Mutation of a gene encoding a putative ribokinase leads to reduced salt tolerance under potassium limitation in Bacillus subtilis.

The Bacillus subtilis L-42 mutant strain, which displays limited growth and inability to cope with hyperosmotic shock in a defined medium with a K+ concentration of < 1 mmol/L, was isolated by non-specific transposon insertional mutagenesis followed by an enrichment selection in media with K+ concentration < 0.5 mmol/L. The growth rate (as the main physiological characteristic) was determined to test the viability of the isolated mutant in media with various concentrations of K+, different values of osmolarity and pH. The mutant revealed a significant decrease in growth rate when cultivated in media with K+ concentration < 1 mmol/L and at hyperosmolarity. Localization of the insertional mutation was provided, based on genetic characteristics of the used transposon. Only 1 insertion of recombinant transposon was found in the mutant chromosome, localized into the yxkO gene (a putative ribokinase with unknown biological function).

Osmoregulation in Bacillus subtilis under potassium limitation: a new inducible K+-stimulated, VO4(3-)-inhibited ATPase.

Bacillus subtilis exhibited an inducible K+-transporting ATPase activity with apparent Km and maximum velocity Vmax of 12.9 microM and 25.1 micromol x min(-1) x (g cell protein)(-1), respectively, when cultivated on a synthetic medium containing less than 400 microM K+. Due to this enzyme, the growth rate of the bacterium in synthetic medium was not changed down to 115 microM K+, and the bacterium was able to grow down to 20 microM K+. The limiting K+ concentration was higher in media with osmolarity increased by NaCl or sucrose. The ATPase was inhibited by micromolar concentrations of vanadate (Ki = 1.6 microM). The ATPase activity was not stimulated by any other monovalent cation. The subunit of this ATPase, with an Mr of 52000, covalently bound the gamma phosphate group of ATP. This phosphorylated intermediate was unstable in neutral and basic pH as well as in the presence of potassium and was stable in acid pH. The enzyme did not show immunological cross-reactivity with antibody against Kdp ATPase of Escherichia coli.

Improved method for rapid purification of protein kinase from streptomycetes.

Protein kinase from Streptomyces lincolnensis was purified nearly to homogeneity using a high performance liquid chromatography (HPLC) and a Pharmacia FPLC system. The procedure used employed column chromatography on DE-53, followed by FPLC affinity chromatography with serine- or threonine-Sepharose (prepared as described in this paper) and gel filtration using a Superose 12 or TSK G3000SW column. Starting with 3.5 g of mycelial proteins, approximately 1 mg of pure enzyme was obtained. The procedure is simple and highly reproducible. The protein kinase thus obtained was nearly pure by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein kinase phosphorylated substrate proteins at the seryl residues.

In vitro measurement of translation accuracy of ribosomes isolated from streptomycin-resistant mutant of Streptomyces granaticolor.

Accuracy of activity of ribosome isolated from UV-light-induced streptomycin-resistant R-21 mutant of Streptomyces granaticolor was measured in an E. coli-derived system translating poly(U) with a high rate and accuracy. Ribosomes from the R-21 mutant strain were shown to be resistant to streptomycin and about two-fold more accurate than those from the wild type. The mutant strain was found to be resistant to 1000 mg/L streptomycin (Stm) during vegetative growth while it sporulated on agar plates containing only up to 200 mg/L of Stm. The growth rate of the R-21 mutant in complex liquid medium was indistinguishable from that of the wild-type strain.

Interdependence of K+ and glutamate accumulation during osmotic adaptation of Escherichia coli.

Escherichia coli responds to an increase in medium osmolarity by accumulating K+ and glutamate. At low osmolarity a large fraction of cytoplasmic K+ serves to balance charge on macromolecular anions. That fraction of K+ is here referred to as "bound," as distinguished from "free" K+ that serves to balance charge of small anions. At higher osmolarity where cytoplasmic K+ increases markedly, the bound fraction decreases but the absolute amount of bound K+ expressed per unit of dry weight increases. The increase in bound K+ can be explained largely by the reduction of cytoplasmic putrescine at high osmolarity. At high osmolarity, glutamate is the major cytoplasmic anion, equal to at least 70% of free cytoplasmic K+. A sudden increase in the osmolarity of the medium stimulates glutamate synthesis with a lag of only about a minute; glutamate synthesis is almost totally dependent on K+ uptake. The high rate of flow of nitrogen through the glutamate pool under control conditions of growth at low osmolarity indicates that glutamate accumulation immediately after shift to high osmolarity must be due to inhibition of utilization of glutamate in the synthesis of other nitrogen-containing compounds rather than stimulation of glutamate synthesis. In agreement with this reasoning we find the kinetics of glutamate accumulation to be independent of the specific path of synthesis, whether by glutamate dehydrogenase or by glutamate synthase. Synthesis of glutamate appears to be required to attain normal values of the electrical membrane potential after shift to high osmolarity.

Protein phosphorylation in Streptomyces albus.

The phosphorylated proteins of Streptomyces albus, radioactively labeled with [32P]orthophosphate have been analyzed by gel electrophoresis and autoradiography. More than 10 protein species were found to be phosphorylated. With [32P]ATP as substrate cell free extracts phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo. From cell extract which exhibited active phosphorylated in vitro, a protein kinase has been partially purified. The kinase activity was identified in fractions corresponding to a 90 kDa protein.

The control of adenylate cyclase activity in Escherichia coli.

Cell-free extract of E. coli possessed an inhibited adenylate cyclase activity after a previous anaerobic incubation of cells with glucose which is transported and metabolized. The degree of the inhibition depends on incubation conditions. Glucose analogues that are only transported but not metabolized, are not inhibitory. To restore the adenylate cyclase activity, the cells have to be cultivated aerobically prior to disintegration for a defined period of time without glucose.

Some properties of DNA-dependent RNA polymerase from Streptomyces granaticolor.

RNA nucleotidyltransferase from Streptomyces granaticolor was purified and some of its properties were investigated. The temperature optimum of the enzyme is 35 degrees C, pH optimum 7.8-8.4. Antibodies against the beta subunit of the enzyme from Bacillus subtilis immunologically cross-react with the beta subunit of the enzyme from S. granaticolor. Antibodies against the beta' subunit of the enzyme from B. subtilis immunologically cross-react with both beta and beta' subunits of the enzyme from S. granaticolor.

The effect of glucose on cellobiose uptake and beta-D-glucosidase activity in Streptomyces granaticolor.

Glucose inhibits the inducible synthesis of beta-D-glucosidase in Streptomyces granaticolor. Neither cAMP nor cGMP influence the inhibitory effect of glucose. Glucose also inhibits the inducible synthesis of the cellobiose uptake system but has no effect on its activity. This may be the mechanism underlying glucose inhibition of induction of beta-D-glucosidase in S. granaticolor.

Induction of beta-D-glucosidase in Streptomyces granaticolor.

beta-D-glucosidase in Streptomyces granaticolor is an inducible enzyme. Methyl-beta-D-glucoside or cellobiose, added to a glycerol-containing medium, are most suitable inducers. The activity of beta-D-glucosidase in a culture fully induced by cellobiose is 50 times higher than the basal level of the enzyme. beta-D-glucosidase is an intracellular enzyme, whose inducibility differ with culture age and reaches its maximum in a 10-h-old mycelium. The enzyme synthesis begins 2 h after the addition of the induced and reaches its maximum after a 10-h-induction.

Purification of DNA-dependent RNA polymerase from Streptomyces granaticolor.

RNA nucleotidyltransferase (EC 2.7.7.6) of Streptomyces granaticolor was purified by precipitation with polymin P and ammonium sulphate, affinity chromatography on DNA- cellulose and gell filtration on Biogel A 1.5 m. SDS-polyacrylamide gel electrophoresis revealed 8 protein bands of molar mass ranging from 37 to 130 kg/mol. Proteins of molar mass of 130 and 120 kg/mol were identified to be beta and beta subunits, respectively. The role of other subunits of the enzyme is discussed.

Catabolite repression of different inducible enzymes in Escherichia coli and the effect of cAMP.

Simultaneous induction of two enzymes sensitive to catabolite repression does not lead to an additive decrease of the specific activity of the two. Exogenously added cAMP increases the specific activity of catabolically repressed enzymes, irrespective of whether the enzyme is induced separately or simultaneously with another enzyme. In the presence of 12 different substrates metabolized by inducible enzymes glucose does not bring about catabolite repression. Synthesis of cAMP is identical with that occurring under conditions when glucose brings about catabolite repression.

Catabolite repression during single and multiple induction in Escherichia coli.

Intracellular concentration of cAMP regulates the synthesis of enzymes sensitive to catabolite repression. The relationship between the single and multiple induction of beta-galactosidase (EC 3.2.1.23), L-tryptophanase (EC 4.1.99.1), D-serine deaminase (EC 4.2.1.14), L-asparaginase (EC 3.5.1.1) and L-malate dehydrogenase (EC 1.1.1.37) was studied and the effect of cAMP level on the induction in Escherichia coli Crookes (ATCC 8739) was investigated. A varying degree of catabolite repression was observed during induction of individual enzymes induced separately on different energy sources. The synthesis of l-tryptophanase was most sensitive, whereas l-asparaginase was not influenced at all. Exogenous cAMP was found to overcome partially the catabolite repression of beta-galactosidase and D-serine deaminase, both during single induction. The synthesis of l-malate dehydrogenase was negatively influenced by the multiple induction even in the presence of cAMP; on the other hand, the synthesis of l-tryptophanase was stimulated, independently of the level of the exogenous cAMP. Similarly, the activity of L-asparaginase slightly but significantly increased during the multiple induction of all five enzymes; here too the activity increase did not depend on exogenous cAMP.

Characterization of adenylate cyclase from Escherichia coli.

Adenylate cyclase activity was detected and characterized in cell-free preparations of different strains of Escherichia coli; it was localized not only in the membrane fraction but also in the cytoplasm, the localization differing from strain to strain. The adenylate cyclase activity is highly dependent on the method used for disintegration of cells. The best results were obtained when using vortexing of the cell suspension with ballotini beads. The pH optimum of adenylate cyclase in cell-free preparations was found to be 9.0--9.5. The enzyme has an absolute requirement for Mg2+ and is inhibited by sodium fluoride and inorganic diphosphate. Release of adenylate cyclase from the membrane leads to an immediate loss of the activity; it was found that adenylate cyclase is quite labile and hence it could not yet been purified. The method used to determine adenylate cyclase activity and cyclic AMP is described.