Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This communication describes the isolation and characterization of mutants of Rhizobium trifolii which can induce nitrogenase activity in defined liquid medium. Two procedures were used for the isolation of these mutants from R. trifolii strain DT-6: (1) following chemical mutagenesis, slow growing mutants were selected which were unable to utilize NH+4 as sole source of nitrogen; (2) as spontaneous mutants resistant to the glutamate analogue L-methionine-DL-sulfoximine. Mutants (DT-71, DT-125) isolated by these procedures induced nitrogenase activity in the free-living state, whereas the parent strain lacked this property. Induction of nitrogenase activity in these mutants occurred during the late exponential phase of growth when the rate of protein synthesis was decreasing. The addition of NH+4 to a medium containing glutamate as the nitrogen-source resulted in a 50--70% reduction (repression?) of nitrogenase activity; in contrast, the rate of protein synthesis or the rate of respiration was not influenced by exogenous NH+4. Biochemical analysis showed that these mutants (strains DT-71 and DT-125) have defects in both nitrogen and carbon metabolism. The levels of glutamate synthase (both NADP+ -and NAD+ -dependent activities) and glutamate dehydrogenase (NAD+-dependent activity) were markedly lower. In addition, the mutants were found to have no detectable ribitol dehydrogenase or beta-galactosidase activity. These findings are discussed in relation to a mechanism of regulation of symbiotic nitrogen fixation.
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PMID:Regulation of nitrogen fixation in Rhizobium spp. Isolation of mutants of Rhizobium trifolii which induce nitrogenase activity. 58 92

The global regulator Lrp (leucine-responsive regulatory protein), in some cases modulated by its co-regulator leucine, has been shown to regulate more than 40 genes and operons in Escherichia coli. Leucine modulates Lrp regulation of leucine-responsive operons. The level of sensitivity of these operons to leucine varies greatly, but the basis for this variation is only partially understood. One operon controlled by Lrp that is relatively insensitive to leucine is gltBDF, which includes genes specifying the large (GltB) and small (GltD) subunits of glutamate synthase. Earlier gel mobility shift assays have demonstrated that Lrp binds to a fragment of DNA containing the gltBDF promoter region. To further define the nature of this Lrp-gltBDF interaction, DNase I footprinting experiments were performed. The results indicate that Lrp binds cooperatively to three sites quite far upstream, spanning the region from -140 to -260 base-pairs relative to the start of transcription. Phased hypersensitivity is observed throughout the entire binding region, suggesting that Lrp bends the DNA. To determine the relative importance of these three sites for the transcriptional activation of gltBDF, a series of site-directed mutations was generated. The effects of these mutations on Lrp binding were determined both by DNase I footprinting and by quantitative mobility shift assays, while their effects on transcription in vivo were examined by measuring beta-galactosidase activity levels of chromosomal gltB::lacZ operon fusions. Our results indicate that all three sites are required for maximal gene expression, as is the proper phasing of the sites with one another and with the start of transcription. Our results suggest that Lrp binds a central palindromic site, interacting predominantly with the major groove of its DNA target, and that additional dimers bind to flanking sites to form a nucleoprotein activation complex.
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PMID:A nucleoprotein activation complex between the leucine-responsive regulatory protein and DNA upstream of the gltBDF operon in Escherichia coli. 923 18

The leucine-responsive regulatory protein (Lrp) binds to three sites centered 252, 216, and 152 bp upstream of the transcription start site of the Escherichia coli glutamate synthase operon (gltBDF) and activates transcription. Activators of sigma(70)-dependent promoters usually bind closer to the -35 hexamer of the core promoter sequence. To study the mechanism by which Lrp-dependent activation occurs over this relatively large distance, the gltBDF upstream region was sequentially replaced with corresponding portions from the well-characterized sigma(70)-dependent promoter lacZYAp. The glt-lac promoter hybrids were placed upstream of lacZ, allowing transcriptional activity to be monitored via beta-galactosidase assays. Even replacing all gltBDF sequences downstream of and including the -35 hexamer did not eliminate Lrp-dependent activation of transcription. When a 91-bp region between the -35 hexamer and the proximal Lrp binding site (-48 to -128) was replaced with heterologous DNA of the same length, transcription was reduced nearly 40-fold. Based on the presence of a consensus binding sequence, this region seemed likely to be a binding site for integration host factor (IHF). Experiments to study the effects of a himD mutant on expression of a gltB::lacZ transcriptional fusion, gel mobility shift analyses, and DNA footprinting assays were used to confirm the direct participation of IHF in gltBDF promoter regulation. Based on these results, we suggest that IHF plays a crucial architectural role, bringing the distant Lrp complex in close proximity to the promoter-bound RNA polymerase.
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PMID:Activation from a distance: roles of Lrp and integration host factor in transcriptional activation of gltBDF. 1139 54

The role of glutamate as osmoprotector was investigated through the study of a mutation in its biosynthetic pathway. A glt::Tn917-lacZ-cat insertion mutant (N1) conferring glutamate auxotrophy and enhanced beta-galactosidase expression on high-salt media was selected. Co-transformation experiments and PCR analysis allowed locating the insertion into the gltB gene corresponding to the small unit of the glutamate synthase (GOGAT). The N1 mutant strain presented a glutamate requirement for growth and a tenfold decrease in GOGAT activity. Transcriptional activity of GOGAT, measured as beta-galactosidase from the transposon fusion, correlated with enzymatic activity; expression was enhanced at the stationary phase and in high-ionic-strength media. However, osmotolerance of cultures of N1 mutant were as wild-type (wt), at least in semi-rich medium. In contrast, sporulation was slightly reduced (75% of wt), and spores were less resistant to UV, heat, and osmolarity, properties linked to the content of small, acid-soluble proteins (SASP). The content of these proteins was, in fact, reduced, in particular the SASP-gamma type. The peptidoglycan-cortex, however, was not impaired since spores maintained lysozyme resistance. Addition of glutamate during sporulation partially rescued spore resistance, but germination and outgrowth remained impaired. Deficiencies in germination and outgrowth were also observed with spores from a gltA mutant strain. Taken together, these results pointed to the importance of GOGAT activity during sporulation, in particular for the synthesis SASPs.
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PMID:Effect of glutamate synthase (GOGAT) activity on Bacillus subtilis spore properties. 1457 Feb 71

The arginine regulatory protein of Pseudomonas aeruginosa, ArgR, is essential for induction of operons that encode enzymes of the arginine succinyltransferase (AST) pathway, which is the primary route for arginine utilization by this organism under aerobic conditions. ArgR also induces the operon that encodes a catabolic NAD(+)-dependent glutamate dehydrogenase (GDH), which converts l-glutamate, the product of the AST pathway, in alpha-ketoglutarate. The studies reported here show that ArgR also participates in the regulation of other enzymes of glutamate metabolism. Exogenous arginine repressed the specific activities of glutamate synthase (GltBD) and anabolic NADP-dependent GDH (GdhA) in cell extracts of strain PAO1, and this repression was abolished in an argR mutant. The promoter regions of the gltBD operon, which encodes GltBD, and the gdhA gene, which encodes GdhA, were identified by primer extension experiments. Measurements of beta-galactosidase expression from gltB::lacZ and gdhA::lacZ translational fusions confirmed the role of ArgR in mediating arginine repression. Gel retardation assays demonstrated the binding of homogeneous ArgR to DNA fragments carrying the regulatory regions for the gltBD and gdhA genes. DNase I footprinting experiments showed that ArgR protects DNA sequences in the control regions for these genes that are homologous to the consensus sequence of the ArgR binding site. In silica analysis of genomic information for P. fluorescens, P. putida, and P. stutzeri suggests that the findings reported here regarding ArgR regulation of operons that encode enzymes of glutamate biosynthesis in P. aeruginosa likely apply to other pseudomonads.
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PMID:The arginine regulatory protein mediates repression by arginine of the operons encoding glutamate synthase and anabolic glutamate dehydrogenase in Pseudomonas aeruginosa. 1517 98