The tryptophan synthetase gene TRP1 of Nodulisporium sp.: molecular characterization and its relation to nodulisporic acid A production.

Nodulisporic acid A (NAA), an insecticidal indole diterpene, is produced by the fungus Nodulisporium sp. Since indole-3-glycerolphosphate is the precursor of the indole moiety of NAA, it is suggested that the activity of tryptophan synthetase may play a role in NAA biosynthesis. To investigate this hypothesis, the tryptophan synthetase gene TRP1 of Nodulisporium sp. was cloned and characterized. The gene consists of three introns of 146, 68, and 57 bp. The four exons encode a protein of 712 amino acids, the sequence of which is highly homologous to that of other fungal tryptophan synthetase proteins. The transcription initiation site was mapped 66 bp upstream to the ATG, and the polyA tail attachment site is 169 bp downstream to the translation stop codon. Replacement of the N-terminal half of the gene with a hygromycin selection marker yielded mutants with the tryptophan auxotroph/hygromycin-resistance (trp(-)/hyr) phenotype. The TRP1 mutants required a high concentration of tryptophan supplement in solid medium (10 mM) to sustain minimal growth and failed to produce NAA in the production medium (FFL-CAM) supplemented with high concentrations of tryptophan.

Distinct effects of the antiestrogen Faslodex on the stability of estrogen receptors-alpha and -beta in the breast cancer cell line MCF-7.

The effects of estrogen receptor (ER) ligands on the stability and transcriptional activity of ERbeta in the breast cancer cell lines MCF-7 and HeLa were examined. We found that ERbeta was degraded in the presence of 17beta-estradiol. Tamoxifen and Faslodex (ICI 182,780) prevented ERbeta receptor destabilization. In contrast to ERalpha, ERbeta degradation was not abolished by inhibitors of the proteasome-mediated protein degradation pathway. Furthermore, single point mutations in helix 12 of the receptor dramatically affected the stability and subsequent transcriptional activation of ERbeta.

Sequence analysis of the GntII (subsidiary) system for gluconate metabolism reveals a novel pathway for L-idonic acid catabolism in Escherichia coli.

The presence of two systems in Escherichia coli for gluconate transport and phosphorylation is puzzling. The main system, GntI, is well characterized, while the subsidiary system, GntII, is poorly understood. Genomic sequence analysis of the region known to contain genes of the GntII system led to a hypothesis which was tested biochemically and confirmed: the GntII system encodes a pathway for catabolism of L-idonic acid in which D-gluconate is an intermediate. The genes have been named accordingly: the idnK gene, encoding a thermosensitive gluconate kinase, is monocistronic and transcribed divergently from the idnD-idnO-idnT-idnR operon, which encodes L-idonate 5-dehydrogenase, 5-keto-D-gluconate 5-reductase, an L-idonate transporter, and an L-idonate regulatory protein, respectively. The metabolic sequence is as follows: IdnT allows uptake of L-idonate; IdnD catalyzes a reversible oxidation of L-idonate to form 5-ketogluconate; IdnO catalyzes a reversible reduction of 5-ketogluconate to form D-gluconate; IdnK catalyzes an ATP-dependent phosphorylation of D-gluconate to form 6-phosphogluconate, which is metabolized further via the Entner-Doudoroff pathway; and IdnR appears to act as a positive regulator of the IdnR regulon, with L-idonate or 5-ketogluconate serving as the true inducer of the pathway. The L-idonate 5-dehydrogenase and 5-keto-D-gluconate 5-reductase reactions were characterized both chemically and biochemically by using crude cell extracts, and it was firmly established that these two enzymes allow for the redox-coupled interconversion of L-idonate and D-gluconate via the intermediate 5-ketogluconate. E. coli K-12 strains are able to utilize L-idonate as the sole carbon and energy source, and as predicted, the ability of idnD, idnK, idnR, and edd mutants to grow on L-idonate is altered.

Positive and negative transcriptional regulation of the Escherichia coli gluconate regulon gene gntT by GntR and the cyclic AMP (cAMP)-cAMP receptor protein complex.

The gntT gene of Escherichia coli is specifically induced by gluconate and repressed via catabolite repression. Thus, gluconate is both an inducer and a repressor of gntT expression since gluconate is a catabolite-repressing sugar. In a gntR deletion mutant, the expression of a chromosomal gntT::lacZ fusion is both high and constitutive, confirming that GntR is the negative regulator of gntT. Indeed, GntR binds to two consensus gnt operator sites; one overlaps the -10 region of the gntT promoter, and the other is centered at +120 with respect to the transcriptional start site. The binding of GntR to these sites was proven in vitro by gel redardation assays and in vivo by site-directed mutagenesis of the binding sites. Binding of GntR to the operators is eliminated by gluconate and also by 6-phosphogluconate at a 10-fold-higher concentration. Interestingly, when gntR deletion strains are grown in the presence of gluconate, there is a twofold decrease in gntT expression which is independent of catabolite repression and binding of GntR to the operator sites. This novel response of gntR mutants to the inducer is termed ultrarepression. Transcription of gntT is activated by binding of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex to a CRP binding site positioned at -71 upstream of the gntT transcription start site.

Molecular genetic characterization of the Escherichia coli gntT gene of GntI, the main system for gluconate metabolism.

The Escherichia coli gntT gene was subcloned from the Kohara library, and its expression was characterized. The cloned gntT gene genetically complemented mutant E. coli strains with defects in gluconate transport and directed the formation of a high-affinity gluconate transporter with a measured apparent Km of 6 microM for gluconate. Primer extension analysis indicated two transcriptional start sites for gntT, which are separated by 66 bp and which give rise to what appears on a Northern blot to be a single, gluconate-inducible, 1.42-kb gntT transcript. Thus, it was concluded that gntT is monocistronic and is regulated by two promoters. Both of the promoters have - 10 and -35 sequence elements typical of sigma70 promoters and catabolite gene activator protein binding sites in appropriate locations to exert glucose catabolite repression. In addition, two putative gnt operator sites were identified in the gntT regulatory region. A search revealed the presence of nearly identical palindromic sequences in the regulatory regions of all known gluconate-inducible genes, and these seven putative gnt operators were used to derive a consensus gnt operator sequence. A gntT::lacZ operon fusion was constructed and used to examine gntT expression. The results indicated that gntT is maximally induced by 500 microM gluconate, modestly induced by very low levels of gluconate (4 microM), and partially catabolite repressed by glucose. The results also showed a pronounced peak of gntT expression very early in the logarithmic phase, a pattern of expression similar to that of the Fis protein. Thus, it is concluded that GntT is important for growth on low concentrations of gluconate, for entry into the logarithmic phase, and for cometabolism of gluconate and glucose.

Characterization of a novel transporter family that includes multiple Escherichia coli gluconate transporters and their homologues.

The nucleotide sequences of seven Escherichia coli genes that encode members of the gluconate permease (GntP) family have recently become available. These genes include gntP, gntU, gntW, ORf449, dsdX, and ORFo454. The deduced amino acid sequences of all seven E. coli genes are homologous to the gntP gene products from Bacillus subtilis and B. licheniformis as well as two additional gene products from Haemophilus influenzae. These 11 proteins are not demonstrably homologous to members of the major facilitator superfamily or other recognized permease families. Four of the E. coli gluconate transporter genes have been cloned and shown to encode gluconate transporters with apparent affinities ranging from 6 to 212 microM. These studies serve to characterize a novel family of bacterial permeases.

Kinetics of Sugar Transport and Phosphorylation Influence Glucose and Fructose Cometabolism by Zymomonas mobilis.

The competitive inhibition of fructokinase by glucose has been proposed as the mechanism by which Zymomonas mobilis preferentially consumes glucose from mixtures of glucose and fructose and accumulates fructose when growing on sucrose. In this study, incorporation of radioactive fructose into biomass was used as a measure of fructose catabolism. It was determined that the rate of fructose incorporation by Z. mobilis CP4 was somewhat lower in the presence of an equimolar concentration of glucose but that the inhibition of fructokinase by glucose was not nearly as severe in vivo as was predicted from in vitro studies. Interestingly, addition of glucose to a culture of Z. mobilis CP4-M2, a glucokinaseless mutant, resulted in an immediate and nearly complete inhibition of fructose incorporation. Furthermore, addition of nonmetabolizeable glucose analogs had a similar effect on fructose catabolism by the wild-type Z. mobilis CP4, and fructose uptake by Z. mobilis CP4-M2 was shown to be severely inhibited by equimolar amounts of glucose. These results suggest that competition for fructose transport plays an important role in preferential catabolism of glucose from sugar mixtures. Indeed, the apparent K(infm) values for sugar uptake by Z. mobilis CP4 were approximately 200 mM for fructose and 13 mM for glucose. Other experiments supported the conclusion that a single facilitated diffusion transport system, encoded by the glf gene, is solely responsible for the uptake of both glucose and fructose. The results are discussed with regard to the hypothesis that the kinetics of sugar transport and phosphorylation allow the preferential consumption of glucose and accumulation of fructose, making the fructose available for the enzyme glucose-fructose oxidoreductase, which forms sorbitol, an important osmoprotectant for Z. mobilis when growing in the presence of high sugar concentrations.

The gluEMP operon from Zymomonas mobilis encodes a high-affinity glutamate carrier with similarity to binding-protein-dependent transport systems.

The nucleotide sequence downstream of the grp gene, encoding the glutamate uptake regulatory protein of Zymomonas mobilis, was determined. Three clustered genes (gluE, gluM, and gluP) close to ghe grp gene, but on the opposite strand, were identified. These genes encode a high-affinity transport system for glutamate and aspartate. The gluP gene product is a polypeptide of 25.4 kDa and contains segments with significant similarity to the ATP-binding proteins of binding-protein-dependent transport systems. The GluM polypeptide (22.9 kDa) is highly hydrophobic and consists of four potential membrane-spanning domains. The hydrophilic gluE gene product, with a molecular mass of 22.1 kDa, contains a region with sequence similarity to some of the periplasmic binding proteins and a sequence motif of a signal peptide for periplasmic localization. The transport system could not be functionally expressed in Z. mobilis. However, when heterologously expressed in Escherichia coli, it catalyzed uptake of glutamate, which was characterized kinetically. Our results suggest that the glutamate transport system encoded by the gluEMP operon is repressed in Z. mobilis by the regulatory protein Grp.

The glutamate uptake regulatory protein (Grp) of Zymomonas mobilis and its relation to the global regulator Lrp of Escherichia coli.

After being expressed in Escherichia coli JC5412, which is defective in glutamate transport, a Zymomonas mobilis gene which enabled this strain to grow on glutamate was cloned. This gene encodes a protein with 33% amino acid identity to the leucine-responsive regulatory protein (Lrp) of E. coli. Although overall glutamate uptake in E. coli was increased, the protein encoded by the cloned fragment repressed the secondary H+/glutamate transport system GltP by interaction with the promoter region of the gltP gene. It also repressed the secondary, H(+)-coupled glutamate uptake system of Z. mobilis, indicating that at least one role of this protein in Z. mobilis is to regulate glutamate transport. Consequently, it was designated Grp (for glutamate uptake regulatory protein). When expressed in E. coli, Grp repressed the secondary H+/glutamate transport system GltP by binding to the regulatory regions of the gltP gene. An lrp mutation in E. coli was complemented in trans with respect to the positive expression regulation of ilvIH (coding for acetohydroxy acid synthase III) by a plasmid which carries the grp gene. The expression of grp is autoregulated, and in Z. mobilis, it depends on growth conditions. The putative presence of a homolog of Grp in E. coli is discussed.

Structure of the gluABCD cluster encoding the glutamate uptake system of Corynebacterium glutamicum.

To assess the mechanism and function of the glutamate uptake system of gram-positive Corynebacterium glutamicum, a mutant deficient in glutamate uptake was isolated and was then used to isolate a DNA fragment restoring this deficiency. In a low-copy-number vector, this fragment resulted in an increased glutamate uptake rate of 4.9 nmol/min/mg (wild type, 1.5 nmol/min/mg). In addition, carbon source-dependent regulation of the glutamate uptake system was determined with the fragment, showing that the entire structures required for expression and control reside on the fragment isolated. Sequencing of 3,977 bp revealed the presence of a four-gene cluster (gluABCD) with deduced polypeptide sequences characteristic of a nucleotide-binding protein (GluA), a periplasmic binding protein (GluB), and integral membrane proteins (GluC and GluD), identifying the glutamate transporter as a binding protein-dependent system (ABC transporter). This identification was confirmed by the kinetic characteristics obtained for cells grown in the presence of globomycin, which exhibited an increased Km of 1,400 microM (without globomycin, the Km was 1.5 microM) but a nearly unaltered maximum velocity. By applying gene-directed mutagenesis, a strain with the entire cluster deleted was constructed. With this mutant, the glutamate uptake rate was reduced from 1.4 to less than 0.1 nmol/min/mg, which is proof that this system is the only relevant one for glutamate uptake. With this strain, the glutamate excretion rate was unaffected (18 nmol/min/mg), showing that no component of gluABCD is involved in export but rather that a specific machinery functions for the latter purpose.