Optimization of a medium and the role of its various components for the biosynthesis of Mycobacterium cyaneum exopolysaccharide

The effect of some components of the medium on the growth of Mycobacterium cyaneum B-646 and on the biosynthesis of an exoglycan by the culture was studied. A medium in which the M. cyaneum M variant produced up to 2.2 g of the exopolysaccharide per litre, which was nearly 4 times more than in the original medium, was proposed. The new medium differed from the original one in an elevated content of carbon, iron and potassium (11.2, 5.0 and 1.4 times, respectively) and in a lower phosphorus content (6.7 times). The exopolysaccharide produced by the culture in this medium contained glucose, galactose, fucose and uronic acid. Therefore, its monomeric composition did not depend on the medium used for growing the culture which produced the exopolysaccharide.     

Mechanism of penetration and of action of local anesthetics in Escherichia coli cells

Escherichia coli cells were used to study the mechanism of penetration of local anesthetics and the relationship between permeation and functional properties. We show that both the neutral and the protonated form of dibucaine can be accumulated in the cells. Accumulation of the protonated form occurs in response to a transmembrane electrical potential (negative inside) and results in high trapped concentrations (70 mM). Accumulation can lead to an alkalinization of the internal pH. Low concentrations of dibucaine stimulate the respiration, increase the transmembrane electrical potential and raise the accumulation of solutes. Inhibition of these functions occurs at higher concentrations of the drug. Furthermore, the drug concentration required to inhibit these functions is smaller at alkaline external pH than at acidic external pH, suggesting that the inhibition is mainly due to the neutral form of the anesthetics. Other hydrophobic amines also stimulate and inhibit different membrane functions, their efficiency being correlated to their lipophilicity.     

Studies on the mechanism of action of local anesthetics on proton translocating ATPase from Mycobacterium phlei

We have measured the inhibitory potencies of local anesthetics (procaine, lidocaine, tetracaine and dibucaine) on ATP-mediated H+-translocation, Ca2+-transport and ATPase activity in membrane vesicles from Mycobacterium phlei. Procaine and lidocaine up to 1 mM concentration did not inhibit ATP-dependent H+-translocation, Ca2+-transport and ATPase activity. However, tetracaine and dibucaine at 0.2 mM concentration caused dissipation of the proton gradient, measured by the reversal of the quenching of fluorescence of quinacrine, and inhibition of active Ca2+-transport. Tetracaine (1 mM) inhibited membrane-bound ATPase activity without affecting solubilized F1-ATPase activity. Studies show that these local anesthetics do not prevent the inactivation of F0-F1 ATPase by dicyclohexylcarbodiimide (DCCD). Binding of [14C]DCCD to F0-proteolipid component remained unchanged in the presence of tetracaine indicating that DCCD and tetracaine do not share common binding sites on the F0-proteolipid sector. The inhibition of H+-translocation and membrane-bound ATPase activity by tetracaine was substantially additive in the presence of vanadate.     

Effect of culture conditions on Mycobacterium cyaneum growth and the formation of extracellular polysaccharide

The source of carbon and nitrogen as well as pH of medium influence the possibility of synthesis of the exocellular heteropolysaccharide of Mycobacterium cyaneum and the quantitative harvest of glucan but not its monomer composition. The conditions of mycobacterial growth and glucan synthesis usually do not coincide.     

Mechanism of action of nalidixic acid on Escherichia coli. IV. Effects on the stability of cellular constituents

Cook, Thomas M. (Sterling-Winthrop Research Institute, Rensselaer, N.Y.), William H. Deitz, and William A. Goss. Mechanism of action of nalidixic acid on Escherichia coli. IV. Effects on the stability of cellular constituents. J. Bacteriol. 91:774-779. 1996.-Treatment of Escherichia coli 15TAU with nalidixic acid resulted in degradation of the nucleic acids of the cells, whereas protein was unaffected. Deoxyribonucleic acid (DNA) degradation appeared to be more extensive than ribonucleic acid degradation during periods of comparable bactericidal action. The onset of DNA degradation was evident prior to a measurable bactericidal effect. However, within the range of 2 to 20%, DNA degradation was accompanied by a decrease in viable cell numbers. Degradation of DNA to acid-soluble material occurred only under conditions permitting the bactericidal action of nalidixic acid. Arrest of the bactericidal action of nalidixic acid by the addition of dinitrophenol or chloramphenicol also inhibited DNA degradation. The acid-soluble products, which were excreted into the medium, have not been characterized completely, but probably were not phosphorylated.     

Mechanism of the reaction catalyzed by isoaspartyl dipeptidase from Escherichia coli

Isoaspartyl dipeptidase (IAD) is a member of the amidohydrolase superfamily and catalyzes the hydrolytic cleavage of beta-aspartyl dipeptides. Structural studies of the wild-type enzyme have demonstrated that the active site consists of a binuclear metal center positioned at the C-terminal end of a (beta/alpha)(8)-barrel domain. Steady-state kinetic parameters for the hydrolysis of beta-aspartyl dipeptides were obtained at pH 8.1. The pH-rate profiles for the hydrolysis of beta-Asp-Leu were obtained for the Zn/Zn-, Co/Co-, Ni/Ni-, and Cd/Cd-substituted forms of IAD. Bell-shaped profiles were observed for k(cat) and k(cat)/K(m) as a function of pH for all four metal-substituted forms. The pK(a) of the group that must be unprotonated for catalytic activity varied according to the specific metal ion bound in the active site, whereas the pK(a) of the group that must be protonated for catalytic activity was relatively independent of the specific metal ion present. The identity of the group that must be unprotonated for catalytic activity was consistent with the hydroxide that bridges the two divalent cations of the binuclear metal center. The identity of the group that must be protonated for activity was consistent with the free alpha-amino group of the dipeptide substrate. Kinetic constants were obtained for the mutant enzymes at conserved residues Glu77, Tyr137, Arg169, Arg233, Asp285, and Ser289. The catalytic properties of the wild-type and mutant enzymes, coupled with the X-ray crystal structure of the D285N mutant complexed with beta-Asp-His, are consistent with a chemical reaction mechanism for the hydrolysis of dipeptides that is initiated by the polarization of the amide bond via complexation to the beta-metal ion of the binuclear metal center. Nucleophilic attack by the bridging hydroxide is facilitated by abstraction of its proton by the side chain carboxylate of Asp285. Collapse of the tetrahedral intermediate and cleavage of the carbon-nitrogen bond occur with donation of a proton from the protonated form of Asp285.     

Mechanism of the physiological reaction catalyzed by tryptophan synthase from Escherichia coli

The physiological synthesis of L-tryptophan from indoleglycerol phosphate and L-serine catalyzed by the alpha 2 beta 2 bienzyme complex of tryptophan synthase requires spatial and dynamic cooperation between the two distant alpha and beta active sites. The carbanion of the adduct of L-tryptophan to pyridoxal phosphate accumulated during the steady state of the catalyzed reaction. Moreover, it was formed transiently and without a lag in single turnovers, and glyceraldehyde 3-phosphate was released only after formation of the carbanion. These and further data prove first that the affinity for indoleglycerol phosphate and its cleavage to indole in the alpha subunit are enhanced substantially by aminoacrylate bound to the beta subunit. This indirect activation explains why the turnover number of the physiological reaction is larger than that of the indoleglycerol phosphate cleavage reaction. Second, reprotonation of nascent tryptophan carbanion is rate limiting for overall tryptophan synthesis. Third, most of the indole generated in the active site of the alpha subunit is transferred directly to the active site of the beta subunit and only insignificant amounts pass through the solvent. Comparison of the single turnover rate constants with the known elementary rate constants of the partial reactions catalyzed by the alpha and beta active sites suggests that the cleavage reaction rather than the transfer of indole or its condensation with aminoacrylate is rate limiting for the formation of nascent tryptophan.     

Mechanism of D-cycloserine action: alanine racemase from Escherichia coli W

The antibiotic d-cycloserine is an effective inhibitor of alanine racemase. The lack of inhibition by l-cycloserine of alanine racemase from Staphylococcus aureus led Roze and Strominger to formulate the cycloserine hypothesis. This hypothesis states that d-cycloserine has the conformation required of the substrates on the enzyme surface and that l-cycloserine cannot have this conformation. Alanine racemase from Escherichia coli W has been examined to establish whether these observations are a general feature of all alanine racemases. The enzyme (molecular weight = 95,000) has Michaelis-Menten constants of 4.6 x 10(-4)m and 9.7 x 10(-4)m for d- and l-alanine, respectively. The ratio of V(max) in the d- to l-direction is 2.3. The equilibrium constant calculated from the Haldane relationship is 1.11 +/- 0.15. Both d- and l-cycloserine are competitive inhibitors with constants (K(i)) of 6.5 x 10(-4)m and 2.1 x 10(-3)m, respectively. The ratio of K(m)d-alanine to K(i)d-cycloserine is 0.71, and the ratio of K(m)l-alanine to K(i)l-cycloserine is 0.46. Since l-cycloserine is an effective inhibitor, it is concluded that the cycloserine hypothesis does not apply to the enzyme from E. coli W.     

The cellular immune response to Mycobacterium tuberculosis infection in the guinea pig

Pulmonary tuberculosis in guinea pigs is an extremely useful model for drug and vaccine testing due to the fact that its pathological disease process is similar to that present in humans. Progress in this field has been hindered because the tools necessary to undertake a complete immunological analysis of the guinea pig cellular immune response against Mycobacterium tuberculosis have been lacking. In this study, we combined a new flow cytometric gating strategy with immunohistochemistry to track T cells, B cells, and the MIL4 Ab, which detects both guinea pig heterophils (neutrophils) and eosinophils, to provide the first documentation of the kinetics of influx and positioning of these cell populations. The results show that the responding T cells are mostly CD4 cells and that after day 30 of the infection numbers of these cells in the lungs drops dramatically. These appear to be replaced by a steady increase in B cells and granulocytes which was associated with worsening lung pathology. These data reveal new information about the cellular phenotypes which mediate protective immunity or host immunopathogenesis during M. tuberculosis infection in this key animal model.     

Mechanism for the pyridoxal neutralization of isoniazid action of Mycobacterium tuberculosis

In Sauton's synthetic liquid medium, 10 mug of pyridoxal per ml completely protected Mycobacterium tuberculosis (H37R(a)) from the effects of a minimal inhibitory concentration of isoniazid (0.01 mug/ml). (14)C-labeled isoniazid was employed to study the nature of this protective effect. Uptake of the drug by cells in a Sauton environment containing 0.01 mug of (14)C-isoniazid per ml was inhibited 20 to 40% by 10 mug of pyridoxal per ml during the early hours of drug exposure. A stronger inhibition of uptake resulted when labeled isoniazid and pyridoxal were increased to 0.1 mug/ml and 50 to 100 mug/ml, respectively. Further studies revealed that certain Sauton nutrients are required to achieve this effect. When l-asparagine or salts (MgSO(4) and ferric ammonium citrate) or both were deleted from the menstruum, pyridoxal did not inhibit isoniazid incorporation by the tubercle bacilli. Pyridoxal also failed to inhibit uptake when (NH(4))(2)SO(4) was substituted for l-asparagine. Growth experiments in Sauton's medium modified to contain (NH(4))(2)SO(4) instead of l-asparagine were consistent with the latter finding. Pyridoxal did not prevent isoniazid growth inhibition in this medium. It is postulated that a large excess of pyridoxal in Sauton's medium protects tubercle bacilli from the effects of isoniazid through formation of an extracellular complex involving drug, vitamin, and certain medium constituents, thereby reducing the level of isoniazid available to the cells.     

Mechanism of the radioprotecting action of chemical compounds on Escherichia coli cells.

Summary The effect of radioprotection of indolylakylamines (5-methoxytryptamine) and aminothiols (cysteamine) on E. coli cells is practically absent if the cells have genetic defects in the repair systems. This means that the explanation of radioprotection by scavenging of free radicals is invalid and that specific repair mechanisms may be involved. In order to explain the radioprotective mechanism it was suggested that the radioprotectors interact with the damaged sites in DNA so that they become partly screened from repairing endonucleases. Under these conditions the reduction of incision rate results in diminished enzymatic induction of lethal double-strand breaks in DNA, this being important only in wild type cells.To prove this hypothesis an experimental procedure was developed using bacterial cells carryng plasmids (ColE1). This procedure enabled to determine the in vivo rate of enzymatic incision of -sites. It was found that the protectors did not change the total amount of -damages in DNA but reduced the rate of enzymatic incision.