Autolysis of Bacillus subtilis induced by monovalent cations

Summary Resting cell suspensions of seven Nocardia species catalyzed the production of 10-hydroxystearic acid from oleic acid. Nocardia cholesterolicum NRRL 5767 gave a good yield with optimum conditions at pH 6.5 and 40°C. Yields exceeding 90% can be obtained within 6 h with 0.1 g cells (dry weight) and 178 mg oleic acid in 10 ml of 0.05 M sodium phosphate buffer (pH 6.5). In addition, minor amounts of 10-ketostearic acid were formed as a by-product. The reaction proceeded via hydration of the double bond as shown by labeling experiments with deuterium oxide and ¹⁸O-labeled water. The system was specific for fatty acids with cis unsaturation at the 9 position.A part of this paper was presented at a poster session at the World Conference on Biotechnology for the Fats and Oils Industry, Hamburg, Federal Republic of Germany, September 1987, and at the 194th National American Chemical Society Meeting, New Orleans, September 1987RetiredThe mention of firm names or trade products does not imply that they are endorsed or recommended by USDA over other firms or similar products not mentioned     

Glyphosate sensitivity of 5-enol-pyruvylshikimate-3-phosphate synthase from Bacillus subtilis depends upon state of activation induced by monovalent cations

The 5-enol-pyruvylshikimate-3-phosphate (EPSP) synthase from Bacillus subtilis was activated by monovalent cations, catalytic activity being negligible in the absence of monovalent cations. The order of cation effectiveness (NH4+ greater than K+ greater than Rb+ greater than Na+ = Cs+ = Li+) indicated that the extent of activation was directly related to the unhydrated cation radius. Ammonium salts, at physiological concentrations, were dramatically more effective than other cations. Activation by ammonium was instantaneous, was not influenced by the counter ion, and gave a hyperbolic saturation curve. Hill plots did not show detectable cooperativity in the binding of ammonium. Double-reciprocal plots indicated that ammonium increases the maximal velocity and decreases the apparent Michaelis constants of EPSP synthase with respect to both phosphoenol pyruvate (PEP) and shikimate 3-phosphate (S3P). A direct relationship between sensitivity to inhibition by glyphosate and the activation state of EPSP synthase was demonstrated. Hill plots indicated a single value for glyphosate binding throughout the range of ammonium activation. Double-reciprocal plots of substrate saturation data obtained with ammonium-activated enzyme in the presence of glyphosate showed glyphosate to behave as a competitive inhibitor with respect to PEP and as a mixed-type inhibitor relative to S3P. The increased glyphosate sensitivity of ammonium-activated EPSP synthase is attributed to a lowering of the inhibitor constant of glyphosate with respect to PEP. Erroneous underestimates of sensitivities of some bacterial EPSP synthases to inhibition by glyphosate may result from failure to recognize cation requirements of EPSP synthases.     

Effects of monovalent cations on red cell shape and size

Human erythrocytes were incubated in isotonic solutions of different monovalent cations. The apparent size of the red cells measured on scanning electron microscopic pictures decreases in the order Li+ greater than Na+ = K+ greater than Rb+. These differences in size are abolished after pretreatment with trypsin, which removes a large part of the charges associated with membrane glycoproteins. Shape alterations are also observed. Normal biconcave shapes are visible after Na+ or K+ incubation, whereas Li+ leads to flabby, flattened cells with a certain tendency to crenation, and Rb+ causes more pronounced biconcavity with a certain tendency to cupping. The overall effects of pretreatment with trypsin are similar to those of Li+. Our results provide evidence that the electrostatic repulsion of glycoproteins and other charged membrane components may play an essential role in maintaining red cell shape.     

Effects of monovalent cations on red cell shape and size

Human erythrocytes were incubated in isotonic solutions of different monovalent cations. The apparent size of the red cells measured on scanning electron microscopic pictures decreases in the order Li⁺>Na⁺=K⁺>Rb⁺. These differences in size are abolished after pretreatment with trypsin, which removes a large part of the charges associated with membrane glycoproteins. Shape alterations are also observed. Normal biconcave shapes are visible after Na⁺ or K⁺ incubation, whereas Li⁺ leads to flabby, flattened cells with a certain tendency to crenation, and Rb⁺ causes more pronounced biconcavity with a certain tendency to cupping. The overall effects of pretreatment with trypsin are similar to those of Li⁺. Our results provide evidence that the electrostatic repulsion of glycoproteins and other charged membrane components may play an essential role in maintaining red cell shape.     

Effect of monovalent cations on glutamate metabolism in rat brain]

Glutamate oxidation in vitro via deamination and transamination during gramicidin C-induced transport of K+ and Na+ in rat nervous tissue mitochondria was studied. An increase in ammonium production, i.e. in glutamate oxidation due to deamination, was shown to occur with maximal increase of oxygen consumption in the presence of cations. It was found that 1.5 mM Na+ activate oxygen consumption by 15% and accelerate ammonium production from glutamate (by 17%). No changes in aspartate production were observed. 15 mM K+ increase oxygen consumption by 29% and ammonium production by 11% during a decrease in aspartate production as compared to glutamate oxidation in the presence of a lower (10 mM) concentration of K+ in the samples.     

Influence of glutamic acid on the endogenous respiration of Bacillus subtilis

Clifton, C. E. (Stanford University, Stanford, Calif.), and John Cherry. Influence of glutamic acid on the endogenous respiration of Bacillus subtilis. J. Bacteriol. 91:546-550. 1966.-Amino acids serve as the major initial endogenous substrate for Bacillus subtilis. The endogenous activity of freshly harvested washed cells is high and falls off rapidly with time of shaking at 30 C to lower but still significant levels. The rate of O(2) consumption after the addition of glutamic acid also decreases as the cells age, but more slowly than noted for endogenous respiration. When cells were fed glutamate as soon as possible after harvesting, an apparent stimulation of endogenous respiration was noted. However, endogenous activity was inhibited if the cell suspensions were shaken for at least 1 hr before addition of the glutamate. Similar results were obtained with glycerol or glucose as exogenous substrates. Variation in rates of respiration with age of the cells, inherent instability of B. subtilis, and possible utilization of substances initially excreted by the cells appear to account for the variations noted regarding the influence of an exogenous substrate on endogenous respiration.     

Regulation of Na, K-ATPase activity by monovalent cations]

A deviation from optimal conditions of the Na, K-ATPase reaction results in a drastic change in the plot: enzyme activity versus Na/K ratio. Acidification of the medium and a decrease in Mg2+ concentration and temperature results in two peaks on the curve at Na/K ratio of about 1 and at Na/K ratio greater than 4. The enhancement of pH of the medium and increase in Mg2+ concentration decreases the first peak and increases the second one. A comparison of these curves for hydrolysis of ATP, UTP and p-nitrophenylphosphate and temperature dependence of the hydrolysis of the substrates suggest that the anomalies observed may be accounted for the Na+ effect on the K-sites or K+ effect on the Na-sites under conditions when cation-binding sites are heterogeneous.     

Regulators of aerobic and anaerobic respiration in Bacillus subtilis.

Two Bacillus subtilis genes, designated resD and resE, encode proteins that are similar to those of two-component signal transduction systems and play a regulatory role in respiration. The overlapping resD-resE genes are transcribed during vegetative growth from a very weak promoter directly upstream of resD. They are also part of a larger operon that includes three upstream genes, resABC (formerly orfX14, -15, and -16), the expression of which is strongly induced postexponentially. ResD is required for the expression of the following genes: resA, ctaA (required for heme A synthesis), and the petCBD operon (encoding subunits of the cytochrome bf complex). The resABC genes are essential genes which encode products with similarity to cytochrome c biogenesis proteins. resD null mutations are more deleterious to the cell than those of resE. resD mutant phenotypes, directly related to respiratory function, include streptomycin resistance, lack of production of aa3 or caa3 terminal oxidases, acid accumulation when grown with glucose as a carbon source, and loss of ability to grow anaerobically on a medium containing nitrate. A resD mutation also affected sporulation, carbon source utilization, and Pho regulon regulation. The data presented here support an activation role for ResD, and to a lesser extent ResE, in global regulation of aerobic and anaerobic respiration i B.subtilis.     

Monovalent cations enable cell wall turnover of the turnover-deficient lyt-15 mutant of Bacillus subtilis.

A lyt-15 mutant reported to be unable to turn over the cell wall exhibited the same rate of wall turnover as the standard strain if the medium contained 0.2 M NaCl, which did not affect growth. Cell wall autolysis was also optimal at 0.2 M NaCl.     

Transfer of monovalent cations across the isolated human amniotic membrane]

When the conductance and the bi-ionic potential are measured in vitro, monovalent cations are transferred according to a simple diffusion mechanism across the amnion (from the inside to the outside of the amniotic cavity or in the reverse direction) in channels with neutral or negative fixed sites. For equal concentration, the permeability to K+ is superior to that of Na+.     

Serine protease of Bacillus subtilis R]

Properties of a protease preparation obtained by ethanol precipitation of a concentrate of the culture fluid of Bacillus subtilis R (1 : 4 v/v; 4 degrees C) grown under the conditions of deep cultivation were studied. The use of specific inhibitors, EDTA and phenylmethylsulfonyl fluoride, made it possible to show that the enzyme belongs to the group of serine proteases. The preparation exhibited high stability in alkaline medium and thermostability; it hydrolyzed protein substrates and retained catalytic properties in the presence of a multicomponent detergent system. The preparation is recommended for use in those branches of industry where proteolysis is required and in the production of detergents (as a biological additive).     

Effects of lipophilic cations on motility and other physiological properties of Bacillus subtilis.

Lipophilic cations (tetraphenylarsonium, tetraphenylphosphonium, and triphenylmethylphosphonium) caused a number of major changes in the physiology of Bacillus subtilis. Macromolecular synthesis was inhibited, adenosine 5'-triphosphate concentration increased, swimming speed was reduced, tumbling was suppressed, and the capacity to take up the cations was greatly enhanced; respiration was not significantly altered. The effects occurred at lipophilic cation concentrations in the range commonly employed for measurement of membrane potential. Neither the enhancement of cation uptake nor the motility inhibition was a consequence of alteration of membrane potential, since both effects were still seen in the presence of valinomycin, with the extent of 86Rb+ uptake indicating a constant potential. Because suppression of tumbling accompanied speed reduction, as has also been found when protonmotive force is reduced, it is likely that lipophilic cations are perturbing the process of conversion of proton energy into work, rather than simply causing structural damage.     

A Bacillus subtilis locus encoding several gene products affecting transport of cations

A 3.6-kb DNA fragment from Bacillus subtilis was found to complement the K⁺ uptake-deficient Escherichia coli strain TK2420. Transformation with a pKLO61 plasmid harboring this fragment conferred the capacity to grow on a minimal medium containing only 10 mM K⁺. Insertional mutagenesis and subcloning identified a single gene responsible for the complementation. This gene coded for an apparent homolog of E. coli TrkA. Sequence analysis of the cloned region also revealed three additional open reading frames. These included: a gene encoding a homolog to the czcD gene product of Alcaligenes eutrophus, a lysR-type regulatory gene which was found to enhance Na⁺ resistance in E. coli NM81 (ΔnhaA) in a separate complementation test, and an orfD with no significant similarity to sequences deposited in Genbank.     

Growth of the Bacillus subtilis cell surface

A double mutant strain, Bacillus subtilis div IV B1 dal trp, has been constructed which grows as filamentous cells and minicells and requires d-alanine for growth. Removal of d-alanine from a growing population of cells resulted in cell bulging 25% of the cell length from one cell pole, followed by cell lysis. Little ultrastructural change in the cell envelope could be detected during bulge formation.     

枯草芽孢杆菌噬菌体载体的构建

以φ0105DI：It为原始株构建的重组噬菌体φ105S35和φ10 5S36具有自主侵染能力和溶源化特征。其基因组内插入的lkb片段上的cat,基因赋予二者所在宿主以氯霉素抗性,在两株噬菌体中插入位点相同,即原φ105DI ：It的smal酶切片段D、E之间,但插入片段在二者中的定向相反。与cat基因同时引入的单一BamHI和Xbal位点提供了外源DNA的插入位置。重组噬菌体DNA可高效转染枯草芽孢杆菌原生质体。因此φ105S35和币φ105S36可作为枯草芽孢杆随系统的载体而被利用。