Binding-protein-dependent glucose transport by Agrobacterium radiobacter grown in glucose-limited continuous culture

Agrobacterium radiobacter NCIB 11883 was grown in glucose-limited continuous culture at low dilution rate. Whole cells transported glucose using an energy-dependent mechanism which exhibited an accumulation ratio greater than 2000. Three major periplasmic proteins were purified and their potential role as glucose-binding proteins (GBP) were investigated using equilibrium dialysis. Two of these, GBP1 (Mr 36,500) and GBP2 (Mr 33,500), bound D-glucose with high affinity (KD 0.23 and 0.07 microM respectively), whereas the third protein (Mr 30,500) showed no binding ability. Competition experiments using various analogues showed that those which differed from glucose at C-6 (e.g. 6-chloro-6-deoxy-D-glucose and 6-deoxy-D-glucose) variably decreased the binding of glucose to both GBP1 and GBP2, whereas those which differed at C-4 (e.g. D-galactose) were only effective with GBP1. The rate of glucose uptake and the concentration of the glucose-binding proteins increased in parallel during prolonged growth under glucose-limitation due to the emergence of new strains in which GBP1 (e.g. strain AR18) or GBP2 (e.g. strain AR9), but not both, was hyperproduced and accounted for at least 27% of the total cell protein. It is concluded that A. radiobacter synthesizes two distinct periplasmic binding proteins which are involved in glucose transport, and that these proteins are maximally derepressed during growth under glucose limitation.     

The relationship between glucose transport and the production of succinoglucan exopolysaccharide by Agrobacterium radiobacter

Agrobacterium radiobacter NCIB 11883 was grown in ammonia-limited continuous culture at low dilution rate with glucose as the carbon source. Under these conditions the organism produced an extracellular succinoglucan polysaccharide and transported glucose using the same periplasmic glucose-binding proteins (GBP1 and GBP2) as during glucose-limited growth. Transition from glucose- to ammonia-limited growth was accompanied by a very rapid decrease in glucose uptake capacity, whereas the glucose-binding proteins were diluted out much more slowly (t1/2 approximately 1 h and 14 h respectively). Although the rate of glucose uptake and the concentrations of GBP1 and GBP2 were much lower during ammonia limitation, the activities of enzymes involved in the early stages of glucose metabolism and in the production of succinoglucan precursors were essentially unchanged. Glucose transport was also investigated in two new strains of A. radiobacter which had been isolated following prolonged growth under glucose limitation. Glucose uptake by strain AR18 was significantly less repressed during ammonia limitation compared with either the original parent strain or strain AR9, and this was reflected both in its relatively high concentration of GBP1 and in its significantly higher rate of succinoglucan synthesis. Flux control analysis using 6-chloro-6-deoxy-D-glucose as an inhibitor of glucose transport showed that the latter was a major kinetic control point for succinoglucan production. It is concluded that glucose uptake by A. radiobacter, particularly via the GBP1-dependent system, is only moderately repressed during ammonia-limited growth and that the organism avoids the potentially deleterious effects of accumulating excess glucose by converting the surplus into succinoglucan.     

The Agrobacterium tumefaciens virulence gene chvE is part of a putative ABC-type sugar transport operon.

The Agrobacterium tumefaciens virulence determinant ChvE is a periplasmic binding protein which participates in chemotaxis and virulence gene induction in response to monosaccharides which occur in the plant wound environment. The region downstream of the A. tumefaciens chvE gene was cloned and sequenced for nucleotide and expression analysis. Three open reading frames transcribed in the same direction as chvE were revealed. The first two, together with chvE, encode putative proteins of a periplasmic binding protein-dependent sugar uptake system, or ABC-type (ATP binding cassette) transporter. The third open reading frame encodes a protein of unknown function. The deduced transporter gene products are related on the amino acid level to bacterial sugar transporters and probably function in glucose and galactose uptake. We have named these genes gguA, -B, and -C, for glucose galactose uptake. Mutations in gguA, gguB, or gguC do not affect virulence of A. tumefaciens on Kalanchoe diagremontiana; growth on 1 mM galactose, glucose, xylose, ribose, arabinose, fucose, or sucrose; or chemotaxis toward glucose, galactose, xylose, or arabinose.     

Isolation of dicarboxylic acid- and glucose-binding proteins from Pseudomonas aeruginosa.

Inducible binding proteins for C4-dicarboxylic acids (DBP) and glucose (GBP) were isolated from Pseudomonas aeruginosa by extraction of exponential-phase cells with 0.2 M MgC12 (pH 8.5) and by an osmotic shock procedure without affecting cell viability. DBP synthesis was induced by growth on aspartate, alpha-ketoglutarate, succinate, fumarate, malate, and malonate but not by growth on acetate, citrate, pyruvate, or glucose. Binding of succinate by DBP was competitively inhibited by 10-fold concentrations of fumarate and malate but not by a variety of related substances. GBP synthesis and transport of methyl alpha-glucoside by whole cells were induced by growth on glucose or pyruvate plus galactose, 2-deoxyglucose, or methyl alpha-glucoside but not by growth on gluconate, succinate, acetate, or pyruvate. The binding of radioactive glucose by GBP was significantly inhibited by 10-fold concentrations of glucose, galactose, and glucose-1-phosphate but not by the other carbohydrates tested. The binding of glucose by GBP or succinate by DBP did not result in any chemical alteration of the substrates.     

Functional survey for heterologous sugar transport proteins, using Saccharomyces cerevisiae as a host

Molecular transport is a key process in cellular metabolism. This step is often limiting when using a nonnative carbon source, as exemplified by xylose catabolism in Saccharomyces cerevisiae. As a step toward addressing this limitation, this study seeks to characterize monosaccharide transport preference and efficiency. A group of 26 known and putative monosaccharide transport proteins was expressed in a recombinant Saccharomyces cerevisiae host unable to transport several monosaccharides. A growth-based assay was used to detect transport capacity across six different carbon sources (glucose, xylose, galactose, fructose, mannose, and ribose). A mixed glucose-and-xylose cofermentation was performed to determine substrate preference. These experiments identified 10 transporter proteins that function as transporters of one or more of these sugars. Most of these proteins exhibited broad substrate ranges, and glucose was preferred in all cases. The broadest transporters confer the highest growth rates and strongly prefer glucose. This study reports the first molecular characterization of the annotated XUT genes of Scheffersomyces stipitis and open reading frames from the yeasts Yarrowia lipolytica and Debaryomyces hansenii. Finally, a phylogenetic analysis demonstrates that transporter function clusters into three distinct groups. One particular group comprised of D. hansenii XylHP and S. stipitis XUT1 and XUT3 demonstrated moderate transport efficiency and higher xylose preferences.     

Binding-protein-dependent lactose transport in Agrobacterium radiobacter.

Agrobacterium radiobacter NCIB 11883 was grown in lactose-limited continuous culture at a dilution rate of 0.045/h. Washed cells transported [14C]lactose and [methyl-14C]beta-D-thiogalactoside, a nonmetabolisable analog of lactose, at similar rates and with similar affinities (Km for transport, less than 1 microM). Transport was inhibited to various extents by the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, by unlabeled beta-galactosides and D-galactose, and by osmotic shock. The accumulation ratio for methyl-beta-D-thiogalactoside was greater than or equal to 4,100. An abundant protein (molecular weight, 41,000) was purified from osmotic-shock fluid and shown by equilibrium dialysis to bind lactose and methyl-beta-D-thiogalactoside, the former with very high affinity (binding constant, 0.14 microM). The N-terminal amino acid sequence of this lactose-binding protein exhibited some homology with several other sugar-binding proteins from bacteria. Antiserum raised against the lactose-binding protein did not cross-react with two glucose-binding proteins from A. radiobacter or with extracts of other bacteria grown under lactose limitation. Lactose transport and beta-galactosidase were induced in batch cultures by lactose, melibiose [O-alpha-D-galactoside-(1----6)alpha-D-glucose], and isopropyl-beta-D-thiogalactoside and were subject to catabolite repression by glucose, galactose, and succinate which was not alleviated by cyclic AMP. We conclude that lactose is transported into A. radiobacter via a binding protein-dependent active transport system (in contrast to the H+ symport and phosphotransferase systems found in other bacteria) and that the expression of this transport system is closely linked to that of beta-galactosidase.     

Influence of cosubstrate concentration on xylose conversion by recombinant, XYL1-expressing Saccharomyces cerevisiae: a comparison of different sugars and ethanol as cosubstrates.

Conversion of xylose to xylitol by recombinant Saccharomyces cerevisiae expressing the XYL1 gene, encoding xylose reductase, was investigated by using different cosubstrates as generators of reduced cofactors. The effect of a pulse addition of the cosubstrate on xylose conversion in cosubstrate-limited fed-batch cultivation was studied. Glucose, mannose, and fructose, which are transported with high affinity by the same transport system as is xylose, inhibited xylose conversion by 99, 77, and 78%, respectively, reflecting competitive inhibition of xylose transport. Pulse addition of maltose, which is transported by a specific transport system, did not inhibit xylose conversion. Pulse addition of galactose, which is also transported by a specific transporter, inhibited xylose conversion by 51%, in accordance with noncompetitive inhibition between the galactose and glucose/ xylose transport systems. Pulse addition of ethanol inhibited xylose conversion by 15%, explained by inhibition of xylose transport through interference with the hydrophobic regions of the cell membrane. The xylitol yields on the different cosubstrates varied widely. Galactose gave the highest xylitol yield, 5.6 times higher than that for glucose. The difference in redox metabolism of glucose and galactose was suggested to enhance the availability of reduced cofactors for xylose reduction with galactose. The differences in xylitol yield observed between some of the other sugars may also reflect differences in redox metabolism. With all cosubstrates, the xylitol yield was higher under cosubstrate limitation than with cosubstrate excess.     

Characterization of sugar mixtures utilization by an Escherichia coli mutant devoid of the phosphotransferase system

Due to catabolite repression in microorganisms, sugar mixtures cannot be metabolized in a rapid and efficient manner. Therefore, the development of mutant strains that avoid this regulatory system is of special interest to fermentation processes. In the present study, the utilization of sugar mixtures by an Escherichia coli mutant strain devoid of the phosphotransferase system (PTS) was characterized. This mutant can transport glucose (PTS- Glucose+ phenotype) by a non-PTS mechanism as rapidly as its wild-type parental strain. In cultures grown in minimal medium supplemented with glucose-xylose or glucose-arabinose mixtures, glucose repressed arabinose- or xylose-utilization in the wild-type strain. However, under the same culture conditions with the PTS- Glucose+ mutant, glucose and arabinose were co-metabolized, but glucose still exerted a partial repressive effect on xylose consumption. In cultures growing with a triple mixture of glucose-arabinose-xylose, the wild-type strain sequentially utilized glucose, arabinose and finally, xylose. In contrast, the PTS- Glucose+ strain co-metabolized glucose and arabinose, whereas xylose was utilized after glucose-arabinose depletion. As a result of glucose-arabinose co-metabolism, the PTS- Glucose+ strain consumed the total amount of sugars contained in the culture medium 16% faster than the wild-type strain. [14C]-Xylose uptake experiments showed that in the PTS- Glucose+ strain, galactose permease increases xylose transport capacity and the observed partial repression of xylose utilization depends on the presence of intracellular glucose.     

Phylogenetic assignment and mechanism of action of a crop growth promoting <Emphasis Type="Italic">Rhizobium radiobacter</Emphasis> strain used as a biofertiliser on graminaceous crops in Russia

The taxonomic position of “Agrobacterium radiobacter strain 204,” used in Russia as a cereal crop growth promoting inoculant, was derived by a polyphasic approach. The phenotypic analyses gave very similar biochemical profiles for strain 204, Rhizobium radiobacter NCIMB 9042 (formerly the A. radiobacter type strain) and R. radiobacter NCIMB 13307 (formerly the Agrobacterium␣tumefaciens type strain). High percentage similarities, above the species separation level, were observed between the 16S rRNA, fusA and rpoB housekeeping gene sequences of these three strains, and the genomic DNA–DNA hybridisation of strain 204 against the type strain of R. radiobacter NCIMB 9042 was over 70%. Strain 204 is not phytopathogenic and it does not fix atmospheric N₂ or form a physical association with the roots of barley. Strain 204 culture and culture supernatant stimulated the rate of mobilisation of seed reserves of barley in darkness and promoted its shoot growth in the light. Gibberellic acid (GA) concentration was 1.3 μM but indole acetic acid was undetectable (<50 nM) in cultures of strain 204. It is concluded that strain 204 is phenotypically and genotypically very similar to the current R. radiobacter type strain and that the mechanism of its effect on growth of cereals is via the production of plant growth promoting substances. GA is likely to play an important role in the strain 204 stimulation of early growth of barley.     

Molecular analysis of the lac operon encoding the binding-protein-dependent lactose transport system and β-galactosidase in Agrobacterium radiobacter

The genes coding for the binding-protein-dependent lactose transport system and beta-galactosidase in Agrobacterium radiobacter strain AR50 were cloned and partially sequenced. A novel lac operon was identified which contains genes coding for a lactose-binding protein (lacE), two integral membrane proteins (lacF and lacG), an ATP-binding protein (lacK) and beta-galactosidase (lacZ). The operon is transcribed in the order lacEFGZK. The operon is controlled by an upstream regulatory region containing putative -35 and -10 promoter sites, an operator site, a CRP-binding site probably mediating catabolite repression by glucose and galactose, and a regulatory gene (lacl) encoding a repressor protein which mediates induction by lactose and other galactosides in wild-type A. radiobacter (but not in strain AR50, thus allowing constitutive expression of the lac operon). The derived amino acid sequences of the gene products indicate marked similarities with other binding-protein-dependent transport systems in bacteria.     

Production of xylitol from D-xylose by recombinant Lactococcus lactis

The D-xylose reductase from Pichia stipitis CBS 5773 and the xylose transporter from Lactobacillus brevis ATCC 8287 were expressed in active form in Lactococcus lactis NZ9800. Xylitol production was investigated using non-growing recombinant cells in high cell-density under microaerobic conditions in the presence of xylose and glucose. Besides xylose, the recombinant strain with xylose reductase activity reduced l-arabinose and D-ribose in significant extent to the corresponding pentitols. The ratio of xylitol produced per glucose consumed was almost 10-fold higher under glucose limitation than the ratio in the presence of excess initial glucose. The co-expression of the xylose transporter with the xylose reductase did not increase the efficiency of xylitol production appreciably when compared to the strain in which only the xylose reductase gene was expressed. A fed-batch experiment with high initial xylose concentration (160 gl(-1)) under glucose limitation was carried out using the strain co-expressing xylose reductase and xylose transporter genes. The xylitol yield from xylose was 1.0 mol mol(-1) and the ratio of xylitol produced per glucose consumed was 2.5 mol mol(-1). The volumetric productivity was 2.72 gl(-1)h(-1) at 20 h. Of the xylose initially present, 34% was consumed. Analysis of the fermentation metabolites revealed a shift from homolactic to mixed acid fermentation at early stages of the experiment.     

Carbohydrate utilization patterns and substrate preferences in Bacteroides thetaiotaomicron

Specific growth rates of Bacteroides thetaiotaomicron NCTC 10582 with either glucose, arabinose, mannose, galactose or xylose as sole carbon sources were 0.42/h, 0.10/h, 0.38/h, 0.38/h and 0.16/h respectively, suggesting that hexose metabolism was energetically more efficient than pentose fermentation in this bacterium. Batch culture experiments to determine whether carbohydrate utilization was controlled by substrate-induced regulatory mechanisms demonstrated that mannose inhibited uptake of glucose, galactose and arabinose, but had less effect on xylose. Arabinose and xylose were preferentially utilized at high dilution rates (D > 0.26/h) in carbon-limited continuous cultures grown on mixtures of arabinose, xylose, galactose and glucose. When mannose was also present, xylose was co-assimilated at all dilution rates. Under nitrogen-limited conditions, however, mannose repressed uptake of all sugars, showing that its effect on xylose utilization was strongly concentration dependent. Studies with individual D-ZU-14C]-labelled substrates showed that transport systems for glucose, galactose, xylose and mannose were inducible. Measurements to determine incorporation of these sugars into trichloroacetic acid-precipitable material indicated that glucose and mannose were the principal precursor monosaccharides. Xylose was only incorporated into intracellular macromolecules when it served as growth substrate. Phosphoenolpyruvate:phosphotransferase systems were not detected in preliminary experiments to elucidate the mechanisms of sugar uptake, and studies with inhibitors of carbohydrate transport showed no consistent pattern of inhibition with glucose, galactose, xylose and mannose. These results indicate the existence of a variety of different systems involved in sugar transport in B. thetaiotaomicron.     

Characterization and regulation of galactose transport in Neurospora crassa

Two galactose uptake systems were found in the mycelia of Neurospora crassa. In glucose-grown mycelia, galactose was transported by a low-affinity (Km = 400 mM) constitutive system which was distinct from the previously described glucose transport system I (R. P. Schneider and W. R. Wiley, J. Bacteriol. 106:479--486, 1971). In carbon-starved mycelia or mycelia incubated with galactose, a second galactose transport activity appeared which required energy, had a high affinity for galactose (Km = 0.7 mM), and was shown to be the same as glucose transport system II. System II also transported mannose, 2-deoxyglucose, xylose, and talose and is therefore a general monosaccharide transport system. System II was derepressed by carbon starvation, completely repressed by glucose, mannose, and 2-deoxyglucose, and partially repressed by fructose and xylose. Incubation with galactose yielded twice as much activity as starvation. This extra induction by galactose required protein synthesis, and represented an increase in activity of system II rather than the induction of another transport system. Glucose, mannose, and 2-deoxyglucose caused rapid degradation of preexisting system II; fructose and xylose caused a slower degradation of activity.     

马克斯克鲁维酵母GX-UN120糖转运蛋白基因的克隆及表征

【背景】马克斯克鲁维酵母(Kluyveromyces marxianus)具有完整的木糖代谢途径,可以高效利用木质纤维素中的木糖,因此对其糖转运蛋白基因的研究或可有效解决酵母木糖转运的相关问题。【目的】根据马克斯克鲁维酵母DMKU3-1042中KLMA＿70145和KLMA＿80101基因位点的功能预测,获得马克斯克鲁维酵母GX-UN120相应的糖转运蛋白基因序列并探究其功能。【方法】将转运蛋白基因分别克隆表达至酿酒酵母EBY.VW4000中考察重组菌株生长特性,以此间接评价对应转运蛋白的转运能力。【结果】Km＿SUT2基因编码的糖转运蛋白可有效提高宿主细胞转运木糖、阿拉伯糖、山梨糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖、果糖、蔗糖和半乳糖。类似地,Km＿SUT3基因编码的糖转运蛋白可提高细胞转运木糖、阿拉伯糖、山梨糖、半乳糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖和果糖。然而在葡萄糖存在的条件下,重组菌株对各种碳源的利用均受抑制,但Km＿SUT3转运木糖和核糖过程中受葡萄糖的抑制作用较小。【结论】马克斯克鲁维酵母GX-UN120中转运蛋白Km＿SUT2和Km＿SUT3可...     

Purification and Properties of the Periplasmic Glucose-Binding Protein of Pseudomonas aeruginosa

A glucose-binding glycoprotein (GBP) from the periplasm of Pseudomonas aeruginosa was purified to homogeneity as judged by polyacrylamide gel electrophoresis, molecular sieve chromatography, and double-diffusion gel precipitation. It had an average molecular weight of 44,500 and an isoelectric point of 4.7. One mole of glucose was bound per mole of GBP with a dissociation constant of 0.35 muM. The binding of radioactive glucose by GBP was not significantly inhibited by 10-fold-higher concentrations of other carbohydrates; however, a number of related compounds were found to compete at 100-fold-higher concentrations. Amino acid analyses revealed predominant amounts of alanine, glutamate, and glycine and a low content of sulfur-containing amino acids. The carbohydrate moiety of GBP, comprising nearly 16% of the total weight, contained galactosamine, glucosamine, fucose, galactose, glucose, and mannose. A GBP-deficient mutant, strain MB723, was found to be defective in both membrane transport and glucose chemotaxis. Strain MB724, a revertant to GBP-positive phenotype, simultaneously recovered normal levels of both membrane functions.     

The influence of hexoses addition on the fermentation of d-xylose in Debaryomyces hansenii under continuous cultivation

The effect of hexoses (glucose and galactose) addition to the feed xylose mineral medium of Debaryomyces hansenii chemostat cultures grown at a constant dilution rate of 0.055 h⁻¹ was studied. Xylitol was the major product detected amongst all tested conditions. The maximal values for xylitol yield and volumetric productivity (0.56 gg⁻¹ xylose and 0.21 gl⁻¹h⁻¹, respectively) were obtained for a glucose/xylose feeding ratio of 10%, showing that the addition of small amounts of glucose, but not galactose, enhanced the xylitol production. A xylitol yield increase of 30%, compared with the sole xylose-containing feed medium, was observed. It was found that the oxygen requirement for D. hansenii growth is lower under glucose compared with xylose. Ethanol and glycerol were only produced for glucose/xylose feeding ratio above 30%. The byproducts accumulation was correlated with glucose metabolism, because a direct relationship between the increase of ethanol (and glycerol) concentration and the increase of glucose in the feed medium was found.     

Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain

We have recently reported about a Saccharomyces cerevisiae strain that, in addition to the Piromyces XylA xylose isomerase gene, overexpresses the native genes for the conversion of xylulose to glycolytic intermediates. This engineered strain (RWB 217) exhibited unprecedentedly high specific growth rates and ethanol production rates under anaerobic conditions with xylose as the sole carbon source. However, when RWB 217 was grown on glucose-xylose mixtures, a diauxic growth pattern was observed with a relatively slow consumption of xylose in the second growth phase. After prolonged cultivation in an anaerobic, xylose-limited chemostat, a culture with improved xylose uptake kinetics was obtained. This culture also exhibited improved xylose consumption in glucose-xylose mixtures. A further improvement in mixed-sugar utilization was obtained by prolonged anaerobic cultivation in automated sequencing-batch reactors on glucose-xylose mixtures. A final single-strain isolate (RWB 218) rapidly consumed glucose-xylose mixtures anaerobically, in synthetic medium, with a specific rate of xylose consumption exceeding 0.9 gg(-1)h(-1). When the kinetics of zero trans-influx of glucose and xylose of RWB 218 were compared to that of the initial strain, a twofold higher capacity (V(max)) as well as an improved K(m) for xylose was apparent in the selected strain. It is concluded that the kinetics of xylose fermentation are no longer a bottleneck in the industrial production of bioethanol with yeast.     

Recovery from galactose-induced neurotoxicity in the chick by the administration of glucose

Abstract— Chicks fed a diet containing 40% (w/w) galactose demonstrated convulsive activity after 52–54 h. However, they recovered from both physical and biochemical symptoms temporarily, following the intraperitoneal injection of glucose. The previously decreased levels of ATP and elevated levels of AMP in the brains of chicks fed galactose returned to normal within 20 min following the glucose treatment. During the recovery phase, plasma glucose content rose and brain glucose returned to the normal range, whereas the levels of brain galactose and its metabolites, galactitol and galactose 1-phosphate, were unchanged. Moderate plasma hyperasmolality was induced in chicks fed the diet containing galactose and xylose or saline in the drinking water. Neurotoxicity was observed only in the group fed galactose, although brain glucose and glycogen were reduced in chicks fed xylose. In the brains of chicks fed xylose, xylitol was identified by gas-liquid chromatography and mass spectrometry, and the amount was approximately 10 per cent of the amount of xylose simultaneously found in the brain. These studies support the viewpoint that dietary galactose exerts its acute neurotoxicity in chicks primarily by inhibition of glucose transport across the blood-brain barrier.