Glucose Permease Research Articles

In vivo Inhibition of the 3-Dehydroquinate Synthase by 7-Deoxysedoheptulose Depends on Promiscuous Uptake by Sugar Transporters in Cyanobacteria.

7-Deoxysedoheptulose (7dSh) is a bioactive deoxy-sugar actively excreted by the unicellular cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) but also Streptomyces setonensis. In our previous publications we have shown that in S. elongatus, 7dSh is exclusively synthesized by promiscuous enzyme activity from an inhibitory by-product of radical SAM enzymes, without a specific gene cluster being involved. Additionally, we showed that 7dSh inhibits the growth of cyanobacteria, but also the growth of plants and fungi, presumably by inhibiting the 3-dehydroquinate synthase (DHQS), the second enzyme of the shikimate pathway, as the substrate of this enzyme strongly accumulates in cells treated with 7dSh. In this study, by using purified DHQS of Anabaena variabilis ATCC 29413 (A. variabilis) we biochemically confirmed that 7dSh is a competitive inhibitor of this enzyme. By analyzing the effect of 7dSh on a subset of cyanobacteria from all the five subsections, we identified different species whose growth was inhibited by 7dSh. We also found that in some of the susceptible cyanobacteria import of 7dSh is mediated by structurally different and promiscuous transporters: 7dSh can be taken up by the fructose ABC-transporter in A. variabilis and via the glucose permease in Synechocystis sp. PCC 6803 (Synechocystis sp.). In both cases, an effective uptake and thereby intracellular enrichment of 7dSh was essential for the inhibitory activity. Importantly, spontaneous mutations in the sugar transporters of A. variabilis and Synechocystis sp. not only disabled growth of the two strains on fructose and glucose, respectively, but also almost abolished their sensitivity to 7dSh. Although we have clearly shown in these examples that the effective uptake plays an essential role in the inhibitory effect of 7dSh, questions remain about how 7dSh resistance works in other (cyano)bacteria. Also, the involvement of a putative ribokinase in 7dSh resistance in the producer strain S. elongatus remained to be further investigated. Overall, these data establish 7dSh as the first allelochemical targeting the shikimate pathway in other cyanobacteria and plants and suggest a role of 7dSh in niche competition.

Improving glucose and xylose assimilation in Azotobacter vinelandii by adaptive laboratory evolution.

Azotobacter vinelandii is a microorganism with biotechnological potential because its ability to produce alginate and polyhydroxybutyrate. Large-scale biotechnological processes are oriented to sustainable production by using biomass hydrolysates that are mainly composed by glucose and xylose. In the present study, it was observed that A. vinelandii was unable to consume xylose as the sole carbon source and that glucose assimilation in the presence of xylose was negatively affected. Adaptive Laboratory Evolution (ALE) was used as a metabolic engineering tool in A. vinelandii, to improve both carbohydrate assimilation. As a result of ALE process, the CT387 strain was obtained. The evolved strain (CT387) grown in shaken flask cultivations with xylose (8gL-1) and glucose (2gL-1), showed an increase of its specific growth rate (µ), as well as of its glucose and xylose uptake rates of 2, 6.45 and 3.57-fold, respectively, as compared with the parental strain. At bioreactor level, the µ, the glucose consumption rate and the relative expression of gluP that codes for the glucose permease in the evolved strain were also higher than in the native strain (1.53, 1.29 and 18-fold, respectively). Therefore, in the present study, we demonstrated the potential of ALE as a metabolic engineering tool for improving glucose and xylose consumption in A. vinelandii.

Role of non-PTS dependent glucose permease (GlcU) in maintaining the fitness cost during acquisition of nisin resistance by Enterococcus faecalis.

Nisin is used for food preservation due to its antibacterial activity. However, some bacteria survive under the prevailing conditions owing to the acquisition of resistance. This study aimed to characterize nisin-resistant Enterococcus faecalis isolated from raw buffalo milk and investigate their fitness cost. FE-SEM, biofilm and cytochrome c assay were used for characterization. Growth kinetics, HPLC, qPCR and western blotting were performed to confer their fitness cost. Results revealed that nisin-resistant E. faecalis were morphologically different from sensitive strain and internalize more glucose. However, no significant difference was observed in the growth pattern of the resistant strain compared to that of the sensitive strain. A non-phosphotransferase glucose permease (GlcU) was found to be associated with enhanced glucose uptake. Conversely, Mpt, a major phosphotransferase system responsible for glucose uptake, did not play any role, as confirmed by gene expression studies and western blot analysis of HPr protein. The phosphorylation of His-15 residue of HPr phosphoprotein was reduced, while that of the Ser-46 residue increased with progression in nisin resistance, indicating that it may be involved in the regulation of pathogenicity. In conclusion, resistance imposes a significant fitness cost and GlcU plays a key role in maintaining the fitness cost in nisin-resistant variants.

The hypoxic expression of the glucose transporter RAG1 reveals the role of the bHLH transcription factor Sck1 as a novel hypoxic modulator in Kluyveromyces lactis

Glucose is the preferred nutrient for most living cells and is also a signaling molecule that modulates several cellular processes. Glucose regulates the expression of glucose permease genes in yeasts through signaling pathways dependent on plasma membrane glucose sensors. In the yeast Kluyveromyces lactis, sufficient levels of glucose induction of the low-affinity glucose transporter RAG1 gene also depends on a functional glycolysis, suggesting additional intracellular signaling. We have found that the expression of RAG1 gene is also induced by hypoxia in the presence of glucose, indicating that glucose and oxygen signaling pathways are interconnected. In this study we investigated the molecular mechanisms underlying this crosstalk. By analyzing RAG1 expression in various K. lactis mutants, we found that the bHLH transcriptional activator Sck1 is required for the hypoxic induction of RAG1 gene. The RAG1 promoter region essential for its hypoxic induction was identified by promoter deletion experiments. Taken together, these results show that the RAG1 glucose permease gene is synergistically induced by hypoxia and glucose and highlighted a novel role for the transcriptional activator Sck1 as a key mediator in this mechanism.

The Small Protein SgrT Controls Transport Activity of the Glucose-Specific Phosphotransferase System.

The bacterial small RNA (sRNA) SgrS has been a fruitful model for discovery of novel RNA-based regulatory mechanisms and new facets of bacterial physiology and metabolism. SgrS is one of only a few characterized dual-function sRNAs. SgrS can control gene expression posttranscriptionally via sRNA-mRNA base-pairing interactions. Its second function is coding for the small protein SgrT. Previous work demonstrated that both functions contribute to relief of growth inhibition caused by glucose-phosphate stress, a condition characterized by disrupted glycolytic flux and accumulation of sugar phosphates. The base-pairing activity of SgrS has been the subject of numerous studies, but the activity of SgrT is less well characterized. Here, we provide evidence that SgrT acts to specifically inhibit the transport activity of the major glucose permease PtsG. Superresolution microscopy demonstrated that SgrT localizes to the cell membrane in a PtsG-dependent manner. Mutational analysis determined that residues in the N-terminal domain of PtsG are important for conferring sensitivity to SgrT-mediated inhibition of transport activity. Growth assays support a model in which SgrT-mediated inhibition of PtsG transport activity reduces accumulation of nonmetabolizable sugar phosphates and promotes utilization of alternative carbon sources by modulating carbon catabolite repression. The results of this study expand our understanding of a basic and well-studied biological problem, namely, how cells coordinate carbohydrate transport and metabolism. Further, this work highlights the complex activities that can be carried out by sRNAs and small proteins in bacteria.IMPORTANCE Sequencing, annotation and investigation of hundreds of bacterial genomes have identified vast numbers of small RNAs and small proteins, the majority of which have no known function. In this study, we explore the function of a small protein that acts in tandem with a well-characterized small RNA during metabolic stress to help bacterial cells maintain balanced metabolism and continue growing. Our results indicate that this protein acts on the glucose transport system, inhibiting its activity under stress conditions in order to allow cells to utilize alternative carbon sources. This work sheds new light on a key biological problem: how cells coordinate carbohydrate transport and metabolism. The study also expands our understanding of the functional capacities of small proteins.

Engineering Corynebacterium glutamicum for fast production of L-lysine and L-pipecolic acid.

The Gram-positive Corynebacterium glutamicum is widely used for fermentative production of amino acids. The world production of L-lysine has surpassed 2 million tons per year. Glucose uptake and phosphorylation by C. glutamicum mainly occur by the phosphotransferase system (PTS) and to lesser extent by inositol permeases and glucokinases. Heterologous expression of the genes for the high-affinity glucose permease from Streptomyces coelicolor and Bacillus subtilis glucokinase fully compensated for the absence of the PTS in Δhpr strains. Growth of PTS-positive strains with glucose was accelerated when the endogenous inositol permease IolT2 and glucokinase from B. subtilis were overproduced with balanced translation initiation rates using plasmid pEKEx3-IolTBest. When the genome-reduced C. glutamicum strain GRLys1 carrying additional in-frame deletions of sugR and ldhA to derepress glycolytic and PTS genes and to circumvent formation of L-lactate as by-product was transformed with this plasmid or with pVWEx1-IolTBest, 18 to 20% higher volumetric productivities and 70 to 72% higher specific productivities as compared to the parental strain resulted. The non-proteinogenic amino acid L-pipecolic acid (L-PA), a precursor of immunosuppressants, peptide antibiotics, or piperidine alkaloids, can be derived from L-lysine. To enable production of L-PA by the constructed L-lysine-producing strain, the L-lysine 6-dehydrogenase gene lysDH from Silicibacter pomeroyi and the endogenous pyrroline 5-carboxylate reductase gene proC were overexpressed as synthetic operon. This enabled C. glutamicum to produce L-PA with a yield of 0.09±0.01gg(-1) and a volumetric productivity of 0.04±0.01gL(-1)h(-1).To the best of our knowledge, this is the first fermentative process for the production of L-PA from glucose.

A transferable plasticity region in Campylobacter coli allows isolates of an otherwise non-glycolytic food-borne pathogen to catabolize glucose.

Thermophilic Campylobacter species colonize the intestine of agricultural and domestic animals commensally but cause severe gastroenteritis in humans. In contrast to other enteropathogenic bacteria, Campylobacter has been considered to be non-glycolytic, a metabolic property originally used for their taxonomic classification. Contrary to this dogma, we demonstrate that several Campylobacter coli strains are able to utilize glucose as a growth substrate. Isotopologue profiling experiments with (13) C-labeled glucose suggested that these strains catabolize glucose via the pentose phosphate and Entner-Doudoroff (ED) pathways and use glucose efficiently for de novo synthesis of amino acids and cell surface carbohydrates. Whole genome sequencing of glycolytic C. coli isolates identified a genomic island located within a ribosomal RNA gene cluster that encodes for all ED pathway enzymes and a glucose permease. We could show in vitro that a non-glycolytic C. coli strain could acquire glycolytic activity through natural transformation with chromosomal DNA of C. coli and C. jejuni subsp. doylei strains possessing the ED pathway encoding plasticity region. These results reveal for the first time the ability of a Campylobacter species to catabolize glucose and provide new insights into how genetic macrodiversity through intra- and interspecies gene transfer expand the metabolic capacity of this food-borne pathogen.

Glycolysis controls plasma membrane glucose sensors to promote glucose signaling in yeasts.

Sensing of extracellular glucose is necessary for cells to adapt to glucose variation in their environment. In the respiratory yeast Kluyveromyces lactis, extracellular glucose controls the expression of major glucose permease gene RAG1 through a cascade similar to the Saccharomyces cerevisiae Snf3/Rgt2/Rgt1 glucose signaling pathway. This regulation depends also on intracellular glucose metabolism since we previously showed that glucose induction of the RAG1 gene is abolished in glycolytic mutants. Here we show that glycolysis regulates RAG1 expression through the K. lactis Rgt1 (KlRgt1) glucose signaling pathway by targeting the localization and probably the stability of Rag4, the single Snf3/Rgt2-type glucose sensor of K. lactis. Additionally, the control exerted by glycolysis on glucose signaling seems to be conserved in S. cerevisiae. This retrocontrol might prevent yeasts from unnecessary glucose transport and intracellular glucose accumulation.

Functional analysis of the GlcP promoter in Streptomyces peucetius var. caesius.

In Streptomyces, carbon utilization is of significant importance for the expression of genes involved in morphological differentiation and antibiotic production. Glucose is mainly transported by GlcP, a membrane protein encoded by glcp. In Streptomyces coelicolor, this protein is encoded by sco5578. However, there is little information about the physiology of the GlcP promoter in Streptomyces. The aim of the present work was to clone and perform a functional analysis of the sp7066 promoter (ortholog of sco5578) from Streptomyces peucetius var. caesius. Hydrophobicity and cellular location analysis of the putative amino acid sequence of the cloned gene predicted SP7066 would be a membrane protein with a topology of six plus six transmembrane segments interrupted by a large cytoplasmic loop. In silico analysis of the upstream region of the sp7066 transcription initiation site predicted the sequences 5'-AGGAATAGT-3' and 5'-TTGACT-3' for regions -10 and -35 of sp7066 promoter. To reflect sp7066 expression, the promoter sequence was amplified, subcloned, and fused to the egfp reporter gene. Immunoblot analysis revealed that D-glucose and its analog 2-deoxyglucose were able to induce sp7066 expression. This effect was not modified by the presence of equimolar concentrations of D-galactose or N-acetylglucosamine. No expression of egfp was detected with the use of other carbon sources such as L-arabinose, D-fructose, and glycerol. Based on these analyses, we conclude that D-glucose is a preferred carbon source in S. peucetius var. caesius and that the sp7066 expression product, a putative non-PTS glucose permease, likely is a H+/symporter, localized to the membrane, and shows a strong specificity for D-glucose for inducing expression.

Comparison of in vivo and in vitro properties of capsulated and noncapsulated variants of Mycoplasma mycoides subsp. mycoides strain Afadé: a potential new insight into the biology of contagious bovine pleuropneumonia.

Mycoplasma mycoides subsp. mycoides (Mmm) strain Afadé had previously been shown to undergo spontaneous phase variations between an opaque capsulated variant and a translucent (TR) variant devoid of a capsule but able to secrete cell-free exopolysaccharides. This phase variation is associated with an ON/OFF genetic switch in a glucose permease gene. In this study, in vivo and in vitro assays were conducted to compare the virulence of the two variants and their abilities to resist host defence. Capsulated variants were shown, in a mouse model, to induce longer bacteraemia that was correlated with better serum resistance in vitro. In contrast, TR variants displayed better ability to adhere to an inert support, linked to the absence of a capsule, changes in cell surface hydrophobicity and increased resistance to antimicrobial peptide and hydrogen peroxide. The switch from one variant population to another, which was observed both in vivo and in vitro under stress conditions, is further discussed as a means for Mmm to modulate its interactions with animal hosts during different stages of the disease.

Comparative genomics analysis of Lactobacillus species associated with weight gain or weight protection

BACKGROUND:Some Lactobacillus species are associated with obesity and weight gain while others are associated with weight loss. Lactobacillus spp. and bifidobacteria represent a major bacterial population of the small intestine where lipids and simple carbohydrates are absorbed, particularly in the duodenum and jejunum. The objective of this study was to identify Lactobacillus spp. proteins involved in carbohydrate and lipid metabolism associated with weight modifications.METHODS:We examined a total of 13 complete genomes belonging to seven different Lactobacillus spp. previously associated with weight gain or weight protection. We combined the data obtained from the Rapid Annotation using Subsystem Technology, Batch CD-Search and Gene Ontology to classify gene function in each genome.RESULTS:We observed major differences between the two groups of genomes. Weight gain-associated Lactobacillus spp. appear to lack enzymes involved in the catabolism of fructose, defense against oxidative stress and the synthesis of dextrin, L-rhamnose and acetate. Weight protection-associated Lactobacillus spp. encoded a significant gene amount of glucose permease. Regarding lipid metabolism, thiolases were only encoded in the genome of weight gain-associated Lactobacillus spp. In addition, we identified 18 different types of bacteriocins in the studied genomes, and weight gain-associated Lactobacillus spp. encoded more bacteriocins than weight protection-associated Lactobacillus spp.CONCLUSIONS:The results of this study revealed that weight protection-associated Lactobacillus spp. have developed defense mechanisms for enhanced glycolysis and defense against oxidative stress. Weight gain-associated Lactobacillus spp. possess a limited ability to breakdown fructose or glucose and might reduce ileal brake effects.

The SWI/SNF KlSnf2 Subunit Controls the Glucose Signaling Pathway To Coordinate Glycolysis and Glucose Transport in Kluyveromyces lactis

In Kluyveromyces lactis, the expression of the major glucose permease gene RAG1 is controlled by extracellular glucose through a signaling cascade similar to the Saccharomyces cerevisiae Snf3/Rgt2/Rgt1 pathway. We have identified a key component of the K. lactis glucose signaling pathway by characterizing a new mutation, rag20-1, which impairs the regulation of RAG1 and hexokinase RAG5 genes by glucose. Functional complementation of the rag20-1 mutation identified the KlSNF2 gene, which encodes a protein 59% identical to S. cerevisiae Snf2, the major subunit of the SWI/SNF chromatin remodeling complex. Reverse transcription-quantitative PCR and chromatin immunoprecipitation analyses confirmed that the KlSnf2 protein binds to RAG1 and RAG5 promoters and promotes the recruitment of the basic helix-loop-helix Sck1 activator. Besides this transcriptional effect, KlSnf2 is also implicated in the glucose signaling pathway by controlling Sms1 and KlRgt1 posttranscriptional modifications. When KlSnf2 is absent, Sms1 is not degraded in the presence of glucose, leading to constitutive RAG1 gene repression by KlRgt1. Our work points out the crucial role played by KlSnf2 in the regulation of glucose transport and metabolism in K. lactis, notably, by suggesting a link between chromatin remodeling and the glucose signaling pathway.

Solution Structure of the IIAChitobiose-HPr Complex of the N,N′-Diacetylchitobiose Branch of the Escherichia coli Phosphotransferase System

The solution structure of the complex of enzyme IIA of the N,N'-diacetylchitobiose (Chb) transporter with the histidine phosphocarrier protein HPr has been solved by NMR. The IIA(Chb)-HPr complex completes the structure elucidation of representative cytoplasmic complexes for all four sugar branches of the bacterial phosphoryl transfer system (PTS). The active site His-89 of IIA(Chb) was mutated to Glu to mimic the phosphorylated state. IIA(Chb)(H89E) and HPr form a weak complex with a K(D) of ~0.7 mM. The interacting binding surfaces, concave for IIA(Chb) and convex for HPr, complement each other in terms of shape, residue type, and charge distribution, with predominantly hydrophobic residues, interspersed by some uncharged polar residues, located centrally, and polar and charged residues at the periphery. The active site histidine of HPr, His-15, is buried within the active site cleft of IIA(Chb) formed at the interface of two adjacent subunits of the IIA(Chb) trimer, thereby coming into close proximity with the active site residue, H89E, of IIA(Chb). A His89-P-His-15 pentacoordinate phosphoryl transition state can readily be modeled without necessitating any significant conformational changes, thereby facilitating rapid phosphoryl transfer. Comparison of the IIA(Chb)-HPr complex with the IIA(Chb)-IIB(Chb) complex, as well as with other cytoplasmic complexes of the PTS, highlights a unifying mechanism for recognition of structurally diverse partners. This involves generating similar binding surfaces from entirely different underlying structural elements, large interaction surfaces coupled with extensive redundancy, and side chain conformational plasticity to optimize diverse sets of intermolecular interactions.

Analysis of the capability of ultra-highly diluted glucose to increase glucose uptake in arsenite-stressed bacteria Escherichia coli

Whether ultra-highly diluted homeopathic remedies can affect living systems is questionable. Therefore, this study sees value in the analysis of whether homeopathically diluted glucose 30C has any effect on Escherichia coli exposed to arsenite stress. E. coli were cultured to their log phase in standard Luria-Bertani medium and then treated with either 1 mmol/L or 2 mmol/L sodium arsenite, with or without supplementation of either 1% or 3% glucose, an ultra-highly diluted and agitated ethanolic solution (70%) of glucose (diluted 10(60) times), glucose 30C or 70% ethanol (placebo) in the medium. Glucose uptake, specific activities of hexokinase and glucokinase, membrane potential, intracellular adenosine triphosphate (ATP) and expression of glucose permease in E. coli were analyzed at two different time intervals. Arsenic content in E. coli (intracellular) and in the spent medium (extracellular) was also determined. In arsenite-exposed E. coli, the glucose uptake increased along with decreases in the specific activities of hexokinase and glucokinase, intracellular ATP and membrane potential and an increase in the gene expression level of glucose permease. Glucose uptake increased further by addition of 1%, 3% or ultra-highly diluted glucose in the medium, but not by the placebo. The results demonstrated the efficacy of the ultra-highly diluted and agitated glucose in mimicking the action of actual glucose supplementation and its ability to modulate expressions of hexokinase and glucokinase enzymes and glucose permease genes, thereby validating the efficacy of ultra-high dilutions used in homeopathy.

The effect of context on the folding of β-hairpins

Small β-hairpin peptides have been widely used as models for the folding of β-sheets. But how applicable is the folding of such models to β-structure in larger proteins with conventional hydrophobic cores? Here we present multiple unfolding simulations of three such proteins that contain the WW domain double hairpin β-sheet motif: cold shock protein A (CspA), cold shock protein B (CspB) and glucose permease IIA domain. We compare the behavior of the free motif in solution and in the context of proteins of different size and architecture. Both Csp proteins lost contacts between the double-hairpin motif and the protein core as the first step of unfolding and proceeded to unfold with loss of the third β-strand, similar to the isolated WW domain. The glucose permease IIA domain is a larger protein and the contacts between the motif and the core were not lost as quickly. Instead the unfolding pathway of glucose permease IIA followed a different pathway with β1 pulling away from the sheet first. Interestingly, when the double hairpin motif was excised from the glucose permease IIA domain and simulated in isolation in water it unfolded by the same pathway as the WW domain, indicating that it is tertiary interactions with the protein that alter the motif's unfolding not a sequence dependent effect on its intrinsic unfolding behavior. With respect to the unfolding of the hairpins, there was no consistent order to the loss of hydrogen bonds between the β-strands in the hairpins in any of the systems. Our results show that while the folding behavior of the isolated WW domain is generally consistent with the double hairpin motif's behavior in the cold shock proteins, it is not the case for the glucose permease IIA domain. So, one must be cautious in extrapolating findings from model systems to larger more complicated proteins where tertiary interactions can overwhelm intrinsic behavior.

Phosphotransferase system-independent glucose utilization in corynebacterium glutamicum by inositol permeases and glucokinases.

Phosphoenolpyruvate-dependent glucose phosphorylation via the phosphotransferase system (PTS) is the major path of glucose uptake in Corynebacterium glutamicum, but some growth from glucose is retained in the absence of the PTS. The growth defect of a deletion mutant lacking the general PTS component HPr in glucose medium could be overcome by suppressor mutations leading to the high expression of inositol utilization genes or by the addition of inositol to the growth medium if a glucokinase is overproduced simultaneously. PTS-independent glucose uptake was shown to require at least one of the inositol transporters IolT1 and IolT2 as a mutant lacking IolT1, IolT2, and the PTS component HPr could not grow with glucose as the sole carbon source. Efficient glucose utilization in the absence of the PTS necessitated the overexpression of a glucokinase gene in addition to either iolT1 or iolT2. IolT1 and IolT2 are low-affinity glucose permeases with K(s) values of 2.8 and 1.9 mM, respectively. As glucose uptake and phosphorylation via the PTS differs from glucose uptake via IolT1 or IolT2 and phosphorylation via glucokinase by the requirement for phosphoenolpyruvate, the roles of the two pathways for l-lysine production were tested. The l-lysine yield by C. glutamicum DM1729, a rationally engineered l-lysine-producing strain, was lower than that by its PTS-deficient derivate DM1729Δhpr, which, however, showed low production rates. The combined overexpression of iolT1 or iolT2 with ppgK, the gene for PolyP/ATP-dependent glucokinase, in DM1729Δhpr enabled l-lysine production as fast as that by the parent strain DM1729 but with 10 to 20% higher l-lysine yield.

Identification of mannose uptake and catabolism genes in Corynebacterium glutamicum and genetic engineering for simultaneous utilization of mannose and glucose

Here, focus is on Corynebacterium glutamicum mannose metabolic genes with the aim to improve this industrially important microorganism's ability to ferment mannose present in mixed sugar substrates. cgR_0857 encodes C. glutamicum's protein with 36% amino acid sequence identity to mannose 6-phosphate isomerase encoded by manA of Escherichia coli. Its deletion mutant did not grow on mannose and exhibited noticeably reduced growth on glucose as sole carbon sources. In effect, C. glutamicum manA is not only essential for growth on mannose but also important in glucose metabolism. A double deletion mutant of genes encoding glucose and fructose permeases (ptsG and ptsF, respectively) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was not able to grow on mannose unlike the respective single deletion mutants with mannose utilization ability. A mutant deficient in ptsH, a general PTS gene, did not utilize mannose. These indicate that the glucose-PTS and fructose-PTS are responsible for mannose uptake in C. glutamicum. When cultured with a glucose and mannose mixture, mannose utilization of manA-overexpressing strain CRM1 was significantly higher than that of its wild-type counterpart, but with a strong preference for glucose. ptsF-overexpressing strain CRM2 co-utilized mannose and glucose, but at a total sugar consumption rate much lower than that of the wild-type strain and CRM1. Strain CRM3 overexpressing both manA and ptsF efficiently co-utilized mannose and glucose. Under oxygen-deprived conditions, high volumetric productivity of organic acids concomitant with the simultaneous consumption of the mixed sugars was achieved by the densely packed growth-arrested CRM3 cells.

Hal4 and Hal5 Protein Kinases Are Required for General Control of Carbon and Nitrogen Uptake and Metabolism

The yeast protein kinases Sat4/Hal4 and Hal5 are required for the plasma membrane stability of the K(+) transporter Trk1 and some amino acid and glucose permeases. The transcriptomic analysis presented here indicates alterations in the general control of the metabolism of both nitrogen and carbon. Accordingly, we observed reduced uptake of methionine and leucine in the hal4 hal5 mutant. This decrease correlates with activation of the Gcn2-Gcn4 pathway, as measured by expression of the lacZ gene under the control of the GCN4 promoter. However, with the exception of methionine biosynthetic genes, few amino acid biosynthetic genes are induced in the hal4 hal5 mutant, whereas several genes involved in amino acid catabolism are repressed. Concerning glucose metabolism, we found that this mutant exhibits derepression of respiratory genes in the presence of glucose, leading to an increased activity of mitochondrial enzymes, as measured by succinate dehydrogenase (SDH) activity. In addition, the reduced glucose consumption in the hal4 hal5 mutant correlates with a more acidic intracellular pH and with low activity of the plasma membrane H(+)-ATPase. As a compensatory mechanism for the low glycolytic rate, the hal4 hal5 mutant overexpresses the HXT4 high-affinity glucose transporter and the hexokinase genes. These results indicate that the hal4 hal5 mutant presents defects in the general control of nitrogen and carbon metabolism, which correlate with reduced transport of amino acids and glucose, respectively. A more acidic intracellular pH may contribute to some defects of this mutant.

Spatial and temporal organization of the E. coli PTS components

The phosphotransferase system (PTS) controls preferential use of sugars in bacteria. It comprises of two general proteins, enzyme I (EI) and HPr, and various sugar-specific permeases. Using fluorescence microscopy, we show here that EI and HPr localize near the Escherichia coli cell poles. Polar localization of each protein occurs independently, but HPr is released from the poles in an EI- and sugar-dependent manner. Conversely, the β-glucoside-specific permease, BglF, localizes to the cell membrane. EI, HPr and BglF control the β-glucoside utilization (bgl) operon by modulating the activity of the BglG transcription factor; BglF inactivates BglG by membrane sequestration and phosphorylation, whereas EI and HPr activate it by an unknown mechanism in response to β-glucosides availability. Using biochemical, genetic and imaging methodologies, we show that EI and HPr interact with BglG and affect its subcellular localization in a phosphorylation-independent manner. Upon sugar stimulation, BglG migrates from the cell periphery to the cytoplasm through the poles. Hence, the PTS components appear to control bgl operon expression by ushering BglG between the cellular compartments. Our results reinforce the notion that signal transduction in bacteria involves dynamic localization of proteins.

The enigmatic lack of glucose utilization in Streptomyces clavuligerus is due to inefficient expression of the glucose permease gene

Streptomyces clavuligerus ATCC 27064 is unable to use glucose but has genes for a glucose permease (glcP) and a glucose kinase (glkA). Transformation of S. clavuligerus 27064 with the Streptomyces coelicolor glcP1 gene with its own promoter results in a strain able to grow on glucose. The glcP gene of S. clavuligerus encodes a 475 amino acid glucose permease with 12 transmembrane segments. GlcP is a functional protein when expressed from the S. coelicolor glcP1 promoter and complements two different glucose transport-negative Escherichia coli mutants. Transcription studies indicate that the glcP promoter is very weak and does not allow growth on glucose. These results suggest that S. clavuligerus initially contained a functional glucose permease gene, like most other Streptomyces species, and lost the expression of this gene by adaptation to glucose-poor habitats.