Glycerol facilitator of Escherichia coli: cloning of glpF and identification of the glpF product.

The glycerol facilitator is known as the only example of a transport protein that catalyzes facilitated diffusion across the Escherichia coli inner membrane. Here we show that the gene encoding the facilitator, glpF, is the first gene in an operon with glpK, encoding glycerol kinase, at 88 min of the E. coli chromosome. The operon is transcribed counterclockwise. We cloned the glpF gene, demonstrated that it complemented a chromosomal glycerol transport-minus mutation, and identified the gene product. The GlpF protein appeared in the membrane fraction of plasmid-bearing strains and had an apparent Mr of 25,000.     

Utilisation of glycerol and glycerol 3-phosphate is differently affected by the phosphotransferase system in Bacillus subtilis

Glycerol and glycerol 3-phosphate uptake in Bacillus subtilis does not involve the phosphotransferase system. In spite of this, B. subtilis mutants defective in the general components of the phosphotransferase system, EnzymeI or Hpr, are unable to grow with glycerol as sole carbon and energy source. Here we show that a Hpr mutant can grow on glycerol 3-phosphate and that glycerol 3-phosphate, but not glycerol, can induce glpD encoding glycerol-3-phosphate dehydrogenase. Induction of glpD also requires the glpP gene product which is a regulator of all known glp genes. Thus the phosphotransferase system general components do not interfere with the overall regulation of the glp regulon. Revertants of a Hpr mutant which can grown on glycerol carry mutations closely linked to the glp region at 75 degrees on the B. subtilis chromosomal map. This region contains the glpP, the glpFK and the glpD operons. The glpFK operon encodes the glycerol uptake facilitator (glpF) and glycerol kinase (glpK). The present results demonstrate that one of these genes, or their gene products, is the target for phosphotransferase system control of glycerol utilisation. Furthermore we conclude that utilisation of glycerol and glycerol 3-phosphate is differently affected by the phosphotransferase system in B. subtilis.     

A single‐component multidrug transporter of the major facilitator superfamily is part of a network that protects Escherichia coli from bile salt stress

Resistance to high concentrations of bile salts in the human intestinal tract is vital for the survival of enteric bacteria such as Escherichia coli. Although the tripartite AcrAB–TolC efflux system plays a significant role in this resistance, it is purported that other efflux pumps must also be involved. We provide evidence from a comprehensive suite of experiments performed at two different pH values (7.2 and 6.0) that reflect pH conditions that E. coli may encounter in human gut that MdtM, a single‐component multidrug resistance transporter of the major facilitator superfamily, functions in bile salt resistance in E. coli by catalysing secondary active transport of bile salts out of the cell cytoplasm. Furthermore, assays performed on a chromosomal ΔacrB mutant transformed with multicopy plasmid encoding MdtM suggested a functional synergism between the single‐component MdtM transporter and the tripartite AcrAB–TolC system that results in a multiplicative effect on resistance. Substrate binding experiments performed on purified MdtM demonstrated that the transporter binds to cholate and deoxycholate with micromolar affinity, and transport assays performed on inverted vesicles confirmed the capacity of MdtM to catalyse electrogenic bile salt/H⁺ antiport.     

Two glycerol uptake systems contribute to the high osmotolerance of Zygosaccharomyces rouxii

The accumulation of glycerol is essential for yeast viability upon hyperosmotic stress. Here we show that the osmotolerant yeast Zygosaccharomyces rouxii has two genes, ZrSTL1 and ZrSTL2, encoding transporters mediating the active uptake of glycerol in symport with protons, contributing to cell osmotolerance and intracellular pH homeostasis. The growth of mutants lacking one or both transporters is affected depending on the growth medium, carbon source, strain auxotrophies, osmotic conditions and the presence of external glycerol. These transporters are localised in the plasma membrane, they transport glycerol with similar kinetic parameters and besides their expected involvement in the cell survival of hyperosmotic stress, they surprisingly both contribute to an efficient survival of hypoosmotic shock and to the maintenance of intracellular pH homeostasis under non‐stressed conditions. Unlike STL1 in Sa. cerevisiae, the two Z. rouxii STL genes are not repressed by glucose, but their expression and activity are downregulated by fructose and upregulated by non‐fermentable carbon sources, with ZrSTL1 being more influenced than ZrSTL2. In summary, both transporters are highly important, though Z. rouxii CBS 732^T cells do not use external glycerol as a source of carbon.     

A gene of the major facilitator superfamily encodes a transporter for enterobactin (Enb1p) in Saccharomyces cerevisiae

While in fungi iron transport via hydroxamate siderophores has been amply proven, iron transport via enterobactin is largely unknown. Enterobactin is a catecholate-type siderophore produced by several enterobacterial genera grown in severe iron deprivation. By using the KanMX disruption module in vector pUG6 in a fet3 background of Saccharomyces cerevisiae we were able to disrupt the gene YOL158c Sce of the major facilitator super family (MFS) which has been previously described as a gene encoding a membrane transporter of unknown function. Contrary to the parental strain, the disruptant was unable to utilize ferric enterobactin in growth promotion tests and in transport assays using ⁵⁵Fe-enterobactin. All other siderophore transport properties remained unaffected. The results are evidence that in S. cerevisiae the YOL158c Sce gene of the major facilitator super family, now designated ENB1, encodes a transporter protein (Enb1p), which specifically recognizes and transports enterobactin.     

Identification of Positive Regulators of the Yeast Fps1 Glycerol Channel

The yeast Fps1 protein is an aquaglyceroporin that functions as the major facilitator of glycerol transport in response to changes in extracellular osmolarity. Although the High Osmolarity Glycerol pathway is thought to have a function in at least basal control of Fps1 activity, its mode of regulation is not understood. We describe the identification of a pair of positive regulators of the Fps1 glycerol channel, Rgc1 (Ypr115w) and Rgc2 (Ask10). An rgc1/2Δ mutant experiences cell wall stress that results from osmotic pressure associated with hyper-accumulation of glycerol. Accumulation of glycerol in the rgc1/2Δ mutant results from a defect in Fps1 activity as evidenced by suppression of the defect through Fps1 overexpression, failure to release glycerol upon hypo-osmotic shock, and resistance to arsenite, a toxic metalloid that enters the cell through Fps1. Regulation of Fps1 by Rgc1/2 appears to be indirect; however, evidence is presented supporting the view that Rgc1/2 regulate Fps1 channel activity, rather than its expression, folding, or localization. Rgc2 was phosphorylated in response to stresses that lead to regulation of Fps1. This stress-induced phosphorylation was partially dependent on the Hog1 MAPK. Hog1 was also required for basal phosphorylation of Rgc2, suggesting a mechanism by which Hog1 may regulate Fps1 indirectly.     

Comparative analysis of FUR regulons in gamma-proteobacteria

Iron is an essential element for the survival and pathogenesis of bacteria. The strict control of iron homeostasis is mediated by the FUR repressor, which is highly conserved among various bacterial species. Here we apply the comparative genomics approach to analyze candidate Fur-binding sites in the genomes of Escherichia coli (K12 and O157:H7), Salmonella typhi, Yersinia pestis and Vibrio cholerae. We describe a number of new loci encoding siderophore biosynthesis and transport proteins. A new regulator of iron-acquisition systems was found in S.typhi. We predict FUR regulation for several virulence systems. We also predict FUR regulation for the chemotaxis system of V.cholerae that is probably involved in the process of pathogenesis.     

Trichome regulator SlMIXTA‐like directly manipulates primary metabolism in tomato fruit

Trichomes are storage compartments for specialized metabolites in many plant species. In trichome, plant primary metabolism is significantly changed, providing substrates for downstream secondary metabolism. However, little is known of how plants coordinate trichome formation and primary metabolism regulation. In this report, tomato (Solanum lycopersicum) trichome regulator SlMIXTA‐like is indicated as a metabolic regulation gene by mGWAS analysis. Overexpression of SlMIXTA‐like in tomato fruit enhances trichome formation. In addition, SlMIXTA‐like can directly bind to the promoter region of gene encoding 3‐deoxy‐7‐phosphoheptulonate synthase (SlDAHPS) to activate its expression. Induction of SlDAHPS expression enhances shikimate pathway activities and provides substrates for downstream secondary metabolism. Our data provide direct evidence that trichome regulator can directly manipulate primary metabolism, in which way plants can coordinate metabolic regulation and the formation of storage compartments for specialized metabolites. The newly identified SlMIXTA‐like can be used for future metabolic engineering.     

Transketolase activity modulates glycerol‐3‐phosphate levels in Escherichia coli

Transketolase activity provides an important link between the metabolic pathways of glycolysis and pentose phosphate shunt and catalyzes inter‐conversions between pentose phosphates and glycolytic intermediates. It is widely conserved in life forms. A genetic screen for suppression of the growth defect of Escherichia coli tktA tktB mutant in LB medium revealed two mutations, one that rendered the glpK expression constitutive and another that inactivated deoB. Characterizing these mutations aided in uncovering the role of ribose‐5‐P (a transketolase substrate) as an inhibitor of glycerol assimilation and de novo glycerol‐3‐P synthesis. Using lacZ fusions, we show that ribose‐5‐P enhances GlpR–mediated repression of the glpFKX operon and inhibits glycerol assimilation. Electrophoretic Mobility Shift Assay (EMSA) showed ribose‐5‐P made the DNA‐GlpR complex less sensitive to the inducer glycerol‐3‐P. In addition to inhibition of glycerol assimilation, obstruction of ribose‐5‐P metabolism retards growth from glycerol‐3‐P limitation. Glucose helps to overcome this limitation through a mechanism involving catabolite repression. To our knowledge, this report is the first to show ribose‐5‐P can modulate glycerol‐3‐P concentration in the cell by regulation of glycerol assimilation as well as its de novo synthesis. This regulation could be prevalent in other organisms.     

The Arabidopsis<Emphasis Type="Italic"> GNARLED</Emphasis> gene encodes the NAP125 homolog and controls several actin-based cell shape changes

In plants many aspects of cell shape regulation are controlled by actin-dependent processes. The ARP2/3 complex has been recognized as a regulator of actin organization. Mutations in genes encoding components of the ARP2/3 complex lead to cell shape defects in several cell types, including trichomes, epidermal pavement cells and hypocotyl cells. We show here that mutations in the GNARLED ( GRL) gene cause a similar range of phenotypes. The GRL gene encodes the Arabidopsis homolog of NAP125, which in animals is known to act as one regulator of the ARP2/3-regulating complex WAVE-HSPC300. As an HSPC300 homolog is present in the Arabidopsis genome but no WAVE homolog has yet been found, the existence of a related regulation pathway was doubtful. Our finding that GRL encodes a putative regulator of the WAVE-HSPC300 complex, NAP125, combined with the phenotypic similarity between arp2/3 and grl mutants, provides evidence that the ARP2/3 complex is indeed regulated by the above mentioned pathway in Arabidopsis.Communicated by G. JürgensThe first two authors contributed equally to the work     

The response regulator BcSkn7 is required for vegetative differentiation and adaptation to oxidative and osmotic stresses in Botrytis cinerea

The high‐osmolarity glycerol pathway plays an important role in the responses of fungi to various environmental stresses. Saccharomyces cerevisiae Skn7 is a response regulator in the high‐osmolarity glycerol pathway, which regulates the oxidative stress response, cell cycle and cell wall biosynthesis. In this study, we characterized an Skn7 orthologue BcSkn7 in Botrytis cinerea. BcSKN7 can partly restore the growth defects of S. cerevisiae SKN7 mutant and vice versa. The BcSKN7 mutant (ΔBcSkn7‐1) revealed increased sensitivity to ionic osmotic and oxidative stresses and to ergosterol biosynthesis inhibitors. In addition, ΔBcSkn7‐1 was also impaired dramatically in conidiation and sclerotial formation. Western blot analysis showed that BcSkn7 positively regulated the phosphorylation of BcSak1 (the orthologue of S. cerevisiae Hog1) under osmotic stress, indicating that BcSkn7 is associated with the high‐osmolarity glycerol pathway in B. cinerea. In contrast with BcSak1, BcSkn7 is not involved in the regulation of B. cinerea virulence. All of the phenotypic defects of ΔBcSkn7‐1 are restored by genetic complementation of the mutant with the wild‐type BcSKN7. The results of this study indicate that BcSkn7 plays an important role in the regulation of vegetative differentiation and in the response to various stresses in B. cinerea.     

Deletion of the <Emphasis Type="Italic">CgTPI</Emphasis> Gene Encoding Triose Phosphate Isomerase of <Emphasis Type="Italic">Candida glycerinogenes</Emphasis> Inhibits the Biosynthesis of Glycerol

The yeast Candida glycerinogenes produces a high yield of glycerol only in response to a medium-osmotic stress, but little is known about the relationship between osmoadaptation and glycerol metabolism. The CgTPI gene encoding triose phosphate isomerase of C. glycerinogenes was cloned and sequenced, and its functionality was confirmed by complementation of Saccharomyces cerevisiae tpi1 Δ. The roles of CgTpip in the glycerol biosynthesis and the osmoadaptation were investigated. Unlike S. cerevisiae tpi1 Δ and Klyuveromyces lactis tpi1 Δ, the mutant lacking CgTPI significantly decreased the rate of glucose consumption and the glycerol yield. Furthermore, the mutants decreased osmotolerance to glucose and NaCl. The results suggest that CgTPI might be crucial for a high yield of glycerol by C. glycerinogenes. The inhibition of glycerol biosynthesis might be related to the reduced ability of osmoadaptation to high external osmolarity. To our knowledge, this is the first report that inactivation of a yeast TPI gene inhibits the biosynthesis of glycerol.     

Glycerol kinase of Escherichia coli is activated by interaction with the glycerol facilitator.

Glycerol transport is commonly cited as the only example of facilitated diffusion across the Escherichia coli cytoplasmic membrane. Two proteins, the glycerol facilitator and glycerol kinase, are involved in the entry of external glycerol into cellular metabolism. The glycerol facilitator is thought to act as a carrier or to form a selective pore in the cytoplasmic membrane, whereas the kinase traps the glycerol inside the cell as sn-glycerol-3-phosphate. We found that the kinetics of glycerol uptake in a facilitator-minus strain are significantly different from the kinetics of glycerol uptake in the wild type. Free glycerol was not observed inside wild-type cells transporting glycerol, and diffusion of glycerol across the cytoplasmic membrane was not the rate-limiting step for phosphorylation in facilitator-minus mutants. Therefore, the kinetics of glycerol phosphorylation are different, depending on the presence or absence of the facilitator protein. We conclude that there is an interaction between the glycerol facilitator protein and glycerol kinase that stimulates kinase activity, analogous to the hexokinase- and glycerol kinase-porin interactions in mitochondria.