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.     

Regulatory Mutations Affecting the Gluconate System in Escherichia coli

A spontaneously arising regulatory mutant of the gluconate system in Escherichia coli was isolated. This mutant became constitutive, probably in one step, for gluconate high-affinity transport, gluconokinase, and gluconate-6-P dehydrase. The mutation involved (gntR18) is cotransducible with asd. Pseudorevertants, derived from a mutant (M2) that shows a long lag for growth on gluconate mineral medium, were also isolated and characterized. They give constitutive levels of gluconokinase and gluconate-6-P dehydrase but lack high-affinity transport function. Genetic experiments performed with one of these pseudorevertants (M4) indicate that it carries a secondary mutation in the gntR gene. The M4 phenotype is thus the result of the interaction of expression of a constitutive mutation (gntR4) with the mutation of strain M2 (gntM2).     

Nucleotide sequence of the uhp region of Escherichia coli.

The Escherichia coli uhp region encodes the transport system that mediates the uptake of a number of sugar phosphates as well as the regulatory components that are responsible for induction of this transport system by external glucose 6-phosphate. Four uhp genes have been identified by analysis of the complementation behavior and polypeptide coding capacity of plasmids carrying subcloned regions or transposon insertions. The nucleotide sequence of a 6.5-kilobase segment that contains the 3' end of the ilvBN operon and the entire uhp region was determined. Four open reading frames were identified in the locations expected for the various uhp genes; all were oriented in the same direction, counterclockwise relative to the genetic map. The properties of the polypeptides predicted from the nucleotide sequence were consistent with their observed features. The 196-amino-acid UhpA polypeptide has the composition characteristic of a soluble protein and bears homology to the DNA-binding regions of many regulatory activators and repressors. The 518-amino-acid UhpB and the 199-amino-acid UhpC regulatory proteins contain substantial segments of hydrophobic character. Similarly, the 463-amino-acid UhpT transporter is a hydrophobic protein with numerous potential transmembrane segments. The UhpC regulatory protein has substantial sequence homology to part of UhpT, suggesting that this regulatory protein might have evolved by duplication of the gene for the transporter and that its role in transmembrane signaling may involve sugar-phosphate-binding sites and transmembrane orientations similar to those of the transport protein.     

Mutations affecting gluconate catabolism in Escherichia coli. Genetic mapping of the locus for the thermosensitive gluconokinase

An Escherichia coli strain unable to use gluconate was isolated by spontaneous curing of lambda cI857 s7 xis6 b515 b519, lambda cI857 s7 delta(A-att) dargI valS lysogens. Two lesions, linked to asd and pyrB markers, respectively, were necessary to produce this phenotype. The asd-linked mutation gnt-17, of regulatory type, seems to affect the expression of the major system of gluconate utilization (min 75) as well as that of 6-phosphogluconate dehydratase (gene edd, min 41), the first enzyme of the Entner-Doudoroff pathway. A closely linked suppressor of gnt-17 causes constitutivity of these activities; this suppressor resembles gntR, which is also in the asd region. Hence, it is possible that gnt-17 is a super-repressing allele of gntR, rather than a positive controlling element. Lesion gnt-17 alone does not prevent the utilization of gluconate; for this, the mutation gnt-18 at 96.9 min is also necessary. This mutation abolishes the thermosensitive gluconokinase activity and thus eliminates the subsidiary ability to catabolize gluconate. Accordingly, gnt-18 seems to be allelic with gntV, the locus postulated as being in the pyrB region specifying the thermosensitive gluconokinase.     

Effect of mutations causing gluconate kinase or gluconate permease deficiency on expression of the Bacillus subtilis gnt operon.

The gluconate (gnt) operon contains genes for a repressor of the operon, gluconate kinase, and gluconate permease. A nonleaky kinase mutation (gntK4) induced the gnt operon constitutively through interaction of the repressor with an inducer of gluconate which had been endogenously formed and accumulated in the cell owing to the complete deficiency of the kinase even in the absence of gluconate in the medium. In contrast, a nonleaky permease mutation (gntP9) never induced the operon by gluconate likely because it cannot give rise to its inducing concentration in the cell even in the presence of gluconate in the medium.     

Genes involved in the uptake and catabolism of gluconate by Escherichia coli.

The isolation and properties of a mutant of Escherichia coli K12 that is totally unable to take up and utilize gluconate are described. Genetical analysis shows this phenotype to be associated with two lesions. One phenotype, designated GntM-, is the result of a mutation in a gene co-transducible with malA; the other, designated GNTS-, is the result of a mutation in a gene (GntS) co-transducible with fdp. The GntS--phenotype differs little from that of wild-type cells, but GntM- GntS+ organisms grow on gluconate only after a prolonged lag and form a gluconate uptake system that is strongly repressed by pyruvate. Moreover, such GntM- mutants readily give rise to further mutants that form a gluconate uptake system, gluconate kinase and 6-phosphogluconate dehydratase consititutively; in partial diploids, this constitutivity is recessive to the inducible character. It is postulated that the GntM- phenotype is due to malfunction of a negative control gene gntR, and that gntS+ specifies the activity of a gluconate uptake system.     

Efficient utilization and operation of the gluconate-inducible system of the promoter of the Bacillus subtilis gnt operon in Escherichia coli.

A DNA fragment containing the promoter of the Bacillus subtilis gluconate (gnt) operon and its first gene (gntR) was cloned into Escherichia coli. E. coli recognized this promoter efficiently and precisely. Moreover, the gluconate-inducible system of this operon operated even in E. coli.     

Overexpression, purification, and characterization of ATP-NAD kinase of Sphingomonas sp. A1

The NAD kinase gene (nadK) of Sphingomonas sp. A1 was cloned and then overexpressed in Escherichia coli, and the gene product (NadK) was purified from the E. coli cells through five steps with a 25% yield of activity. NadK was a homodimer of 32 kDa subunits, utilized ATP or other nucleoside triphosphates, but not inorganic polyphosphates, as phosphoryl donors for the phosphorylation of NAD, most efficiently at pH 8.0 and 50-55 degrees C, and was designated as ATP-NAD kinase (NadK). NadK showed no NADH kinase activity and was slightly inhibited by NADP(H). Precursors for NAD biosynthesis such as quinolinic acid, nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, and nicotinic acid had no effect on the NadK activity, as observed in the cases of the NAD kinases of Micrococcus flavus, Mycobacterium tuberculosis, and E. coli. Taken together with the report that the NAD kinase of Bacillus subtilis is activated by quinolinic acid [J. Bacteriol. 185 (2003) 4844], it is indicated that the regulatory patterns of NAD kinases differ even among bacterial NAD kinases.     

Cloning and characterization of an eukaryotic initiation factor-2alpha kinase from the silkworm,Bombyx mori

Eukaryotic initiation factor 2alpha (eIF-2alpha) kinases are involved in the translational regulations that occur in response to various types of environmental stress, and play an important role in the cellular defense system operating under unfavorable conditions. The identification of additional eIF-2alpha kinases and the elucidation of their functions are necessary to understand how different eIF-2alpha kinases can specifically respond to distinct stimuli. Here, we report a novel eIF-2alpha kinase, termed BeK, from the silkworm, Bombyx mori. This gene encodes 579 amino acids and contains all 11 catalytic domains of protein-serine/threonine kinases. Most notably, it contains an "Ile-Gln-Met-Xaa-Xaa-Cys" motif, which is highly conserved from yeast to mammalian eIF-2alpha kinases. BeK does not show any significant homology in the NH(2) terminal regulatory domain, suggesting a distinct regulatory mechanism of this novel eIF-2alpha kinase. BeK is ubiquitously expressed in the various tissues throughout the final larval stage. Importantly, BeK is activated in Drosophila Schneider cells following heat shock and osmotic stress, and activated-BeK has been shown to phosphorylate an eIF-2alpha subunit at the Ser(50) site. However, other forms of stress, such as immune stress, endoplasmic reticulum stress and oxidative stress, cannot significantly elicit BeK activity. Interestingly, the baculovirus gene product, PK2, can inhibit BeK enzymatic activity, suggesting that BeK may be an endogenous target for a viral gene product. Taken together, these data indicate that BeK is a novel eIF-2alpha kinase involved in the stress response in B. mori.