The glk gene from Bacillus megaterium, which encodes glucose kinase, was isolated and analyzed. Disruption by a transcriptional glk-luxAB fusion indicated that glk is the only glucose kinase gene in that strain but did not affect growth of that mutant on glucose. Determination of luciferase activity under various growth conditions revealed constitutive transcription of glk. Expression of a xylA-lacZ fusion was repressed by glucose in the strain with the glk disruption about twofold less efficiently than in the wild type. The potential contribution of glk expression to glucose repression is discussed.
The full-length 6.14-kb polycistronic glf-zwf-edd-glk mRNA from Zymomonas mobilis appears to be processed by endonucleolytic cleavage, resulting in the formation of several discrete transcripts. Northern analysis and transcript mapping revealed that the processed transcripts correspond to functional mono-, di-, or tricistronic messages. The relative abundance of the gene-specific, functional messages was measured. Expression of zwf and edd correlated well with functional message levels. Disproportionally high levels of the glk-specific mRNAs might compensate for the instability of glucokinase by allowing increased translation. The relative abundance of the discrete transcripts was shown to be a function of their respective decay rates. Northern analysis of the fate of the 6.14-kb transcript after inhibition of transcription by rifampin showed that the abundance of shorter, more stable transcripts increased at the expense of longer, less stable transcripts. This is suggestive of endonucleolytic mRNA processing. The most abundant 5' and 3' transcript ends were found to lie within secondary structures that probably impart stability to the most abundant mRNAs.
Streptococcus pneumonia is the common cause of sepsis and meningitis. Emergence of multiple
antibiotic resistant strains in the community‐acquired bacterium is catastrophic. Glucose kinase (GLK) is a regulatory
enzyme capable of adding phosphate group to glucose in the first step of streptomycin biosynthesis. The activity of glucose
kinase was regulated by the Carbon Catabolite Repression (CCR) system. Therefore, it is important to establish the structure‐function
relation of GLK in S. pneumoniae. However, a solved structure for S. pneumoniae GLK is not available at
the protein data bank (PDB). Therefore, we created a model of GLK from S. pnemoniae using the X‐ray structure
of Glk from E. faecalis as template with MODELLER (a comparative modeling program). The model was validated using
protein structure checking tools such as PROCHECK, WHAT IF and ProSA for reliability. The active site amino acid Asp114 in the template
is retained in S. pneumoniae GLK model (Asp115). Solvent accessible surface area (ASA) analysis of the GLK model showed
that known key residues playing important role in active site for ligand binding and metal ion binding are buried and hence not
accessible to solvent. The information thus discussed provides insight to the molecular understanding of glucose kinase in
Glucose kinase; active site; model; homology; function
Regulation of chlorophyll metabolism comprises strong transcriptional control together with a range of post-translational mechanisms during chloroplast biogenesis. Recently we reported that chlorophyll biosynthesis in Arabidopsis thaliana roots is regulated by auxin/cytokinin signaling via the combination of two transcription factors, LONG-HYPOCOTYL5 (HY5) and GOLDEN2-LIKE2 (GLK2). In this study, we examined the involvement of cis-elements in the expression of chlorophyll biosynthesis genes. Searches for predicted cis-elements in key chlorophyll biosynthesis genes and their co-expressed genes revealed coexistence of the G-box motif and the CCAATC motif, which may be targeted by HY5 and GLK factors, respectively, in their promoter regions. Deletion of the G-box from the promoter of the CHLH gene encoding the H subunit of Mg-chelatase resulted in the absence of its expression in roots but not in shoots, showing a differing involvement of the G-box in CHLH expression between shoots and roots. Our data suggest that transcription factors and cis-elements participating chlorophyll biosynthesis are substantially changed during organ differentiation, which may be linked to the differentiation of plastids.
cis-element; chlorophyll biosynthesis; co-expression network; photosynthesis; transcriptional regulation
A pair of GOLDEN2-LIKE transcription factors is required for normal chloroplast development in land plant species that encompass the range from bryophytes to angiosperms. In the C4 plant maize, compartmentalized function of the two GLK genes in bundle sheath and mesophyll cells regulates dimorphic chloroplast differentiation, whereas in the C3 plants Physcomitrella patens and Arabidopsis thaliana the genes act redundantly in all photosynthetic cells. To assess whether the cell-specific function of GLK genes is unique to maize, we analyzed gene expression patterns in the C4 monocot Sorghum bicolor and C4 eudicot Cleome gynandra. Compartmentalized expression was observed in S. bicolor, consistent with the development of dimorphic chloroplasts in this species, but not in C. gynandra where bundle sheath and mesophyll chloroplasts are morphologically similar. The generation of single and double mutants demonstrated that GLK genes function redundantly in rice, as in other C3 plants, despite the fact that GLK gene duplication in monocots preceded the speciation of rice, maize and sorghum. Together with phylogenetic analyses of GLK gene sequences, these data have allowed speculation on the evolutionary trajectory of GLK function. Based on current evidence, most species that retain single GLK genes belong to orders that contain only C3 species. We therefore propose that the ancestral state is a single GLK gene, and hypothesize that GLK gene duplication enabled sub-functionalization, which in turn enabled cell-specific function in C4 plants with dimorphic chloroplasts. In this scenario, GLK gene duplication preconditioned the evolution of C4 physiology that is associated with chloroplast dimorphism.
Electronic supplementary material
The online version of this article (doi:10.1007/s00425-012-1754-3) contains supplementary material, which is available to authorized users.
Bundle sheath; Chloroplast; Cleome; Mesophyll; Rice; Sorghum
In the genome of Thermoanaerobacter tengcongensis, three genes belonging to ROK (Repressor, ORF, and Kinase) family are annotated as glucokinases (GLKs). Using enzyme assays, the three GLKs were identified as ATP-dependent GLK (ATP-GLK), ADP-dependent GLK (ADP-GLK), and N-acetyl-glucosamine/mannosamine kinase (glu/man-NacK). The kinetic properties of the three GLKs such as Km, Vmax, optimal pH, and temperature were characterized, demonstrating that these enzymes performed the specific functions against varied substrates and under different temperatures. The abundance of ATP-GLK was attenuated when culture temperature was elevated and was almost undetectable at 80°C, whereas the ADP-GLK abundance was insensitive to temperature changes. Using degradation assays, ATP-GLK was found to have significantly faster degradation than ADP-GLK at 80°C. Co-immunoprecipitation results revealed that heat shock protein 60 (HSP60) could interact with ATP-GLK and ADP-GLK at 60 and 75°C, whereas at 80°C, the interaction was only effectively with ADP-GLK but not ATP-GLK. The functions of GLKs in T. tengcongensis are temperature dependent, likely regulated through interactions with HSP60.
The Zymomonas mobilis genes that encode the glucose-facilitated diffusion transporter (glf), glucose-6-phosphate dehydrogenase (zwf), 6-phosphogluconate dehydratase (edd), and glucokinase (glk) are clustered on the genome. The data presented here firmly establish that the glf, zwf, edd, and glk genes form an operon, in that order. The four genes of the operon are cotranscribed on a 6.14-kb mRNA. The site of transcriptional initiation for the polycistronic message was mapped by primer extension and nuclease S1 protection analysis. The glf operon promoter region showed significant homology to other highly expressed Z. mobilis promoters, but not to consensus promoters from other bacteria. The highly expressed Z. mobilis promoter set contains two independent, overlapping, conserved sequences that extend from approximately bp -100 to +15 with respect to the transcriptional start sites. Expression of the glf operon was shown to be subject to carbon source-dependent regulation. The mRNA level was threefold higher in cells grown on fructose than in cells grown on glucose. This increase was not the result of differential mRNA processing when cells were grown on the different carbon sources, nor was it the result of differential transcript stability. Degradation of the 6.14-kb glf operon mRNA was biphasic, with initial half-lives of 11.5 min in fructose-grown cells and 12.0 min in glucose-grown cells. Thus, the higher level of glf operon mRNA in fructose-grown cells is the result of an increased rate of transcription. The importance of increasing glf expression in cells growing on fructose is discussed.
The objective of this study was to investigate the potential role of the Toll-like receptor 7 (TLR7) signaling pathway in the pathogenesis of adult-onset Still's disease (AOSD).
Frequencies of TLR7-expressing precursor of myeloid dendritic cells (pre-mDCs) and mDCs in 28 AOSD patients, 28 patients with systemic lupus erythematosus (SLE) and 12 healthy controls (HC) were determined by flow cytometry analysis. Transcript and protein levels of TLR7 signaling molecules in peripheral blood mononuclear cells (PBMCs) were evaluated by quantitative PCR and western blotting respectively. Serum cytokines levels were measured by ELISA.
Significantly higher median frequencies of TLR7-expressing pre-mDCs and mDCs were observed in AOSD patients (65.5% and 14.9%, respectively) and in SLE patients (60.3% and 14.4%, respectively) than in HC (42.8% and 8.8%, respectively; both P <0.001). Transcript and protein levels of TLR7-signaling molecules, including MyD88, TRAF6, IRAK4 and IFN-α, were upregulated in AOSD patients and SLE patients compared with those in HC. Disease activity scores were positively correlated with the frequencies of TLR7-expressing mDCs and expression levels of TLR7 signaling molecules in both AOSD and SLE patients. TLR7 ligand (imiquimod) stimulation of PBMCs resulted in significantly enhanced levels of interleukin (IL)-1β, IL-6, IL-18 and IFN-α in AOSD and SLE patients. Frequencies of TLR7-expressing mDCs and expression levels of TLR7 signaling molecules significantly decreased after effective therapy.
Elevated levels of TLR7 signaling molecules and their positive correlation with disease activity in AOSD patients suggest involvement of the TLR7 signaling pathway in the pathogenesis of this disease. The overexpression of TLR7 MyD88-dependent signaling molecules may be a common pathogenic mechanism for both AOSD and SLE.
Glucose transport kinetics and mRNA levels of different glucose transporters were determined in Saccharomyces cerevisiae strains expressing different sugar kinases. During exponential growth on glucose, a hxk2 null strain exhibited high-affinity hexose transport associated with an elevated transcription of the genes HXT2 and HXT7, encoding high-affinity transporters, and a diminished expression of the HXT1 and HXT3 genes, encoding low-affinity transporters. Deletion of HXT7 revealed that the high-affinity component is mostly due to HXT7; however, a previously unidentified very-high-affinity component (Km = 0.19 mM) appeared to be due to other factors. Expression of genes encoding hexokinases from Schizosaccharomyces pombe or Yarrowia lipolytica in a hxk1 hxk2 glk1 strain prevented derepression of the high-affinity transport system at high concentrations of glucose.
An Arabidopsis thaliana mutant, cbd (carotenoid biosynthesis deficient), was recovered from a mutant population based on its yellow cotyledons, yellow-first true leaves, and stunted growth. Seven-day-old seedlings and mature seeds of this mutant had lower chlorophyll and total carotenoids than the wild type (WT). Genetic and molecular characterization revealed that cbd was a recessive mutant caused by a T-DNA insertion in the gene cpSRP54 encoding the 54kDa subunit of the chloroplast signal recognition particle. Transcript levels of most of the main carotenoid biosynthetic genes in cbd were unchanged relative to WT, but expression increased in carotenoid and abscisic acid catabolic genes. The chloroplasts of cbd also had developmental defects that contributed to decreased carotenoid and chlorophyll contents. Transcription of AtGLK1 (Golden 2-like 1), AtGLK2, and GUN4 appeared to be disrupted in the cbd mutant suggesting that the plastid-to-nucleus retrograde signal may be affected, regulating the changes in chloroplast functional and developmental states and carotenoid content flux. Transformation of A. thaliana and Brassica napus with a gDNA encoding the Arabidopsis cpSRP54 showed the utility of this gene in enhancing levels of seed carotenoids without affecting growth or seed yield.
Accumulation, Arabidopsis thaliana, biosynthesis, Brassica napus, carotenoids, gene expression
Follicular helper T cells (TFH) represent a distinct subset of CD4+ T cells specialized in providing help to B lymphocytes, which may play a central role in autoimmune diseases having a major B cell component such as systemic lupus erythematosus. Recently, TFH subsets that share common phenotypic and functional characteristics with TFH cells from germinal centers, have been described in the peripheral blood from healthy individuals. The aim of this study was to analyze the distribution of such populations in lupus patients. Circulating TFH cell subsets were defined by multicolor flow cytometry as TFH17 (CXCR3-CCR6+), TFH1 (CXCR3 + CCR6-) or TFH2 (CXCR3-CCR6-) cells among CXCR5 + CD45RA-CD4+ T cells in the peripheral blood of 23 SLE patients and 23 sex and age-matched healthy controls. IL-21 receptor expression by B cells was analyzed by flow cytometry and the serum levels of IL-21 and Igs were determined by ELISA tests. We found that the TFH2 cell subset frequency is strongly and significantly increased in lupus patients with an active disease (SLEDAI score>8), while the TFH1 cell subset percentage is greatly decreased. The TFH2 and TFH1 cell subset frequency alteration is associated with the presence of high Ig levels and autoantibodies in patient’s sera. Moreover, the TFH2 cell subset enhancement correlates with an increased frequency of double negative memory B cells (CD27-IgD-CD19+ cells) expressing the IL-21R. Finally, we found that IgE levels in lupus patients’ sera correlate with disease activity and seem to be associated with high TFH2 cell subset frequency. In conclusion, our study describes for the first time the distribution of circulating TFH cell subsets in lupus patients. Interestingly, we found an increased frequency of TFH2 cells, which correlates with disease activity. Our results suggest that this subset might play a key role in lupus pathogenesis.
glk, the structural gene for glucokinase of Escherichia coli, was cloned and sequenced. Overexpression of glk resulted in the synthesis of a cytoplasmic protein with a molecular weight of 35,000. The enzyme was purified, and its kinetic parameters were determined. Its Km values for glucose and ATP were 0.78 and 3.76 mM, respectively. Its Vmax was 158 U/mg of protein. A chromosomal glk-lacZ fusion was constructed and used to monitor glk expression. Under all conditions tested, only growth on glucose reduced the expression of glk by about 50%. A fruR mutation slightly increased the expression of glk-lacZ, whereas the overexpression of plasmid-encoded fruR+ weakly decreased expression. A FruR consensus binding motif was found 123 bp upstream of the potential transcriptional start site of glk. Overexpression of glk interfered with the expression of the maltose system. Repression was strongest in strains that exhibited constitutive mal gene expression due to endogenous induction and, in the absence of a functional MalK protein, the ATP-hydrolyzing subunit of the maltose transport system. It was least effective in wild-type strains growing on maltose or in strains constitutive for the maltose system due to a mutation in malT rendering the mal gene expression independent of inducer. This demonstrates that free internal glucose plays an essential role in the formation of the endogenous inducer of the maltose system.
Systemic lupus erythematosus is a poorly understood autoimmune disease, characterized by autoantibodies to nuclear antigens and immune complex deposition in organs like the kidney. Current evidence indicates that a pathologic CD4+T cell subset, characterized by impaired extracellular signal-regulated kinase (ERK) pathway signaling, DNA hypomethylation, and consequent aberrant gene expression contributes to disease pathogenesis. Hydralazine is a lupus-inducing drug that also decreases T cell DNA methylation by inhibiting the ERK signaling pathway, replicating the defect found in lupus T cells. These observations suggest that defective ERK pathway signaling alters gene expression in T cells by inhibiting DNA methylation, contributing to lupus pathogenesis. The signaling defect in hydralazine-treated and lupus T cells has now been mapped to protein kinase C δ. Understanding the mechanism causing decreased ERK pathway signaling in lupus may shed light on mechanisms contributing to disease development in genetically predisposed people.
Lupus T cells; epigenetics; DNA methylation; extracellular signal-regulated kinase pathway signaling; Protein Kinase δ
Carbon catabolite repression (CCR) is a widespread phenomenon in many bacteria that is defined as the repression of catabolic enzyme activities for an unfavorable carbon source by the presence of a preferable carbon source. In Streptomyces, secondary metabolite production often is negatively affected by the carbon source, indicating the involvement of CCR in secondary metabolism. Although the CCR mechanism in Streptomyces still is unclear, glucokinase is presumably a central player in CCR. SgGlkA, a glucokinase from S. griseus, belongs to the ROK family glucokinases, which have two consensus sequence motifs (1 and 2). Here, we report the crystal structures of apo-SgGlkA, SgGlkA in complex with glucose, and SgGlkA in complex with glucose and adenylyl imidodiphosphate (AMPPNP), which are the first structures of an ROK family glucokinase. SgGlkA is divided into a small α/β domain and a large α+β domain, and it forms a dimer-of-dimer tetrameric configuration. SgGlkA binds a β-anomer of glucose between the two domains, and His157 in consensus sequence 1 plays an important role in the glucose-binding mechanism and anomer specificity of SgGlkA. In the structures of SgGlkA, His157 forms an HC3-type zinc finger motif with three cysteine residues in consensus sequence 2 to bind a zinc ion, and it forms two hydrogen bonds with the C1 and C2 hydroxyls of glucose. When the three structures are compared, the structure of SgGlkA is found to be modified by the binding of substrates. The substrate-dependent conformational changes of SgGlkA may be related to the CCR mechanism in Streptomyces.
Glucose-6-phosphate (G-6-P) formation in Staphylococcus aureus is catalysed by glucokinase (glkA) gene under high glucose
concentration leading to upregulation of various pathogenic factors; therefore the present study is aimed in the cloning and
characterization of glk A gene from S. aureus ATCC12600. The glk A gene was cloned in the Sma I site of pQE 30, sequenced
(Accession number: JN645812) and expressed in E. coli DH5α. The recombinant glk A expressed from the resultant glk A 1 clone
was purified using nickel metal chelate chromatography, the pure enzyme gave single band in SDS-PAGE with molecular weight
of 33kDa. The rglk A showed very high affinity to glucose Km 5.1±0.06mM with Hill coefficient of 1.66±0.032mM. Analysis of
glucokinase sequence of S. aureus showed presence of typical ATP binding site and ROK motif CNCGRSGCIE. Sequentially and
phylogenetically S. aureus glk A exhibited low identity with other bacterial glk A and 21% homology with human glucokinase
(GCK). Functionally, S. aureus glk A showed higher rate of G-6-P formation compared to human GCK which may have profound
role in the pathogenesis.
glk A; pQE 30; ROK; Km
Glucokinase from S. griseus (SgGlkA) was expressed, purified and crystallized using the sitting-drop vapour-diffusion method. A crystal of SgGlkA in complex with glucose diffracted X-rays to 1.84 Å resolution.
Glucokinase catalyzes the phosphorylation of glucose using ATP to yield glucose 6-phosphate. SgGlkA is a bacterial group III glucokinase from Streptomyces griseus that seems to play a regulatory role in carbon catabolite repression in this organism. SgGlkA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method at 293 K. A crystal of SgGlkA in complex with glucose was obtained using a reservoir solution consisting of 0.9 M sodium/potassium tartrate, 0.2 M NaCl and 0.1 M imidazole pH 8.1 and diffracted X-rays to 1.84 Å resolution. The crystal of SgGlkA in complex with glucose belonged to space group P6222 or P6422, with unit-cell parameters a = b = 109.19, c = 141.18 Å. The crystal contained one molecule in the asymmetric unit.
GlkA; catabolite repression; Streptomyces griseus
Hexokinases are conserved proteins functioning in glucose sensing and signaling. The rice blast fungus Magnaporthe oryzae contains several hexokinases, including MoHxk1 (hexokinase) and MoGlk1 (glucokinase) encoded respectively by MoHXK1 and MoGLK1 genes. The heterologous expression of MoGlk1 and MoHxk1 in Saccharomyces cerevisiae confirmed their conserved functions. Disruption of MoHXK1 resulted in growth reduction in medium containing fructose as the sole carbon source, whereas disruption of MoGLK1 did not cause the similar defect. However, the ΔMoglk1 mutant displayed decreased proton extrusion and a lower biomass in the presence of ammonium, suggesting a decline in the utilization of ammonium. Additionally, the MoGLK1 allele lacking catalytic activity restored growth to the ΔMoglk1 mutant. Moreover, the expression of MoPMA1 encoding a plasma membrane H+-ATPase decreased in the ΔMoglk1 mutant that can be suppressed by glucose and G-6-P. Thus, MoGlk1, but not MoHxk1, regulates ammonium utilization through a mechanism that is independent from its catalytic activity.
A gene for glucokinase (Glk) in Escherichia coli B was cloned onto vector plasmid pBR322, and the hybrid plasmid obtained was designated pGK100. The gene for Glk was located in the central MluI fragment (0.82 megadalton) of the 6.0-megadalton chromosomal DNA inserted in the HindIII site of the vector. The introduction of pGK100 into E. coli 112L having a decreased level of Glk activity resulted in the about 15-fold increase in this enzyme activity. The poor growth rate of 112L cells on glucose or mannose was also improved by the introduction of pGK100. However, removal of some portion near the glk gene prevented the growth of 112L cells, although Glk activity was high enough to support growth. Therefore, some function of Glk may be regulated by a gene(s) near the glk gene.
By transposon Tn917 mutagenesis, 16 mutants of Staphylococcus xylosus were isolated that showed higher levels of beta-galactosidase activity in the presence of glucose than the wild-type strain. The transposons were found to reside in three adjacent locations in the genome of S. xylosus. The nucleotide sequence of the chromosomal fragment affected by the Tn917 insertions yielded an open reading frame encoding a protein with a size of 328 amino acids with a high level of similarity to glucose kinase from Streptomyces coelicolor. Weaker similarity was also found to bacterial fructokinases and xylose repressors of gram-positive bacteria. The gene was designated glkA. Immediately downstream of glkA, two open reading frames were present whose deduced gene products showed no obvious similarity to known proteins. Measurements of catabolic enzyme activities in the mutant strains grown in the presence or absence of sugars established the pleiotropic nature of the mutations. Besides beta-galactosidase activity, which had been used to detect the mutants, six other tested enzymes were partially relieved from repression by glucose. Reduction of fructose-mediated catabolite repression was observed for some of the enzyme activities. Glucose transport and ATP-dependent phosphorylation of HPr, the phosphocarrier of the phosphoenolpyruvate:carbohydrate phosphotransferase system involved in catabolite repression in gram-positive bacteria, were not affected. The cloned glkA gene fully restored catabolite repression in the mutant strains in trans. Loss of GlkA function is thus responsible for the partial relief from catabolite repression. Glucose kinase activity in the mutants reached about 75% of the wild-type level, indicating the presence of another enzyme in S. xylosus. However, the cloned gene complemented an Escherichia coli strain in glucose kinase. Therefore, the glkA gene encodes a glucose kinase that participates in catabolite repression in S. xylosus.
MalT is the central transcriptional activator of all mal genes in Escherichia coli. Its activity is controlled by the inducer maltotriose. It can be inhibited by the interaction with certain proteins, and its expression can be controlled. We report here a novel aspect of mal gene regulation: the effect of cytoplasmic glucose and glucokinase (Glk) on the activity and the expression of MalT. Amylomaltase (MalQ) is essential for the metabolism of maltose. It forms maltodextrins and glucose from maltose or maltodextrins. We found that glucose above a concentration of 0.1 mM blocked the activity of the enzyme. malQ mutants when grown in the absence of maltodextrins are endogenously induced by maltotriose that is derived from the degradation of glycogen. Therefore, the fact that glk malQ+ mutants showed elevated mal gene expression finds its explanation in the reduced ability to remove glucose from MalQ-catalyzed maltodextrin formation and is caused by a metabolically induced MalQ− phenotype. However, even in mutants lacking glycogen, Glk controls endogenous induction. We found that overexpressed Glk due to its structural similarity with Mlc, the repressor of malT, binds to the glucose transporter (PtsG), releasing Mlc and thus increasing malT repression. In addition, even in mutants lacking Mlc (and glycogen), the overexpression of glk leads to a reduction in mal gene expression. We interpret this repression by a direct interaction of Glk with MalT concomitant with MalT inhibition. This repression was dependent on the presence of either maltodextrin phosphorylase or amylomaltase and led to the inactivation of MalT.
The Zymomonas mobilis genes encoding the glucose facilitator (glf), glucokinase (glk), or fructokinase (frk) were cloned and expressed in a lacIq-Ptac system using Escherichia coli K-12 mutants deficient in uptake and phosphorylation of glucose and fructose. Growth on glucose or fructose was restored when the respective genes (glf-glk or glf-frk) were expressed. In E. coli glf+ strains, both glucose and fructose were taken up via facilitated diffusion (Km, 4.1 mM for glucose and 39 mM for fructose; Vmax at 15 degrees C, 75 and 93 nmol min-1 mg-1 [dry weight] for glucose and fructose, respectively). For both substrates, counterflow maxima were observed.
For systematic and genome-wide analyses of rice gene functions, we took advantage of the full-length cDNA overexpresser (FOX) gene-hunting system and generated >12 000 independent FOX-rice lines from >25 000 rice calli treated with the rice-FOX Agrobacterium library. We found two FOX-rice lines generating green calli on a callus-inducing medium containing 2,4-D, on which wild-type rice calli became ivory yellow. In both lines, OsGLK1 cDNA encoding a GARP transcription factor was ectopically overexpressed. Using rice expression-microarray and northern blot analyses, we found that a large number of nucleus-encoded genes involved in chloroplast functions were highly expressed and transcripts of plastid-encoded genes, psaA, psbA and rbcL, increased in the OsGLK1-FOX calli. Transmission electron microscopy showed the existence of differentiated chloroplasts with grana stacks in OsGLK1-FOX calli cells. However, in darkness, OsGLK1-FOX calli did not show a green color or develop grana stacks. Furthermore, we found developed chloroplasts in vascular bundle and bundle sheath cells of coleoptiles and leaves from OsGLK1-FOX seedlings. The OsGLK1-FOX calli exhibited high photosynthetic activity and were able to grow on sucrose-depleted media, indicating that developed chloroplasts in OsGLK1-FOX rice calli are functional and active. We also observed that the endogenous OsGLK1 mRNA level increased synchronously with the greening of wild-type calli after transfer to plantlet regeneration medium. These results strongly suggest that OsGLK1 regulates chloroplast development under the control of light and phytohormones, and that it is a key regulator of chloroplast development.
Chloroplast development • FOX hunting system • GARP transcription factor • OsGLK1 • Oryza sativa • Rice
Crystals of the enzyme hexokinase 1 from the yeast K. lactis (KlHxk1) suitable for X-ray analysis were obtained in various space groups.
Glucose acts as both a carbon source and a hormone-like regulator of gene expression in eukaryotic organisms from yeast to man. Phosphorylation of glucose is executed by hexokinases, which represent a class of multifunctional enzymes that, in addition to their contribution to the uptake and initiation of metabolism of glucose, fructose and mannose, are involved in glucose signalling. The genome of the budding yeast Kluyveromyces lactis encodes a single hexokinase (KlHxk1) and a single glucokinase (KlGlk1). KlHxk1 exists in a monomer–homodimer equilibrium which is presumed to play a role in metabolic regulation. In order to evaluate the physiological significance of KlHxk1 dimerization on a molecular level, the enzyme was crystallized and subjected to X-ray structure analysis. Crystallization employing ammonium sulfate, diammonium phosphate or polyethylene glycol 6000 at pH values of 8.0–9.5 gave seven different crystal forms of KlHxk1. Crystallographic data to 1.66 Å resolution were obtained using synchrotron radiation. Structure determination of KlHxk1 in various packing environments will reveal the full architecture of the homodimeric enzyme and complete our mechanistic understanding of the catalytic and regulatory functions of the enzyme.
glycolysis; hexokinases; KlHxk1; Kluyveromyces lactis; yeast
The glucose analog 2-deoxyglucose (2dGlc) inhibits the growth and multicellular development of Myxococcus xanthus. Mutants of M. xanthus resistant to 2dGlc, designated hex mutants, arise at a low spontaneous frequency. Expression of the Escherichia coli glk (glucokinase) gene in M. xanthus hex mutants restores 2dGlc sensitivity, suggesting that these mutants arise upon the loss of a soluble hexokinase function that phosphorylates 2dGlc to form the toxic intermediate, 2-deoxyglucose-6-phosphate. Enzyme assays of M. xanthus extracts reveal a soluble hexokinase (ATP:d-hexose-6-phosphotransferase; EC 126.96.36.199) activity but no phosphotransferase system activities. The hex mutants have lower levels of hexokinase activities than the wild type, and the levels of hexokinase activity exhibited by the hex mutants are inversely correlated with the ability of 2dGlc to inhibit their growth and sporulation. Both 2dGlc and N-acetylglucosamine act as inhibitors of glucose turnover by the M. xanthus hexokinase in vitro, consistent with the finding that glucose and N-acetylglucosamine can antagonize the toxic effects of 2dGlc in vivo.
Two classes of cell lines heterozygous at the galactokinase (glk) locus have been isolated from Chinese hamster ovary cells. Class I, selected by plating nonmutagenized wild-type cells at low density in medium containing 2-deoxygalactose at a partially selective concentration, underwent subsequent mutation to the glk-/- genotype at a low frequency (approximately 10(-6) per cell), which was increased by mutagenesis. Class II heterozygotes, isolated by sib selection from mutagenized wild-type cells, had a higher spontaneous frequency of mutation to the homozygous state (approximately 10(-4) per cell), which was not affected by mutagenesis. About half of the glk-/- mutants derived from a class II heterozygote, but not the heterozygote itself, were functionally hemizygous at the syntenic thymidine kinase (tk) locus. Similarly, a tk+/- heterozygote with characteristics analogous to the class II glk+/- cell lines underwent high-frequency mutation to tk-/-, and most of these mutants, but not the tk+/- heterozygote, were functionally hemizygous at the glk locus. A model is proposed, similar to that for the mutational events at the adenine phosphoribosyl transferase locus (W. E. C. Bradley and D. Letovanec, Somatic Cell Genet. 8:51-66, 1982), of two different events, high and low frequency, being responsible for mutation at either of the linked loci tk and glk. The low-frequency event may be a point mutation, but the high-frequency event, in many instances, involves coordinated inactivation of a portion of a chromosome carrying the two linked alleles. Class II heterozygotes would be generated as a result of a low-frequency event at one allele, and class I heterozygotes would be generated by a high-frequency event. Supporting this model was the demonstration that all class I glk+/- lines examined were functionally hemizygous at tk.