The subsidiary GntII system for gluconate metabolism in Escherichia coli: alternative induction of the gntV gene.

Two systems are involved in the transport and phosphorylation of gluconate in Escherichia coli. GntI, the main system, consists of high and low-affinity gluconate transporters and a thermoresistant gluconokinase for its phosphorylation. The corresponding genes, gntT, gntU and gntK at 76.5 min, are induced by gluconate. GntII, the subsidiary system, includes IdnT and GntV, which duplicate activities of transport and phosphorylation of gluconate, respectively. Gene gntV at 96.8 min is divergently transcribed from the idnDOTR operon involved in L-idonate metabolism. These genetic elements are induced by the substrate or 5-keto-D-gluconate. Because gntV is also induced in cells grown in gluconate, it was of interest to investigate its expression in this condition. E. coli gntK, idnOokan mutants were constructed to study this question. These idnO kan-cassete inserted mutants, unable to convert gluconate to 5-keto-D-gluconate, permitted examining gntV expression in the absence of this inducer and demonstrating that it is not required when the cells grow in gluconate. The results suggest that E. coli gntV gene is alternatively induced by 5-keto-D-gluconate or gluconate in cells cultivated either in idonate or gluconate. In this way, the control of gntV expression would seem to be involved in the efficient utilization of these substrates.

The subsidiary GntII system for gluconate metabolism in Escherichia coli: Alternative induction of the gntV gene

Two systems are involved in the transport and phosphorylation of gluconate in Escherichia coli. GntI, the main system, consists of high and low-affinity gluconate transporters and a thermoresistant gluconokinase for its phosphorylation. The corresponding genes, gntT, gntU and gntK at 76.5 min, are induced by gluconate. GntII, the subsidiary system, includes IdnT and GntV, which duplicate activities of transport and phosphorylation of gluconate, respectively. Gene gntV at 96.8 min is divergently transcribed from the idnDOTR operon involved in L-idonate metabolism. These genetic elements are induced by the substrate or 5-keto-D-gluconate. Because gntV is also induced in cells grown in gluconate, it was of interest to investigate its expression in this condition. E. coli gntK, idnOokan mutants were constructed to study this question. These idnO kan-cassete inserted mutants, unable to convert gluconate to 5-keto-D-gluconate, permitted examining gntV expression in the absence of this inducer and demonstrating that it is not required when the cells grow in gluconate. The results suggest that E. coli gntV gene is alternatively induced by 5-keto-D-gluconate or gluconate in cells cultivated either in idonate or gluconate. In this way, the control of gntV expression would seem to be involved in the efficient utilization of these substrates.

Gluconate as suitable potential reduction supplier in Corynebacterium glutamicum: cloning and expression of gntP and gntK in Escherichia coli.

Corynebacterium glutamicum is widely used in the industrial production of amino acids. We have found that this bacterium grows exponentially on a mineral medium supplemented with gluconate. Gluconate permease and Gluconokinase are expressed in an inducible form and, 6-phosphogluconate dehydrogenase, although constitutively expressed, shows a 3-fold higher specific level in gluconate grown cells than those grown in fructose under similar conditions. Interestingly, these activities are lower than those detected in the strain Escherichia coli M1-8, cultivated under similar conditions. Additionally, here we also confirmed that this bacterium lacks 6-phosphogluconate dehydratase activity. Thus, gluconate must be metabolized through the pentose phosphate pathway. Genes encoding gluconate transport and its phosphorylation were cloned from C. glutamicum, and expressed in suitable E. coli mutants. Sequence analysis revealed that the amino acid sequences obtained from these genes, denoted as gntP and gntK, were similar to those found in other bacteria. Analysis of both genes by RT-PCR suggested constitutive expression, in disagreement with the inducible character of their corresponding activities. The results suggest that gluconate might be a suitable source of reduction potential for improving the efficiency in cultures engaged in amino acids production. This is the first time that gluconate specific enzymatic activities are reported in C. glutamicum.

Gluconate as suitable potential reduction supplier in Corynebacterium glutamicum: Cloning and expression of gntP and gntK in Escherichia coli

Corynebacterium glutamicum is widely used in the industrial production of amino acids. We have found that this bacterium grows exponentially on a mineral médium supplemented with gluconate. Gluconate permease and Gluconokinase are expressed in an inducible form and, 6-phosphogluconate dehydrogenase, although constituvely expressed, shows a 3-fold higher specific level in gluconate grown cells than those grown in fructose under similar conditions. Interestingly, these activities are lower than those detected in the strain Escherichia coli Ml-8, cultivated under similar conditions. Additionally, here we also confirmed that this bacterium lacks 6-phosphogluconate dehydratase activity. Thus, gluconate must be metabolized through the pentose phosphate pathway. Genes encoding gluconate transport and its phosphorylation were cloned from C. glutamicum, and expressed in suitable E. coli mutants. Sequence analysis revealed that the amino acid sequences obtained from these genes, denoted as gntP and gntK, were similar to those found in other bacteria. Analysis of both genes by RT-PCR suggested constitutive expression, in disagreement with the inducible character of their corresponding activities. The results suggest that gluconate might be a suitable source of reduction potential for improving the efficiency in cultures engaged in amino acids production. This is the first time that gluconate specific enzymatic activities are reported in C. glutamicum.

The metabolism of gluconate in Escherichia coli: Physiological evidences of a regulatory effect of idnR on the expression of the gntR regulon operons

Uptake and phosphorylation initiate the catabolism of gluconate in E. coli. Such activities conform two systems,GntI and GntII, encoded by two sets of genes differently located on the E. coli chromosome and under different regulation. gntT, gntU and gntK (minute 76) encode for high and low affinity gluconate transports and for a thermoresistant gluconokinase respectively, that conform GntI; the mentioned genes and those of the edd-eda operon (minute 41) are negatively regulated by the gntR gene product conforming the gntR regulon. idnT and gntV (minute 96), encode for another gluconate transport and a thermosensitive gluconokinase, conforming GntII. These genes are presumably positively controlled by IdnR. IdnT also functions as a permease for idonate; the corresponding gene is included in the idnDOTR operon, responsible for idonate metabolism, in which gluconate is an intermediary. Here we report a regulatory action of IdnR on the operons of the gntR regulon; i.e., gntT, gntKU and edd-eda. The expression of these operons, was diminished in a gntR mutant complemented with a clone of idnR and also in E. coli mutants in which the idnDOTR operon is expressed in a gluconate dependent inducibility. This is the first report of a regulatory effect of IdnR on edd-eda operon expression.

El transporte y la fosforilación inician el catabolismo del gluconato en E. coli. Estas actividades conforman dossistemas, GntI y GntII, codificados por dos grupos de genes, diferentemente regulados y ubicados en distintos sitios del cromosoma. Los genes gntT, gntU y gntK (minuto 76), codifican distintas proteínas para transportes de alta y baja afinidad para gluconato y una gluconoquinasa termoresistente respectivamente, que forman el sistema GntI; los genes respectivos junto con los del operón edd-eda (minuto 41), son regulados negativamente por el producto de gntR (minuto76) constituyendo el regulón gntR. Los genes IdnT y gntV (minuto 96), codifican otra permeasa para gluconato y una gluconoquinasa termosensible que forman GntII. IdnT funciona también como permeasa para idonato; el gen correspondiente es parte del operón idnDOTR, regulado positivamente por IdnR y responsable del metabolismo del idonato en el que gluconato es un intermediario. Se reporta un efecto regulatorio de IdnR sobre los operones del regulón gntR; i.e., gntT, gntKU and edd-eda. La expresión de estos operones resultó disminuida en una mutante gntR complementada con un clon de idnR y también en mutantes de E. coli en la que la expresión del operón idnDOTR se induce en presencia de gluconato. Este es el primer reporte de la acción regulatoria de IdnR sobre la expresión del operón eddeda.

Localización molecular mediante PCR inverso del sitio de inserción de un transposon TN 10 ligado a gntS en mutantes de Escherichia coli / Molecular location by inverse PCR of the insertion site of a Tn10 transposón ligated to gntS in Escherichia coli mutants

La vecindad del sitio de inserción del transposón Tn10, portado por la mutante de Escherichia coli DF601, contiene el gene gntS, un presunto regulador positivo involucrado en el metabolismo del gluconato en E. coli. Aunque el análisis molecular de la región del minuto 95.3, señalado originalmente como el sitio de inserción del transposón, reveló el ORF f251 con características de regulador, transformaciones con éste y otros ORFs de la región, una vez clonados, no complementaron la función perdida en mutantes gntS. El presente trabajo racionaliza la causa de tales resultados. Con base a la secuencia nucleotídica suministrada por GenBank y la aplicación de la técnica de PCR inverso, se encontró que el sitio exacto de inserción del transposón Tn10, portado por la mencionada mutante y sus derivadas TetR, es la posición 4442377, la cual interrumpe el ORF ytfN en la región del minuto 95.8 del mapa genético y no en la del minuto 95.3, como fue originalmente establecido. Los resultados, además de señalar sin ambigüedad la región cromosómica a investigar para lograr los fines propuestos, indican la conveniencia de aplicar la técnica sencilla de PCR inverso, para ubicar elementos genéticos antes de emplearlos en estudios moleculares.

The vicinity of the Tn10 transposon insertion site, carried by the Escherichia. coli mutant DF601, contains the genegntS, a putative positive regulator involved in the metabolism of the gluconate in E. coli. Although the molecular analysis of the 95.3 minute region, originally reported as the transposon insertion site, revealed the ORF f251 as one with regulator characteristics, transformations with this and other ORFs associated with the region, once cloned, did not complement the lost function in gntS mutants. The present work rationalizes on the cause of such results. Based on the nucleotide sequence provided by GenBank and application of the inverse PCR technique, it was found that the exact site of the Tn10 transposon insertion is in the position 4442377, interrupting the ytfN ORF at the minute 95.8 of the E. coli genetic map and not at minute 95.3, as it was originally established. The results indicate the precise chromosomal region to investigate in order to obtain the initially proposed aims and the convenience of applying the simple technique of inverse PCR to locate genetic elements as well.

A mutation effecting gluconate catabolism in Escherichia coli: The locus for the main high affinity transport / Una mutación que afecta el catabolismo del gluconato en Escherichia coli: el locus para el transporte principal de alta afinidad

The bioH-malA region of the E.coli chromosome (min 75.5) includes the gntT gene which encodes a high affinity transport for gluconate. Other gnt loci have not been characterized in this region; nevertheless, because lesion in it affect severely the utilization of gluconate, it has been suggested as being more complex. This region was investigated with respect to gluconate catabolism through the characterization of suitable E.coli strains lysogenized with a specialized transducing phage carrying the bioH-malA region of the bacterial chromosome (cI857st68h80d2bioH-malA). It was found that the region transduced by this phage while includes the gntT gene lacks other gnt loci that might code additional activities for transport of gluconate or its phosphorylation. Moreover, the pleiotropic lesion gntM2, previously mapped into this region and suggested as altering gntT or a presumptive regulator gene that might be involved in this catabolism, resulted recessive in lysogens (partial diploids) containing the defective prophage. The results obtained supported the idea that gntM2 is an allele of gntT; consequently those results suggested the precise position of this gene on the cromosomic map and the central role that its product might have in the initial incorporation of gluconate in E.coli.

Selection of lacZ operon fusions in genes of gluconate metabolism in E. coli: Characterization of A gntT::lacZ fusion / Selección de fusiones lacZ de operon en genes del metabolismo del gluconato en E.coli: Caracterización de una fusión gntT::lacZ

The initial steps involved in the utilization of gluconate by E.coli, its incorporation into the cell and susequent phosphorylation to gluconate 6-phosphate, conform two system that duplicate activities. These system, GntI and GntII, are specified by two sets of genes distinctly regulated and located respectively at the malA-asd (75 min) and fdp-valS (96 min) regions of the bacterial chromosome. The precence of duplicate activities in the metabolism of gluconates of E.coli, has made difficult the study of the expression and participation of the GntI and GntII system. In order to advance in these respec, the phage *placMu53 was used to select operon gnt::lacZ fusion in a E.coli strain (edd-zwf), (gnd-his), lac. Here we report the study of a gntT::lacZ fusion. This transductan allowed to differentiate the inducible expresion of the gntT and gntU genes. its characteristic, in agreement with previous report, support the central role of the gntT gene in this metabolism