot plant study. The carbon source costs for reducing the concentration of nitrate nitrogen in the Bromma plant from 12 to 8 mg/l in the effluent are 117,400 EUR and 147,400 EUR with methanol and ethanol, respectively, as a carbon source.

Acetates↗

Dbf2 is implicated in a Cbt1-dependent pathway following a shift from glucose to galactose or non-fermentable carbon sources in Saccharomyces cerevisiae.

Dbf2, a cell cycle-regulated protein kinase, has been shown recently to be part of the CCR4 transcriptional regulatory complex in the yeast Saccharomyces cerevisiae. We report here that temperature-sensitive (ts) dhf2-2 mutant cells can be rescued by overexpression of CDC14, which encodes a dual-specificity protein phosphatase, when grown on glucose-containing medium, as reported previously, but not on galactose. Screening of two S. cerevisiae cDNA libraries led to the identification of CBT1 as a gene which, if overexpressed simultaneously with CDC14, results in the rescue of the dbJ2-2 mutation at restrictive temperature on galactose-based medium, as well as on media containing non-fermentable carbon sources such as glycerol, lactate and acetate. Cbt1 has been shown previously to be essential for formation of cytochrome b in the mitochondria. On the other hand, the ts defects of ccr4delta and caf1delta mutants on glycerol medium at 37 degrees C (Ccr4 and Caf1/Pop2 are two other members of the CCR4 complex) could not be complemented by simultaneous overexpression of CBT1 and CDC14. CCR4 and CAF1 have been shown to play an essential role in activating the expression of genes for non-fermentative growth. Our results strongly suggest that, within the CCR4 complex, Dbf2 is directly involved in some mitochondrial function that depends on cytochrome b or on one of its main regulators, Cbt1. Therefore, Dbf2 may be required not only during mitosis but also during growth on non-fermentable carbon sources.

Carbon↗

Influence of Three Contrasting Detrital Carbon Sources on Planktonic Bacterial Metabolism in a Mesotrophic Lake.

Abstract Lakes receive organic carbon from a diversity of sources which vary in their contribution to planktonic microbial food webs. We conducted a mesocosm study to test the effects of three different detrital carbon sources (algae, aquatic macrophytes, terrestrial leaves) on several measures of microbial metabolism in a small meso-eutrophic lake (DOC approximately 5 mg/L). Small DOC additions (DeltaC < 1 mg/L) affected bacterial numbers, growth, and pathways of carbon acquisition. Macrophyte and leaf detritus significantly increased TDP and color, but bacterial densities initially (+12 h) were unaffected. After 168 h, densities in systems amended with terrestrial detritus were 60% less than in controls, while production rates in mesocosms with macrophyte detritus were 4-fold greater. Detritus treatments resulted in greater per-cell production rates either through stable cell numbers and greater growth rates (macrophyte-C) or lower densities with stable production rates (terrestrial-C). After only 12 h, rates of leucine aminopeptidase (LAPase) activity were 2.5x greater in macrophyte-C systems than in controls, but LAPase and beta-N-acetylglucosamindase activities in systems amended with terrestrial-C were only 50% of rates in controls. After 168 h, beta-xylosidase rates were significantly greater in communities with terrestrial and phytoplankton detritus. Microbial utilization of >20% of 102 carbon sources tested were affected by at least one detritus addition. Macrophyte-C had positive (6% of substrates) and negative (14%) effects on substrate use; terrestrial detritus had mainly positive effects. An ordination based on carbon-use profiles (+12 h) revealed a cluster of macrophyte-amended communities with greater use of psicose, lactulose, and succinamic acid; controls and algal-detritus systems were more effective in metabolizing two common sugars and cellobiose. After 168 h, communities receiving terrestrial detritus were most tightly clustered, exhibiting greater use of raffinose, pyroglutamic acid, and sebacic acid. Results suggest that pelagic bacterial communities respond to changes in organic carbon source rapidly and by different routes, including shifts in per-cell production rates and variations in degradation of a variety of compounds comprising the DOC pool.

Journal Article↗

Production of polysaccharidases in different carbon sources by Leucoagaricus gongylophorus Möller (Singer), the symbiotic fungus of the leaf-cutting ant Atta sexdens Linnaeus.

Leucoagaricus gongylophorus, the fungus cultured by the leaf-cutting ant Atta sexdens, produces polysaccharidases that degrade leaf components by generating nutrients believed to be essential for ant nutrition. We evaluated pectinase, amylase, xylanase, and cellulase production by L. gongylophorus in laboratory cultures and found that polysaccharidases are produced during fungal growth on pectin, starch, cellulose, xylan, or glucose but not cellulase, whose production is inhibited during fungal growth on xylan. Pectin was the carbon source that best stimulated the production of enzymes, which showed that pectinase had the highest production activity of all of the carbon sources tested, indicating that the presence of pectin and the production of pectinase are key features for symbiotic nutrition on plant material. During growth on starch and cellulose, polysaccharidase production level was intermediate, although during growth on xylan and glucose, enzyme production was very low. We propose a possible profile of polysaccharide degradation inside the nest, where the fungus is cultured on the foliar substrate.

Agaricales↗

High-osmolarity signalling in Saccharomyces cerevisiae is modulated in a carbon-source-dependent fashion.

High-osmolarity-induced expression of the small heat-shock gene HSP12 is regulated by the HOG (high-osmolarity glycerol) pathway and PKA (protein kinase A). To analyse the regulatory input of both signal transduction pathways, high-salt-induced HSP12 expression in different genetic backgrounds on glucose-, ethanol- and glycerol-based culture media was examined. Upon exposure to high-osmolarity stress, the kinetics of induction of HSP12 in cells growing on the non-fermentable carbon sources are strikingly different from those on glucose. Derepression of HSP12 gene expression under non-stress conditions was observed in cells growing on non-fermentable carbon sources. High-salt challenge resulted in a lower induction of the HSP12 mRNA levels in ethanol-grown cells as compared to glucose-grown cells, whereas in glycerol-grown cells hardly any high-salt induction of HSP12 mRNA levels could be detected. Analysis of signalling through the HOG pathway suggested that glycerol may influence the activity of this signalling route, possible via negative feedback. Furthermore, the cellular level of PKA activity was found to have a great impact on stress-responsive gene transcription. On the basis of the data obtained it was concluded that modulation of PKA activity plays a major role in the stress response. A glucose-dependent, PKA-regulated cellular component is postulated to affect high-osmolarity-induced HSP12 expression.

Carbon↗

Genomic footprinting of Mig1p in the MAL62 promoter. Binding is dependent upon carbon source and competitive with the Mal63p activator.

Mig1p inhibits gene expression in glucose by binding the Cyc8p (Ssn6p)-Tup1p repressor to the promoter of glucose-repressible genes. While the binding properties of Mig1p have been studied in vitro and the ability of Mig1p-Cyc8p (Ssn6p)-Tup1p to repress has been studied in vivo, no experiments have measured the effect of a carbon source on the in vivo binding of Mig1p or the effect of bound MIg1p on activator occupancy of the upstream activation sequence (UAS). To obtain this information, we used genomic footprinting to investigate glucose repression of MAL62, a gene that is also regulated by the Mal63p activator. These experiments show that two interrelated mechanisms are involved in the glucose repression of MAL62: 1) competition between the Mal63p activator and Mig1p for DNA binding and 2) modulation of Mig1p binding by the carbon source. Mig1p affects basal MAL62 expression in the absence of Mal63p by binding to a site in the MAL62 promoter and affects Mal63p-dependent synthesis by also inhibiting the access of Mal63p to site 1 in the UASMAL. The binding of Mig1p is increased in glucose and decreased in nonrepressing sugars, but the increased binding in glucose is not due to an increase in the levels of Mig1p.

Binding, Competitive↗

Cardiolipin is not essential for the growth of Saccharomyces cerevisiae on fermentable or non-fermentable carbon sources.

Cardiolipin is a unique dimeric phospholipid, which is present throughout the eukaryotic kingdom and is specifically localized in mitochondrial membranes. It is widely believed that mitochondria possess an essential requirement for this phospholipid. To determine whether cardiolipin is essential for yeast growth, we generated a cardiolipin synthase null mutant by disrupting the CLS1 gene (open reading frame YDL142c on chromosome IV) of Saccharomyces cerevisiae. Biochemical analysis of the mutant indicated that it had no cardiolipin synthase activity and no cardiolipin in its membranes. The enzyme phosphatidylglycerolphosphate synthase, which catalyses the committed step of the cardiolipin pathway, remained unaffected in the null mutant. Haploid cells containing the null allele are viable in media containing glucose, galactose or glycerol/ethanol as the sole carbon source, although growth in galactose or glycerol/ethanol is somewhat reduced in the mutant compared with the wild type. These results indicate that cardiolipin is not essential for the growth of S. cerevisiae in fermentable or non-fermentable carbon sources.

Amino Acid Sequence↗

Mouse intestine selects nonmotile flhDC mutants of Escherichia coli MG1655 with increased colonizing ability and better utilization of carbon sources.

D-gluconate which is primarily catabolized via the Entner-Doudoroff (ED) pathway, has been implicated as being important for colonization of the streptomycin-treated mouse large intestine by Escherichia coli MG1655, a human commensal strain. In the present study, we report that an MG1655 Deltaedd mutant defective in the ED pathway grows poorly not only on gluconate as a sole carbon source but on a number of other sugars previously implicated as being important for colonization, including L-fucose, D-gluconate, D-glucuronate, N-acetyl-D-glucosamine, D-mannose, and D-ribose. Furthermore, we show that the mouse intestine selects mutants of MG1655 Deltaedd and wild-type MG1655 that have improved mouse intestine-colonizing ability and grow 15 to 30% faster on the aforementioned sugars. The mutants of MG1655 Deltaedd and wild-type MG1655 selected by the intestine are shown to be nonmotile and to have deletions in the flhDC operon, which encodes the master regulator of flagellar biosynthesis. Finally, we show that DeltaflhDC mutants of wild-type MG1655 and MG1655 Deltaedd constructed in the laboratory act identically to those selected by the intestine; i.e., they grow better than their respective parents on sugars as sole carbon sources and are better colonizers of the mouse intestine.

Animals↗

Genome-wide analysis of differentially expressed genes from Penicillium chrysogenum grown with a repressing or a non-repressing carbon source.

Penicillium chrysogenum is an economically important ascomycete used as industrial producer of penicillin. However, with the exception of penicillin biosynthesis genes, little attention has been paid to the genetics of other aspects of the metabolism of this fungus. In this article we describe the first attempt of systematic analysis of expressed genes in P. chrysogenum, using a suppression subtractive hybridization approach to clone and identify sequences of genes differentially expressed in media with glucose or lactose as carbon source (penicillin-repressing or non-repressing conditions). A total of 167 clones were analysed, 95 from the glucose condition and 72 from the lactose condition. Genes differentially expressed in the glucose condition encode mainly proteins involved in the mitochondrial electron transport chain and primary metabolism. Genes expressed differentially in lactose-containing medium include genes for secondary metabolism (pcbC, isopenicillin N synthase), different hydrolases and a gene encoding a putative hexose transporter or sensor. The results provided information on how the metabolism of this fungus adapts to different carbon sources. The expression patterns of some of the genes support the hypothesis that glucose induces higher rates of respiration in P. chrysogenum while repressing secondary metabolism.

Amino Acid Sequence↗

An overlooked carbon source for grassland soils: loss of structural carbon from stubble in response to elevated pCO2 and nitrogen supply.

In grasslands, the loss of structural carbon (C) from nonharvested plant parts is a primary C source for the soil. The amount of input depends not only on the size of structural C pools but also on their loss rates. In the field, we examined the effects of elevated atmospheric partial pressures of CO2 (pCO2) and nitrogen (N) supply on pool size and rates of structural C loss in stubble and roots of perennial ryegrass (Lolium perenne) by using multiple-pulse labelling and steady-state labelling. Stubble retained structural C for roughly half the time it was retained in roots. Elevated pCO2 combined with low N supply enlarged the pools of roots and stubble. These conditions also stimulated the rate of structural C loss from stubble and, thus, the amounts available for further transformation. The potential of multiple-pulse labelling as a field technique is highlighted. The stimulation of structural C loss from stubble by elevated pCO2 at low N provides a missing link between increased C assimilation and low yield response and indicates a potentially higher input of structural C into the soil.

Biomass↗

Coordinate increase in activity of proteinase subunits of the 1300-kDa megaproteinase of Frankia strain BR: role of carbon source depletion and extracellular metabolites.

We have recently described the presence of a high molecular mass multicatalytic proteinase complex (megaproteinase; 28 S, 1300 kDa) in Frankia strain BR. The complex dissociates into 11 low molecular mass proteinase subunits (40-19 kDa) when subjected to sodium dodecyl sulfate - gelatin - polyacrylamide gel electrophoresis. We show here that the activity of these proteinase subunits strongly increased after cessation of growth in stirred BAP-PCM mineral medium. Subsequent addition of either BAP medium components or sodium propionate alone, as carbon source, to a Frankia culture at the end of the exponential growth phase was found to prolong growth for 1 additional day, and to delay the increase in activity of the proteinase subunits for 3 days after cessation of growth. Addition of ammonium chloride alone, as nitrogen source, had no effect. On the other hand, when Frankia cells in the late exponential phase (3 days) were resuspended in a culture filtrate recovered from a 5-day-old culture and supplemented with BAP-PCM medium components, the biomass yield decreased to about 50%. Also, the activity of the proteinase subunits increased as soon as growth ceased. The ability of this culture filtrate to inhibit growth and stimulate the activity of proteinase subunits was partially lost by heating or was largely removed by DEAE-cellulose treatment. Thus, our findings indicate an extracellular control of Frankia megaproteinase activity, suggesting that carbon source depletion and probably accumulation of heat-sensitive growth-inhibiting metabolites in the medium are determining factors.

Carbon↗

A carbon-source-responsive element is required for regulation of the hypoxic ADP/ATP carrier (AAC3) isoform in Saccharomyces cerevisiae.

The mitochondrial ADP/ATP carrier in Saccharomyces cerevisiae is encoded by three genes that are differentially expressed under different physiological conditions. We investigated the transcriptional control of AAC3, an oxygen-repressed isoform. By deletion analysis, DNA electrophoretic mobility-shift assays, DNase I footprinting and site-directed mutagenesis, we have identified a promoter region (upstream repressing sequence 1, URS(1)) involved in a carbon-source-dependent repression of AAC3. It is different from the previously characterized oxygen-dependent ROX1 (regulation by oxygen 1) repressor-binding region (URS(2)). The complex character of URS(1) includes the presence of two different cis-acting sequences: (i) a RAP1 (repressor activator protein 1)-binding site that is capable of binding the RAP1 protein in vitro and (ii) two putative ethanol-repression sequences, the modification of which derepresses the AAC3 gene. These findings demonstrate that the hypoxic AAC3 gene is regulated by two upstream repressor sites; one controlled by oxygen and haem, the other by the carbon source. Both sites function to completely switch off the expression of the AAC3 isoform when ATP is made by oxidative phosphorylation, and they modulate AAC3 expression when import of glycolytic ATP into mitochondria is required.

Base Sequence↗