A mutant Ebg enzyme that converts lactose into an inducer of the lac operon.

Lactose is not itself an inducer of the lac operon, nor is it converted to an inducer by ebg+ beta-galactosidase of Escherichia coli. We report here the isolation of a mutant Ebg beta-galactosidase which is capable of converting lactose into an inducer of the lac operon.     

Genetic fusions defining trp and lac operon regulatory elements

Two procedures for easily isolating deletions that fuse the trp and lac operons are described. Using these procedures, a large number of fusion deletions have been isolated. The lac ends of these deletions extend varying distances into the lacI gene and the lac promoter-operator region. Therefore, contrary to a previous report, there does not appear to be a messenger-termination signal at the C-terminal end of the lacI gene.The trp ends of fusion deletions do not have to extend into the trp structural genes to effect fusion, suggesting that mRNA synthesis initiated at the trp promoter proceeds some distance beyond the trp structural genes before a messenger termination signal is reached. Deletions that extend a short distance into the C-terminus of trpA, the last gene in the trp operon, do not completely abolish activity of the trpA product.The procedures described for isolating fusions of the trp and lac operons can be generalized to other systems.     

Expression of lupin nodulin genes during root nodule development

Seven lupin cDNA clones were used to study the expression of corresponding genes during nodule development by Northern blots analysis. They include six nodulin cDNAs: pLLb (lupin leghemoglobin), pLN 13, pLN 21–27, pLN 281, pLN 50, pLNGS (nodule form of glutamine synthetase GS_n and root form of GS: pGS. The appearance of nodulin mRNAs during lupin nodule development showed that the nodulin sequences analysed represent a group of plant genes involved in the nitrogen fixation process rather than formation of nodule. This is based on the observation that they are activated at the time when the nodule has already been formed, prior to the onset of nitrogenase activity. The products of Lb, nodulin 21–67, the nodulin coded by pLN13 and the nodulin 281 genes appeared between 11 and 13 days after infection, whereas the nodulion coded by pLN50 and the nodule form of GS appeared 18 days after inoculation. Twenty-one days post-infection a dramatic increase in the transciption rates of all nodulin genes is observed. This phenomenon may be related to the onset of nitrogenase activity. The possible mechanism of two-step activation of nodulin genes is discussed.     

Expression of host genes during root nodule development in soybeans

Summary Nine unique nodulin cDNA clones from soybean have been characterized with regard to the size of the RNA and the corresponding protein products. Based on the sequence homology between clones C51 and E27 and the multiple RNA species corresponding to clones D41 and E41, it is suggested that some of the nodulin genes represent members of small gene families. The amino acid sequence deduced from the nucleotide sequence of clones C51 and E27 revealed the presence of a signal peptide and no stop transfer signal, typical of membrane proteins, suggesting that the proteins encoded by these clones are localized in organelles and as such probably involved in ureide biosynthesis (Boland et al. 1982; Schubert and Boland 1984). Based on the timing of appearance of RNA corresponding to the nodulin clones and the pattern of their accumulation, at least three sets of nodulin genes are being represented here. Al1 the nodulin RNAs examined were made in Fix^- nodules formed by strain Ag168 (which does not make Cl component of nitrogenase) at a level comparable to that in Fix⁺ nodules and at a very reduced level in Fix^- nodules formed by strain HS124 (which show very few infected cells). It is concluded that all the nodulin genes examined here are induced independent of nitrogenase activity.     

Structure of the lac operon galactoside acetyltransferase

The galactoside acetyltransferase (thiogalactoside transacetylase) of Escherichia coli (GAT, LacA, EC 2.3.1.18) is a gene product of the classical lac operon. GAT may assist cellular detoxification by acetylating nonmetabolizable pyranosides, thereby preventing their reentry into the cell. The structure of GAT has been solved in binary complexes with acetyl-CoA or CoA and in ternary complexes with CoA and the nonphysiological acceptor substrates isopropyl beta-D-thiogalactoside (IPTG) or p-nitrophenyl beta-D-galactopyranoside (PNPbetaGal). A hydrophobic cleft that binds the thioisopropyl and p-nitrophenyl aglycones of IPTG and PNPbetaGal may discriminate against substrates with hydrophilic substituents at this position, such as lactose, or inducers of the lac operon. An extended loop projecting from the left-handed parallel beta helix domain contributes His115, which is in position to facilitate attack of the C6-hydroxyl group of the substrate on the thioester.     

Expression of nodule-specific glutamine synthetase genes during nodule development in soybeans

Summary A cDNA clone (pcPvNGS-01) to glutamine synthetase (GS) mRNA from root nodules of Phaseolus vulgaris showed cross-hybridization to GS and mRNA from soybean root nodules, thus allowing its use as a probe to study the expression of GS genes during root nodule development in soybeans. Hybrid-select translation of root and nodule RNA of soybean with DNA from pcPvNGS-01, followed by 2D gel electrophoresis, showed six peptides in the root and an additional four peptides in the nodule which represent nodule-specific glutamine synthetase (GS_n) gene products. The GS_n gene products appeared for the first time between day 11 and 12 after infection, either concomitant with the onset of nitrogenase activity or immediately following it. The levels of expression of the GS_n and leghemoglobin genes were not affected in young Fix^- nodules formed by Bradyrhizobium japonicum strains that are defective in nitrogenase activity, suggesting that the induction of these two sets of host genes take place independent of nitrogenase activity. However, in Fix^- nodules that are incapable of maintaining the peribacteroid membrane, GS_n gene products were not detected while 1ba, 1bc₂ and 1bc₃ appeared. In both the timing of appearance during root nodule development and the effect of different bacterial mutations on the expression, GS_n genes differ from most other nodulin genes examined (30), suggesting different regulatory mechanisms.     

A calorimetric investigation of the interaction of the lac repressor with inducer

A calorimetric study has been made of the interaction between the lac repressor and isopropyl-1-thio-beta-D-galactopyranoside (IPTG). The buffer-corrected enthalpy of reaction at 25 degrees C was found to be -15.6, -24.7, -4.6 kJ/mol of bound IPTG at pH 7.0, pH 8.1, and pH 9.0, respectively. This large range of enthalpy values is in contrast to a maximum difference in the free energy of the reaction of only 1.5 kJ/mol of bound IPTG between these pH values. The reaction was found by calorimetric measurements in different buffers to be accompanied by an uptake of 0.29 mol of protons/mol of bound IPTG at pH 8.1. The pH dependency of the reaction enthalpy suggests differences in the extent of protonation of the binding site and the involvement of H bonding with IPTG. The lack of strong hydrophobic contributions in the IPTG binding process is revealed by the absence of any determinable heat capacity change for the reaction at pH 7.0. The presence of phosphate buffer significantly alters the enthalpy of IPTG binding at higher pH values, but has little effect upon the binding constant. This implies that highly negative phosphate species change the nature of the IPTG binding site without any displacement of phosphate upon IPTG binding.