Magnesium Starvation of Aerobacter aerogenes III. Protein Metabolism

The metabolism of the ribosomal and soluble protein components of Aerobacter aerogenes was examined during its incubation in a Mg(++)-deficient medium. Bacteria were exposed to leucine-H(3) during the exponential growth period preceding Mg(++) starvation, and extracts were prepared after intervals of starvation and were centrifuged through gradients of sucrose to separate ribosomal from soluble proteins. Ribosomal proteins synthesized during the preceding exponential growth were slowly lost from the ribosomes; after 8 hr of starvation, few, if any, sedimented with ribosomes. Losses of total protein, together with the known rate of ribosome decay during Mg(++) starvation, suggested that these ribosomal proteins are ultimately degraded to acid-soluble products and account for all protein lost by the starving cells. These conclusions were supported by studies of Mg(++) starvation in a uracil-requiring strain of A. aerogenes: during uracil starvation a smaller fraction of the proteins synthesized were ribosomal, and the fraction of protein which subsequently decayed during Mg(++) starvation was correspondingly less. During recovery from Mg(++) starvation, proteins, lost from disintegrated ribosomes, were not detectably reutilized into new particles even before their degradation to acid-soluble products was complete. Synthesis of soluble proteins continued for more than 24 hr of starvation at a rate per milliliter close to 45% of the instantaneous rate per milliliter of the exponentially growing bacteria at the time Mg(++) was removed. This value agreed with that found previously for synthetic rates of deoxyribonucleic acid, transfer ribonucleic acid, and ribosomal ribonucleic acid during starvation relative to rates during exponential growth.     

Magnesium Starvation of Aerobacter aerogenes II. Rates of Nucleic Acid Synthesis and Methods for Their Measurement

The rates of synthesis of Aerobacter aerogenes nucleic acids were estimated during incubation of the bacteria in a Mg(++)-free medium. Deoxyribonucleic acid (DNA) synthesized during Mg(++) starvation, or in the preceding exponential growth, remained acid-precipitable for 2.5 hr before breaking down to acid-soluble products during a period of many hours. Rates of DNA synthesis were calculated by correcting the net amounts of DNA per milliliter to values that would have appeared had there been no decay. After the first few hours, this rate was constant, the amount of DNA present at the start of Mg(++) starvation being synthesized every 130 min. Rates of synthesis of total ribonucleic acid (RNA) were established in two ways: (i) by measurements of the incorporation of exogeneous uracil and glucose carbon into RNA, and (ii) by the accumulation of transfer RNA (tRNA), since this component is stable during Mg(++) starvation. After the first few hours, this rate was constant, the amount of RNA present at the start of Mg(++) starvation being synthesized about every 120 min. Fractionation by gradient centrifugation revealed that at all times of starvation the ratio of newly synthesized tRNA-rRNA was the same as it was during exponential growth. Furthermore, newly synthesized ribosomal RNA (rRNA) became a part of polysomal structures. Thus, in the absence of Mg(++), DNA, tRNA, and rRNA were synthesized in the same relative proportions as during exponential growth, at rates close to one-half the instantaneous rates of synthesis in the bacteria growing exponentially at the start of starvation.     

Membrane Adenosine Triphosphatase of Escherichia coli: Activation by Calcium Ion and Inhibition by Monovalent Cations

Membrane ghost preparations of Escherichia coli K-12 obtained by osmotic lysis of lysozyme-induced spheroplasts were found to possess both Mg(++)- and Ca(++)-activated adenosine 5'-triphosphatase (ATPase, EC 3.6.1.3) activities. Maximal activities of 1.0 to 1.5 mumoles of orthophosphate released per min per mg of protein were obtained at pH 9.0 with a molar Mg(++) to adenosine 5'triphosphate (ATP) ratio of 2:5 and at pH 9.9 with a molar Ca(++) to ATP ratio of 1:5. These ATPase activities were not altered by ouabain, fluoride, N-ethylmaleimide, 2,4-dinitrophenol, cyanide, or dithionite, but were inhibited by low concentrations of azide, p-chloromercuribenzoate, and pentachlorophenol. Mg(++) ATPase was more susceptible to inhibition by azide than was Ca(++) ATPase. Fifty per cent inactivation of both activities was observed when membrane ghost preparations were preincubated at 66 C for 10 min. The Mg(++) and Ca(++) ATPase activities of these preparations were not additive, but did respond independently to inhibition by monovalent cations. Ca(++) ATPase was found to be very sensitive to inhibition by K(+), Na(+), Li(+), Rb(+), and Cs(+); Mg(++) ATPase was relatively insensitive to these ions. One possible interpretation of the results presented in this paper is that the membrane of E. coli possesses an ATPase which is activated by either Mg(++) or Ca(++) and that activation by Ca(++) increases the susceptibility of this enzyme to inhibition by monovalent cations. Increased susceptibility of E. coli membrane ATPase to inhibition by monovalent cations such as Na(+) and K(+) as a consequence of Ca(++) activation could represent a regulatory mechanism.     

Effect of low-nutrient seawater on morphology,chemical composition,and virulence of Salmonella typhimurium

The response of Salmonella typhimurium to low nutrient levels was determined by measuring the concentrations of lipids, carbohydrates, DNA, RNA, and proteins over a 32-day starvation period. Ultrastructural integrity was observed by transmission electron microscopy. Lipid and carbohydrate content of bacterial cells rapidly declined within the first 16 days, while DNA and proteins exhibited a more gradual decline over the 32 days of starvation. In contrast, RNA content did not decrease appreciably upon nutrient starvation. Structural damage occurred especially after 16 days of starvation. After 32 days of nutrient deprivation, we recorded degenerative cellular forms, a coccoidal cell shape, a decrease in cellular volume, and the loss of the three-layered outer membrane. The morphological and structural alterations correlated with virulence in infected animals. We observed a decrease in virulence of S. typhimurium after 9, 16, and 32 days of starvation, reaching a maximal decrease after 32 days of nutrient deprivation. The decrease in virulence correlated to surface hydrophobicity alterations, adherence to eukaryotic cells, and phagocytosis.Abbreviations BHI brain heart infusion - PBS phosphate-buffered saline - TE Tris-EDTA buffer - F _t phagocytic index - K _t Killing index     

Ribosomal complexes from an extremely halophilic bacterium and the role of cations

Concentrated extracts of Halobacterium cutirubrum were prepared at 0 C by gently disrupting cells with a nonionic detergent in a medium containing 3.0 m KCl, 0.5 m NH(4)Cl, and 0.04 m (or more) magnesium acetate and then treating the gelatinous mass with deoxyribonuclease. On KCl-sucrose gradients containing 0.5 m NH(4)Cl and 0.04 m magnesium acetate, these extracts showed 30S and 50S ribosomal subunits plus a flat profile of faster-sedimenting material up to high S values. Only after frozen storage or brief incubation of the extract were 70S ribosomes and distinct classes of small polyribosomes detected. Digestion with ribonuclease converted faster-sedimenting material to 70S particles. The presence of chloramphenicol during preparation of the extracts did not affect these results. The evidence suggests that ribosomal particles exist in these cells as subunits or as polyribosomes but not as 70S ribosomes. To investigate the function of Mg(++) and NH(4) (+) ions in ribosomal complexes from this halophile, concentrated cell extracts and extracts incubated with (14)C-leucine were examined on KCl-sucrose gradients containing different concentrations of these ions. Polyribosomes and the bulk of 70S ribosomes dissociated reversibly to subunits at about 0.01 m Mg(++), whereas a small fraction of the 70S particles, including those which in vitro incorporated (14)C-leucine into nascent protein, dissociated only below 1 mm Mg(++). Below this concentration of Mg(++), nascent protein remained attached to the 50S subunit even at 0.04 mm Mg(++) in the presence of 0.35 to 0.5 m NH(4)Cl. Nascent protein, presumably as peptidyl-transfer ribonucleic acid, dissociated reversibly from 50S subunits only at 0.04 mm Mg(++) and 0.1 m or less NH(4) (+). Thus, the stability of polyribosomes from H. cutirubrum depends specifically on both Mg(++) and NH(4) (+) ions.     

Regulation of betacyanin efflux from beet root by poly-L-lysine, ca-ion and other substances

Poly-l-lysine, poly-alpha, gamma-diaminobutyric acid and basic proteins cause efflux of betacyanin from beet root tissues to varying degrees. Membrane activities fall in the order: polylysine > poly-alpha, gamma-diaminobutyric acid > polyarginine (protamine), suggesting the importance of steric factors in side-chain to backbone relations. It was also observed that homopolymer activity > heteropolymer activity, using ribonuclease and lysozyme as examples of the latter. Among polylysines, there appears to be an optimal chain length at a molecular weight equal to 50,000. Lowered activity of larger polymers is interpreted in terms of a diffusion barrier, the cell wall.Polylysine and Ca(++) exhibit competitive kinetics, and Ca(++) otherwise is far more active than other cations. It is assumed that polylysine displaces Ca(++) from anionic centers on the membrane, but cannot confer equivalent dimensional stability, rendering the membrane leaky. The possible role of cationic shielding in ionic stabilization of the membrane was also considered. The order of divalent ion activity against polylysine was Ca(++) > Sr(++) > Mg(++), suggesting again a specific size-fit relationship.     

Effect of freezing on the transmembrane distribution of ions in freeze-tolerant larvae of the wood fly Xylophagus cinctus (Diptera, Xylophagidae)

The present study shows that freezing of freeze-tolerant larvae of the wood fly Xylophagus cinctus caused Na(+), K(+) and Mg(++) to move to electrochemical equilibrium across the cell membranes. Na(+) and Mg(++) moved from the haemolymph into the cells, while K(+) moved the opposite way. The original distribution of ions was restored after the larvae were thawed. The transmembrane fluxes of ions were of the same magnitude in the frozen and thawed larvae. The redistribution of ions in the frozen larvae did not give rise to any apparent change in the volume of cells and haemolymph upon thawing, i.e. the redistribution of solutes appeared to be osmotically neutral.     

Haematological and haematopoietic responses to starvation in an air-breathing fish Channapunctatus Bloch

Changes in haematological values (RBC numbers, haemoglobin content, haematocrit value, MCV, MCH, MCHC, TLC and DLC) based on weekly samples from a group of starved fish were investigated. After 8 weeks of starvation, the effects of restoration to a normal diet was evaluated. Parallel studies on haematopoietic tissues were also made. Changes in some biochemical values such as blood glucose, liver and muscle glycogen were also examined to correlate biochemical effects with those of haematological changes. Erythrocytes, thrombocytes and neutrophils were found to be most sensitive to starvation. The initial response to deprivation of food was an increase in RBCs and related values and in total leukocyte population. However, from week 5 onwards a sharp decline in these cell populations was noted. The leukocytes and thrombocytes showed a change parallel to RBC and the total leukocyte counts. However, neutrophils were observed to show a consistent increase throughout the starvation period. A blood glucose level below SOmglOOmh¹ appeared critical in relation to blood cell population. Haematopoietic studies revealed that reticulocytes and mesomyelocytes were unable to keep pace with the changing peripheral blood picture. Other stages in development responded to the changes in the peripheral blood.     

Structural analysis of the Leptospiral sphingomyelinases: in silico and experimental evaluation of Sph2 as an Mg-dependent sphingomyelinase

BACKGROUND: Leptospiral sphingomyelinases are candidate virulence factors present only in pathogenic Leptospira spp. Leptospira interrogans serovar Lai encodes Sph1, Sph2, Sph3, Sph4 and SphH. Except for Sph4, they all possess the exo-endo-phosphatase domain that groups them under the DNase I superfamily. METHODS, RESULTS AND CONCLUSIONS: Modeling of exo-endo-phosphatase domains reveals high-level structural similarity of Sph2 with the crystal structure of SmcL and BC SMase sphingomyelinases from Listeria ivanovii and Bacillus cereus, respectively. A β-hairpin loop, essential for host cell membrane interaction, is absent in leptospiral sphingomyelinases. Instead, several aromatic amino acids were oriented outward from the surface of these molecules and formed clusters of hydrophobic regions that possibly enables the anchoring of these molecules into the host cell membrane, as demonstrated in Sph2 and Sph3. Sph2 is unique and possesses the Mg(++)-binding Glu53 residue in the metal-binding site and two His residues (His151 and His286) in the catalytic site. We demonstrate experimentally the Mg(++)-dependent hemolysis of erythrocytes by rSph2 and its ability to cleave sphingomyelin to ceramide. Anti-Sph2 antibodies neutralized the hemolytic activity of Sph2. In conclusion, we provide evidence showing that Sph2 is a Mg(++)-dependent hemolysin with both sphingomyelinase and hemolytic activities.     

REVERSIBLE AND IRREVERSIBLE CHANGES IN THE FINE STRUCTURE OF NERVOUS TISSUE DURING OXYGEN AND GLUCOSE DEPRIVATION

Rabbit retinas were fixed for electron microscopy immediately after removing the eye and after incubations in a control medium and in three different deprivation media that were identical with the control except for the omission of glucose, oxygen, or both. A systematic comparison was made of the electron microscopic appearance of the different retinas with particular attention to four regions: rod inner segments, rod synapses, bipolar cell bodies, and ganglion cell myelinated axons. Retinas fixed after 1 hour of incubation in the control medium appeared virtually identical with those fixed immediately after ocular removal. Retinas deprived of oxygen and glucose for only 3 minutes showed generalized swelling of mitochondria and alterations in the structure of the synapses with loss of synaptic vesicles. Extending the combined deprivation caused further mitochondrial swelling and synaptic changes and also led to progressive swelling of the Golgi membranes and the granular endoplasmic reticulum. All these changes were almost completely reversible for up to 20 minutes but were irreversible by 30 minutes, at which time multiple discontinuities had appeared in cell and organelle membranes. Anoxia alone produced alterations similar to those found after somewhat shorter periods of the combined deprivation, whereas glucose withdrawal produced only minor changes. These electron microscopic results correlate quite well with previously reported electrophysiological measurements.     

Choline Kinase and Phosphorylcholine Phosphatase in Plants

Choline kinase was present in barley and wheat roots and leaves of barley, wheat, tobacco, spinach and squash plants. The kinase was purified 25-fold from spinach leaves. The enzyme had a broad pH optimum between 7.5 and 10.0. Mg(++) was required for activity and in the presence of Mg(++) the enzyme was relatively stable. Maximum enzyme activity was obtained when the Mg(++): ATP ratio was 1:1. The K(m) was 1 x 10(-4)m. The kinase from leaves was similar to that from rapeseed or from yeast, except that the leaf and seed enzymes were not inhibited by compounds which attach sulfhydryl groups.Only a very slow hydrolysis of phosphorylcholine by similar plant extracts was observed. This phosphatase activity was purified 200- or 300-fold and appeared to be caused by a nonspecific acid phosphatase.The activity of both the kinase and the phosphatase did not seem sufficient to account for the rapid equilibration of the large phosphorylcholine reservoir of plants with exogenous P(32)-labeled orthophosphate.     

Effects in vitro of mercury on rat brain Mg(++)-ATPase.

Mercuric chloride (Hg) in micromolar concentrations inhibited Mg(++)-dependent ATPase activity in rat brain microsomes. Inhibition was higher in oligomycin-sensitive (O.S.) than oligomycin-insensitive (O.I.) Mg(++)-ATPase. Hydrolysis of ATP with 15 and 50 micrograms of microsomal protein for 45 min without and with (2.10(-7M) Hg showed linear rates for 15-20 min. Altered pH vs activity demonstrated comparable inhibitions by Hg in buffered (neutral greater than acidic greater than basic) pH ranges. Inhibition of enzyme activity by Hg was found to be greater at 37 degrees C than at lower temperatures suggesting positive correlation trend. An uncompetitive inhibition with respect to the activation of Mg(++)-ATPase, O.S. Mg(++)-ATPase and O.I. Mg++ ATPase by ATP was indicated by a decrease in apparent Vmax and Km. Mg(++)-activation kinetic studies indicated that Hg causes uncompetitive inhibition of Mg(++)-ATPase and O.I. Mg(++)-ATPase and mixed inhibition of O.S. Mg(++)-ATPase. Inhibition was partially restored by repeated washings. These results indicate that the inhibition of microsomal Mg(++)-ATPase by Hg was pH, temperature, enzyme and Mg++ concentration dependent. Additionally, the data also suggest that O.S. compared to O.I. Mg(++)-ATPase is more sensitive to Hg toxicity.     

Mn、Ca阳离子在菠菜PSII颗粒氧化水中的作用

用Triton X-100处理菠菜叶绿体,获得一个基本不含PSⅠ成分、而具放氧活性的PSⅡ颗粒。最适pH移至6.9,超过pH7.2就发生凝集,在照光下只形成很小或不形成H~+梯度,只有微弱的毫秒延迟荧光发射,老化和解联剂都不加速电子传递。 Mn、Ca阳离子促进PSⅡ颗粒的放氧和H~+释放,两者作用不能叠加。Mn离子只作用于活化的PSⅡ颗粒,对叶绿体和部分失活的PSⅡ颗粒无效。Ca离子对叶绿体、PSⅡ颗粒或部分失活的PSⅡ颗粒,都有相同程度的促进效应。     

Separation and localization of cell wall layers of a gram-negative bacterium

The cell envelope of a marine pseudomonad as seen in thin section by electron microscopy has the double-membrane structure typical of other gram-negative bacteria. Cells washed with a solution containing Na(+), K(+), and Mg(++) at their concentrations in the growth medium, when suspended briefly in 0.5 m sucrose, lost 13% of their hexosamine in a form nonsedimentable by centrifugation at 73,000 x g. Since the resulting cells in thin section appeared unchanged, it was concluded that the material released was derived from a nonstaining, loosely bound outer layer. This same layer could be removed from the cells by washing with 0.5 m NaCl. A second nonsedimentable fraction was released after successive suspension of the cells in 0.5 m sucrose. Since this material was released only when the outer double-track structure had broken, it was concluded that it arose from a layer immediately underlying the latter layer. The three layers differed in their content of hexosamine and protein. None of the layers released contained muramic or diaminopimelic acid. The cell form remaining was rod shaped and appeared in thin section to be bounded only by its cytoplasmic membrane. This form contained all the muramic and diaminopimelic acid in the cell. Treatment with lysozyme released the muramic and diaminopimelic acid and converted the rod form to a protoplast, indicating that in the rod form (mureinoplast) a thin layer of peptidoglycan is located on the outside surface of the cytoplasmic membrane. Thus, five separate layers have been detected in the cell envelope of this marine pseudomonad.     

Synthesis of membrane and periplasmic proteins during starvation of a marine Vibrio sp

Changes in membrane and periplasmic protein profiles induced by starvation conditions in the marine Vibrio sp. S14 were examined by one-dimensional gel electrophoresis. Analysis by densitometry resolved at least six periplasmic proteins, nine outer membrane proteins, and four cytoplasmic membrane proteins induced at various times during 120 h of nutrient and energy starvation. Eight of these were also synthesized by heat- and/or ethanol-shocked cells. Pulse-labelling indicated that the starvation-induced proteins were not products of degradation, and that their synthesis was differently modulated during starvation. The most pronounced changes occurred during the initial hours of nutrient and energy deprivation. The correlation between the initial changes in protein composition and utilization of the intracellular energy reserve poly-beta-hydroxybutyrate is discussed. The rate of proteolysis during the initial hours of starvation was approximately 16 times greater than that during exponential growth.     

Alteration of cylindrospermopsin production in sulfate- or phosphate-starved cyanobacterium Aphanizomenon ovalisporum

The effect of sulfate and phosphate deprivation on cell growth and cylindrospermopsin level was studied in Aphanizomenon ovalisporum ILC-164. Sulfate starvation induced a characteristic reduction of cylindrospermopsin pool size on the basis of cell number and unit of dry mass of culture. Phosphorous starvation of A. ovalisporum cultures induced a lesser reduction of cylindrospermopsin pool size. This divergence in the pool size of cylindrospermopsin may be the consequence of different growth rate. To show the metabolic changes concomitant with reduction of cylindrospermopsin pool size were obtained by measurement of ATP sulfurylase and alkaline phosphatase activity. The present study is the first concerning the cylindrospermopsin content under sulfate starvation and discusses it in relation to phosphorous starvation.     

Changes in Protein Composition of Three Bacterial Isolates from Marine Waters during Short Periods of Energy and Nutrient Deprivation

Two-dimensional gel electrophoresis analysis of and total cell protein determination for three bacterial isolates from marine waters at the onset and after 24 h of energy and nutrient deprivation demonstrated that the three isolates exhibited different pathways of starvation survival. Two strains appeared to synthesize new proteins during starvation.     

Tetracycline inhibition of cell-free protein synthesis. I. Binding of tetracycline to components of the system

Day, L. E. (Chas. Pfizer & Co., Inc., Groton, Conn.). Tetracycline inhibition of cell-free protein synthesis. I. Binding of tetracycline to components of the system. J. Bacteriol. 91:1917-1923. 1966.-Tetracycline, an inhibitor of cell-free protein synthesis, effected the dissociation of Escherichia coli 100S ribosomes to 70S particles in vivo and in vitro, but was not observed to mediate the further degradation of these particles. The antibiotic was bound by both 50S (Svedberg) and 30S subunits of 70S ribosomes and also by E. coli soluble RNA (sRNA), polyuridylic acid (poly U), and polyadenylic acid (poly A). The binding to ribosomal subunits was higher at 5 x 10(-4)m Mg(++) than at 10(-2)m Mg(++). The binding to polynucleotide chains was highest when Mg(++) was not added to the reaction mixture.     

Freezing injury to erythrocytes. II. Morphological alterations of cell membranes.

Morphological alterations of human red blood cell membranes were examined with the cells containing different concentrations of glycerol being subjected to rapid rates of cooling, approximately 10⁴ and 10⁵ °C/min, and subsequent rewarming. Small membrane defects, similar to holes, were observed in specimens frozen with and without 10% glycerol. Various degrees of roughness were found on the surface of the cells at all freezing rates tested. The membrane alterations were reduced with increasing glycerol concentration, although roughness also appeared on the surface of the cells in 30% glycerol suspensions, frozen rapidly, and rewarmed to ?80 or ?60 °C. The cell membrane surface texture correlated with the growth of intra- and extracellular ice particles. There was also a positive correlation between these alterations and post-thaw hemolysis. It is concluded, therefore, that morphological alterations appearing on the erythrocyte membranes may be a manifestation of freezing damage.     

Factors Affecting Deoxycholate Inactivation and Mg++ Reactivation of Bacillus megaterium KM Membrane Nicotinamide Adenine Dinucleotide (Reduced Form) Oxidase

Decay of the reduced nicotinamide adenine dinucleotide oxidase of Bacillus megaterium KM membranes was prevented by storage in 10% (v/v) glycerol or 0.4% bovine serum albumin. Differential rates of solubilization of components of the oxidase system by 0.4% deoxycholate was demonstrable at 4 C. The amount of Mg(++) necessary for maximal oxidase reactivation increased with increasing amounts of deoxycholate-inactivated oxidase. Mg(++) activation of deoxycholate-inactivated oxidase was partially temperature-dependent. Maximal activation was observed at 37 C, but only partial activation took place at 4 C. A small amount of deoxycholate was required for Mg(++) activation of deoxycholate-inactivated oxidase. Two pH optima were found for Mg(++) activation of deoxycholate-inactivated oxidase, pH 5.3 and 7.3. Significant amounts of activation of the inactive oxidase occurred in the absence of Mg(++) with an optimum at pH 5.0, with essentially no Mg(++)-independent activation demonstrable at pH 7.0.