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Elizabeth Langley

Publications and source records attributed to Elizabeth Langley.

6 recordsLinked to original sources
PubMed2005

Sugar uptake and sensitivity to carbon catabolite regulation in Streptomyces peucetius var. caesius.

Streptomyces peucetius var. caesius produces a family of secondary metabolites called anthracyclines. Production of these compounds is negatively affected in the presence of glucose, galactose, and lactose, but the greatest effect is observed under conditions of excess glucose. Other carbon sources, such as arabinose or glutamate, show either no effect or stimulate production. Among the carbon sources that negatively affect anthracycline production, glucose is consumed in greater concentrations. We determined glucose and galactose transport in S. peucetius var. caesius and in a mutant of this strain whose anthracycline production is insensitive to carbon catabolite repression (CCR). In the original strain, incorporation of glucose and galactose was stimulated when the microorganism was grown in media containing these sugars, although we also observed basal galactose incorporation. Both the induced and the basal incorporation of galactose were suppressed when the microorganism was grown in the presence of glucose. Furthermore, adding glucose directly during the transport assay also inhibited galactose incorporation. In the mutant strain, we observed a reduction in both glucose (48%) and galactose (81%) incorporation compared to the original. Galactose transport in this mutant showed reduced sensitivity to the negative effect of glucose; however, it was still sensitive to inhibition. The deficient transport of these sugars, as well as CCR sensitivity to glucose in this mutant was corrected when the mutant was transformed with the SCO2127 region of the Streptomyces coelicolor genome. Our results support a role for glucose as the most easily utilized carbon source capable of exerting the greatest repression on anthracycline biosynthesis. In consequence, glucose also prevented the repressive effect of galactose by suppressing its incorporation. This suggests the participation of an integral regulatory system, which is initiated by an increase in incorporation of repressive sugars and their metabolism as a prerequisite for establishing the phenomenon of CCR in S. peucetius var. caesius.

Anthracyclines↗
PubMed2005

Regulation by glucagon of the rat histidase gene promoter in cultured rat hepatocytes and human hepatoblastoma cells.

Histidase (Hal), the amino acid-degrading enzyme of histidine, is regulated by the protein content of the diet and by hormones such as glucocorticoids and glucagon. However, glucagon can activate the following two possible transduction pathways: protein kinase A (PKA) and protein kinase C (PKC). The aim of this study was to isolate the 5'-flanking region of rat Hal gene to locate possible cAMP- and glucocorticoid-responsive elements and to identify whether the activation of the Hal promoter by glucagon occurs via PKA or PKC. The results showed that glucagon was able to induce Hal expression 1.5-fold in primary hepatocytes. The addition of phorbol 12-myristate,13-acetate (PMA) and forskolin to hepatocytes increased Hal mRNA concentration by 100 and 40%, respectively. To identify the Hal gene regulatory region, a 1248-bp fragment of the 5'-region was obtained. The transcription initiation site was located at 404 bp from ATG. The sequence did not show consensus TATA-like or CAAT-like boxes in the first 100 bp upstream from the transcription start site. The promoter contained six GC rich boxes, seven putative AP1 binding sites, and four glucocorticoid-responsive elements. The putative Hal promoter region was cloned into the pGL3basic vector and transfected into HepG2 cells. Luciferase expression was significantly stimulated by glucagon (0.9-fold), forskolin (0.9-fold), PMA (2.0-fold), and dexamethasone (2.9-fold). This evidence supports that the Hal gene is turned on by glucocorticoids and by glucagon either via PKC or PKA, but prefers the PKA pathway.

Animals↗
PubMed2005

Pleiotropic effect of the SCO2127 gene on the glucose uptake, glucose kinase activity and carbon catabolite repression in Streptomyces peucetius var. caesius.

SCO2127 and SCO2126 (glkA) are adjacent regions located in Streptomyces coelicolor DNA. glkA encodes glucose kinase (Glk), which has been implicated in carbon catabolite repression (CCR) in the genus Streptomyces. In this work, the glkA and SCO2127 genes from S. coelicolor were used, either individually or together, to transform three mutants of Streptomyces peucetius var. caesius resistant to CCR. These mutants present decreased levels of Glk, and deficiency in glucose transport. When the mutants were transformed with a plasmid containing the SCO2127 sequence, glucose uptake and Glk activity values were increased to levels similar to or higher than those of the original strain, and each strain regained sensitivity to CCR. This result was surprising considering that the putative SCO2127 amino acid sequence does not seem to encode a glucose permease or a Glk. In agreement with these results, an increase in glkA mRNA levels was observed in a CCR-resistant mutant transformed with SCO2127 compared with those of the original strain and the CCR-resistant mutant itself. As expected, recombinants containing the glkA sequence reverted Glk to normal activity values, but glucose uptake remained deficient. The data suggest that the SCO2127 gene product enhances transcription of both genes, and support the first specific role for this region in Streptomyces species. The physiological consequence of this effect is an increase in the glucose catabolites that may be involved in eliciting CCR in this genus.

Antimetabolites↗
PubMed2004

Biochemical characterization of the glucose kinase from Streptomyces coelicolor compared to Streptomyces peucetius var. caesius.

Glucose kinase (Glk) from Streptomyces coelicolor was purified, characterized biochemically and kinetically, and compared to Glk from Streptomyces peucetius var. caesius. In both Streptomyces, 96% of the enzyme activity was detected in the cytosolic fraction. The use of a Glk activity gel showed no isoforms of the enzyme in extracts from these microorganisms. The purified S. coelicolor Glk was stable in its tetrameric form, unlike the purified enzyme from S. peucetius var. caesius which easily dissociated into dimers. Tetramer dissociation was prevented by 100 mM d-glucose; however, this effect was not observed with other sugars. The optimum pH and the pI are practically identical for both enzymes. Maximum activity was found at a lower temperature for S. coelicolor Glk (33 compared to 42 degrees C) and its activity was apparently less stable at higher temperatures. Its activation energy was also 40% lower than that of S. peucetius var. caesius. The kinetic mechanism appears to follow a rapid equilibrium-ordered Bi-Bi sequential mechanism in both microbial enzymes, where Glk first binds glucose and then the MgATP(2-) complex, to form a ternary complex (enzyme d-glucose-MgATP(-2)). The Km values for D-glucose and MgATP(2-) were 1.4, 0.5 mM and 1.6, 0.8 mM for the S. coelicolor and S. peucetius var. caesius Glks, respectively. However Vmax of S. coelicolor Glk was higher. In conclusion, the S. coelicolor Glk showed a more stable tetrameric form with better affinity for its substrates and higher Vmax, suggesting greater catalytic efficiency.

Electrophoresis, Polyacrylamide Gel↗
PubMed2004

Glucose kinase alone cannot be responsible for carbon source regulation in Streptomyces peucetius var. caesius.

Using an antibiotic enrichment procedure, eight mutants of Streptomyces peucetius var. caesius were isolated for their sensitivity to the glucose analogue 2-deoxyglucose (DOG), from a DOG-resistant strain (Dog(R)). These mutants (Dog(S)) and their parent strain were examined for growth sensitivity to DOG, glucose kinase (Glk) activity, glucose uptake, and sensitivity to repression by glucose and other catabolites derived from it. No correlation was found between Glk levels or glucose uptake and carbon catabolite repression (CCR) in these strains. However, the ratio of glucose uptake to Glk activity, and thus the flux through glycolysis, seemed responsible for this effect. Among several products of glucose catabolism tested, fructose-1,6-bis-phosphate and phosphoenolpyruvate showed significant repression of anthracycline formation. These compounds also reduced anthracycline formation in a Dog(R) mutant insensitive to glucose repression. Our data suggest that Glk alone is not sufficient to elicit CCR in this microorganism, and gives the first physiological evidence supporting the hypothesis that some products of glucose catabolism are involved in CCR in Streptomyces.

Antimetabolites↗