The synthesis of beta-galactosidase by constitutive and other regulatory mutants of Escherichia coli in chemostat culture.

The synthesis of beta-galactosidase by an E. coli constitutive mutant was examined in a chemostat using glucose-, glycerol-, succinate- or N-limited growth media. Except for glucose-grown bacteria, the steady-state intracellular level of beta-galactosidase was maximal at dilution rates between 0-2 and 0-3 h-1. At higher dilution rates enzyme synthesis was reduced by catabolite repression, which could be relieved by the addition of cyclic AMP. With a catabolite-resistant mutant (UV5c), no decrease in enzyme level at high dilution rates were observed. All mutants examined were constitutive and gave decreased enzyme levels at low dilution rates, with the exception of lac-/F'lac UV5c mutants where the enzyme levels rose at low dilution rates. Hyper-producing mutants were isolated but were unstable. A constitutive mutant growing on glycerol-limited media was considered the most suitable for large-scale production of beta-galactosidase in a chemostat.     

Inducible synthesis of beta-galactosidase in disrupted spheroplast of Escherichia coli

A membrane preparation obtained from osmotic lysate of spheroplasts of Escherichia coli cells showed an activity of synthesizing beta-galactosidase which was dependent upon oxidative phosphorylation. The synthesis was inhibited by the addition of actinomycin D or of chloramphenicol. The beta-galactosidase synthesized in the membrane preparation was completely released into the medium, while that synthesized in the spheroplasts and intact cells remained within the cells. The minimum concentration of the inducer, methyl-beta-d-thiogalactoside, required for the induction of beta-galactosidase was 5 x 10(-5)m for intact cells, 3 x 10(-4)m for spheroplasts and 1 x 10(-3)m for membrane preparation. Incorporation of labeled glucose into insoluble components in membrane preparation was extremely low compared with that in intact cells or in spheroplasts. Based on these and other observations, the nature of this membrane preparation is discussed in relation to the structure of E. coli cells.     

Interactions between mutations affecting ribosome synthesis in Escherichia coli

RNA synthesis was followed during amino acid starvation of strains of Escherichia coli that contained both the relaxed (relA) mutation and a mutation affecting ribosome assembly that results in oversynthesis of RNA. The ribosome mutation did not by itself lead to relaxedness. The relaxed mutation could be expressed in organisms that contained the ribosome mutation.     

Crystallization of beta-galactosidase from Escherichia coli.

Two crystal forms of beta-galactosidase have been obtained from Escherichia coli. One crystal form is hexagonal space group P6222 or enantiomorph, with cell dimensions a = b = 154 A, c = 750 A. The second form is monoclinic, space group P21, with cell dimensions a = 107.9 A, b = 207.5 A, c = 509.9 A, beta = 94.7 degrees. The monoclinic form seems better suited to detailed structural analysis. The crystals are radiation-sensitive, but by using synchrotron radiation in conjunction with a long (400 mm) crystal-to-film distance it was possible to resolve the individual reflections. On the basis of crystal density measurements, there are four tetramers each of molecular weight 465,000 per asymmetric unit. The Patterson function strongly suggests that two of the tetramers are related to the other two by translation. The data are consistent with the tetramers having 222 point symmetry, but this is not proven.     

A rapid and simple chemiluminescent assay for Escherichia coli beta-galactosidase.

We describe a chemiluminescent assay for E. coli beta-galactosidase using Lumi-Gal 530, a commercial formulation containing a stable phenylgalactose-substituted dioxetane as the substrate. Removal of the galactose moiety leads to the generation of an unstable dioxetane which decomposes to provide the observed chemiluminescence which is measured with a luminometer. Advantages of the assay are that it is simple, inexpensive and has 20-fold greater sensitivity than the standard spectrophotometric assay. Additional advantages are that the dioxetane is quite stable in the commercial formulation, and beta-galactosidase functions efficiently and is not degraded during the course of an assay. As luminometers are becoming commonplace in molecular biology laboratories, this assay provides a preferable alternative to the spectrophotometric assay.     

Hyper-recombination in Escherichia coli K-12 mutants constitutive for protein X synthesis.

Genetic recombination was studied in Escherichia coli F- strains in which synthesis of the recA gene product protein X is increased due to mutation in either recA (tif-1) or lexA (spr). When a single donor marker was selected, the recombination proficiency of these strains was not significantly altered in Hfr crosses. However, linkage of unselected, proximal Hfr markers was found to be much reduced among the progeny tested, and more of the progeny showed evidence of multiple exchanges between donor and recipient DNA. These effects were much more apparent when the recipient carried both tif-1 and spr mutations, but in this case recombination proficiency was reduced compared with those strains carrying either mutation alone, particularly in crosses with Hfr Cavalli. A lexA mutation was found to suppress the effect of tif-1 on the recombinant genotype.     

Reduced synthesis of beta-galactosidase in Escherichia coli infected with phage phi X 174.

The synthesis of beta-galactosidase (EC 3.2.1.23;beta-D-galactoside galactohydrolase) in E. coli was repressed as a result of infection with single-stranded DNA phage phi chi 174. Evidence is presented to show that this repression was not due to the restricted entry of the inducer molecules into the infected cells but to some phage-specified product(s). It was further shown that either the infected cells synthesized a fewer number of enzyme-specific mRNA or all such molecules were translated with a reduced efficiency; the half-lives of the mRNA's remained more or less unaffected.     

Metabolic pathway structures for recombinant protein synthesis in Escherichia coli

Escherichia coli is a valuable commercial host for the production of heterologous proteins. We used elementary mode analysis to identify all possible genetically independent pathways for the production of three specific recombinant proteins, green fluorescent protein, savinase and an artificial protein consisting of repeating units of a five-amino-acid cassette. Analysis of these pathways led to the identification of the most efficient pathways for the production of each of these proteins. The results indicate that the amino acid composition of expressed proteins has a profound effect on the number and identity of possible pathways for the production of these proteins. We show that several groups of elementary modes produce the same ratio of biomass and recombinant protein. The pattern of occurrence of these modes is dependent on the amino acid composition of the specific foreign protein produced. These pathways are formed as systemic combinations of other pathways that produce biomass or foreign protein alone after the elimination of fluxes in specific internal reversible reactions or the reversible carbon dioxide exchange reaction. Since these modes represent pathway options that enable the cell to produce biomass and protein without utilizing these reactions, removal of these reactions would constrain the cells to utilize these modes for producing biomass and foreign protein at constant ratios.     

Efflux of beta-galactosidase products from Escherichia coli.

Several different strains of Escherichia coli were grown on a variety of carbon sources under various growth conditions. Lactose was added (usually at mid-log phase), and the concentrations of the products of beta-galactosidase action on this sugar (galactose, glucose, and allolactose) were determined at various times thereafter in the total culture and in the medium. It was found that with each strain, with all carbon sources, and under all of the conditions studied, a very large proportion of the products were found in the medium. Control studies were carried out which showed that these results were not artifacts of the method of separating the cells from the medium. The results also did not arise from the secretion of beta-galactosidase into the medium, from the diffusion of substrates and products into and out of the cells due to leaks in the membrane, or from faults in the method of sugar analysis. In addition, the results showed that there were very high levels of products inside the cells under the conditions used and that the efflux of the products was rapid. The efflux might be energetically advantageous to the cell as well as being a means of storing excess products until needed.     

Regulation of the balanced synthesis of membrane phospholipids. Experimental test of models for regulation in Escherichia coli

In Escherichia coli, highly effective regulation controls the balanced synthesis of membrane phospholipids, important for optimal growth. Regulation is such that normally about 70% of a common pool of cytosine liponucleotide precursor is utilized by phosphatidylserine synthase and eventually converted to phosphatidylethanolamine, while about 30% is utilized by the competing enzyme phosphatidylglycerophosphate synthase and converted to phosphatidylglycerol (25%) plus cardiolipin (5%). Although the ratio of phosphatidylglycerol to cardiolipin may vary with conditions of growth, the sum of these two lipids remains relatively constant at about 30% of the total. Alternative models, postulating coordinate regulation of the two competing enzymes, or independent feedback regulation are proposed. These models were tested in experiments in which phosphatidylglycerol was continuously removed from growing cells treated with arbutin (4-hydroxyphenyl-O-beta-D-glucoside), causing its conversion to arbutinphosphoglycerol (Bohin, J.-P., and Kennedy, E.P. (1984) J. Biol. Chem. 259, 8388-8393.) The synthesis of phosphatidylglycerol was increased by a factor of 7 in cells treated with arbutin, with only small changes in phospholipid composition and with no significant change in the level of phosphatidylglycerophosphate synthase. The synthesis of phosphatidylethanolamine was not significantly increased, decisively eliminating the model that requires coordinate regulation of phosphatidylserine synthase and phosphatidylglycerophosphate synthase, and supporting the model of independent feedback inhibition, sensitive to very small changes in composition of cellular phospholipids.     

Evidence for an active dimer of Escherichia coli beta-galactosidase.

BETA-Galactosidase (EC 3.2.1.23), prepared from strains ML 308 and K12 3300 of Escherichia coli, dissociated into an inactive monomer in the presence of Ag+. When such a monomer preparation is treated with excess of thiol an enzymically active dimer is formed in addition to an active tetramer. It is suggested that Ag+ may be of value in studies on other multimeric proteins as a mild dissociating agent.     

Osmotic regulation of PhoE porin synthesis in Escherichia coli.

In Escherichia coli, adaptation to hyperosmotic conditions alters the expression of the outer membrane porins OmpF and OmpC. The amount of PhoE porin, which is normally induced by phosphate deprivation, was greatly reduced in cells adapted to high-osmolarity conditions. Osmoregulation of PhoE operated independently of the activity of the PhoR phosphate sensor and did not involve cross-talk from the homologous osmosensor EnvZ. PhoE synthesis was partially restored by additional copies of the positive regulator phoB+ and by the osmoprotectant glycine betaine.     

RNA-protein interactions in the Escherichia coli ribosome.

Over the last two decades essentially three different approaches have been used to study the topography of RNA-protein interactions in the ribosome. These are: (a) the analysis of binding sites for individual ribosomal proteins or groups of proteins on the RNA; (b) the determination of protein footprint sites on the RNA by the application of higher order structure analytical techniques; and (c) the localisation of RNA-protein cross-link sites on the RNA. This article compares and contrasts the types of data that the three different approaches provide, and gives a brief and highly simplified summary of the results that have been obtained for both the 16S and 23S ribosomal RNA from E coli.