Mary F. Roberts

Growth inhibition of Clostridium thermocellum by carboxylic acids: A mechanism based on uncoupling by weak acids

SummaryThe inhibition of Clostridium thermocellum strains by acetate and other organic acids (propionate, butyrate) can be explained by a model based on the chemiosmotic theory and uncoupler action. It is proposed that the charged permeant species in the process of anion exclusion is the dimer HA-2. Evidence for this mechanisms is provided by 31P-NMR studies of whole cells and cell extracts.

Sensitivity of phospholipase C (Bacillus cereus) activity to phosphatidylcholine structural modifications

The structural features of a phosphatidylcholine molecule important for binding to phospholipase C (Bacillus cereus) have been examined using kinetic analyses of a series of short-chain phosphatidylcholines and analogues. Lipids examined had varying chain lengths, methyl branched chains, phenyl alkanoate chains, and a single fatty acyl chain (lysophosphatidylcholines). A comparison of Vmax and Km for monomolecularly dispersed dibutyroyl-, dihexanoyl- and diheptanoylphosphatidylcholine indicates that the length of the fatty acyl chains must be at least six carbons for efficient binding of the phosphatidylcholine to the enzyme. Enzymatic rates of hydrolysis for pure short-chain phosphatidylcholine micelles of different chain lengths or detergent mixed micelles with comparable concentrations of short- and long-chain phosphatidylcholines show no dependence on substrate chain length greater than six carbons. Methyl branching of short-chain phosphatidylcholines only inhibits phospholipase C activity when the methyl group is adjacent to the carbonyl (e.g., di(2-methyl)hexanoylphosphatidylcholine). In a similar fashion, phosphatidylcholines with phenylalkanoate chains become poor substrates when the phenyl group is near the acyl linkage. As the phenyl group is moved from C-4 to C-2 a large increase in the micellar apparent Km is observed. Chain specificity (sn-1 and/or sn-2 ester linkages) for binding is not absolute, since phospholipase C will hydrolyze micellar short-chain lysophosphatidylcholines at rates one tenth of phosphatidylcholines. In contrast, substitution of ester linkages with ether moieties yields phosphatidylcholine analogues which are even poorer substrates and not good inhibitors of phospholipase C. These results suggest that the carbonyl group and its immediate environment are important for phospholipid interacting with this water-soluble lipolytic enzyme.

Charged detergents enhance the activity of phospholipase C (Bacillus cereus) towards micellar short-chain phosphatidylcholine

Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) (Bacillus cereus) activity toward diheptanoylphosphatidylcholine is increased 50-100% by low concentrations of both positively and negatively charged detergents. Zwitterionic and nonionic detergents have no such activating effect. This charged detergent activation requires an interface, since comparable detergent concentrations have no effect on the hydrolysis rate of monomeric dihexanoylphosphatidylcholine. From NMR and diacylglycerol solubility studies it is suggested that activation results from detergent interacting with diacylglycerol to accelerate product release from the enzyme.

The cyclohexane moiety of rapamycin is derived from shikimic acid inStreptomyces hygroscopicus

SummaryAlthough the addition of shikimic acid to the medium had no effect on the level of production of rapamycin byStreptomyces hygroscopicus,14C-shikimic acid was incorporated into rapamycin to a very high degree.13C-Shikimic acid was successfully prepared from 1-[13C]-glucose using a mutant ofKlebsiella pneumoniae, and used to label rapamycin. It was found that13C-shikimic acid was incorporated into the cyclohexane moiety of rapamycin, thereby establishing the shikimic acid pathway origin of the seven-carbon starter unit.

The immediate precursor of the nitrogen-containing ring of rapamycin is free pipecolic acid

Abstract Studies were carried out to determine the precursor of the nitrogen-containing ring of rapamycin. The addition of l -lysine and d,l -pipecolate to the medium of Streptomyces hygroscopicus did not affect the specific production of rapamycin. In a basal medium containing no l -lysine except for that in the yeast extract component, l - 14 C-lysine and d,l - 3 H-pipecolate were incorporated to a high degree into rapamycin, d,l - 3 H-pipecolate being incorporated at a higher efficiency. Unlabeled pipecolate reduced the incorporation of radioactive lysine much more than unlabeled lysine decreased the incorporation of radioactive pipecolate. These results indicate that pipecolate is the more direct precursor of rapamycin, lysine being converted to pipecolate and free pipecolate being incorporated directly into rapamycin. Rapamycin samples enriched with 14 C-lysine and 3 H-pipecolate were hydrolyzed and analyzed to provide evidence of specific incorporation of these precursors into the pipecolate moiety .

Vanadium-containing tunicate blood cells are not highly acidic

The intracellular pH of intact blood cells of the tunicate Ascidia nigra was measured by transmembrane equilibration of [14C] methylamine. The pH of unfractionated blood cells is 7.39 +/- 1.10. The pH of vanadocytes, determined in a fractionation study, is 7.2. Previously used methods, in which pH values less than 3.0 are inferred from cell lysis or vital staining experiments, are shown to be unsuitable for intracellular pH determination due to the chemical composition of these vanadium-containing cells.

31P-NMR spectra of methanogens: 2,3-cyclopyrophosphoglycerate is detectable only in methanobacteria strains

The unique compound 2,3-cyclopyrophosphoglycerate occurs at a detectable concentration in the genera Methanobacterium and Methanobrevibacter but not in Methanococcus, Methanospirillum and Methanosarcina, as shown by a 31P-NMR survey of several different methanogens. Metabolic poisons (carbonyl cyanide m-chlorophenylhydrazone and valinomycin) do not decrease the level of the cyclic pyrophosphate in Methanobacterium thermoautotrophicum; therefore, it cannot be a phosphagen, i.e., an energy storage material. 13CO2 is rapidly incorporated into this cyclic compound which represents the major soluble carbon as well as the phosphorus component of this methanobacteria. 13C-NMR analysis demonstrates that the pKa of the 2,3-cyclopyrophosphoglycerate carboxyl group is 2.55. The unusual pseudomurein cell wall structure of methano- and methanobrevibacteria necessitates a high demand on carbohydrate metabolism. For this reason, and the fact that when its concentration is decreased no new phosphorus resonances appear in the high resolution spectra, it is suggested that 2,3-cyclopyrophosphoglycerate has a function in carbohydrate metabolism.

500 MHz 1H-NMR studies of bile salt-phosphatidylcholine mixed micelles and vesicles Evidence for differential motional restraint on bile salt and phosphatidylcholine resonances

Abstract 1 H nuclear magnetic resonance (NMR) spectra at 500 MHz have been obtained for taurocholate/egg phosphatidylcholine mixtures of varying composition. The excellent chemical shift dispersion permits identification of most resonances for each component. This high-resolution character of the NMR spectra is retained until the phosphatidylcholine (PC) mole fraction exceeds 60–70% (the exact limit depends on ionic strength). 1 H linewidths have been monitored as a function of solute composition in order to evaluate trends in local molecular mobility of each component as the distribution of aggregate particles is varied, and to examine the effects of added NaCl in altering micellar size and shape. Although prior light scattering studies (Mazer, N.A., Benedek, G.B. and Carey, M.C. (1980) Biochemistry 19, 601–615) and our own work indicate a 6-fold increase in particle hydrodynamic radius from pure taurocholate micelles to 1 : 1 taurocholate/PC mixtures containing 150 mM NaCl, both lipid components retain substantial motional freedom and exhibit narrow NMR signals in this compositional region. As the solubilization limit for PC is approached (approx. 2:1 PC:taurocholate), differential behavior is observed for the two components: the motion of taurocholate becomes preferentially restricted, while polar portions of the PC remain mobile until large multilayers predominate.

Physiological and 15N-NMR analysis of molecular nitrogen fixation by Methanococcus thermolithotrophicus, Methanobacterium bryantii and Methanospirillum hungatei

Two mesophilic methanogenic bacteria, Methanobacterium bryantii strain MOH and Methanospirillum hungatei strain GP1 were demonstrated, using several different experimental approaches, to fix dinitrogen. Evidence includes (1) growth with N2 as the sole nitrogen source; (2) incorporation of 15N2 into cellular material (both soluble amino acid pools and insoluble cell protein and other macromolecules) detected by 15N-NMR spectroscopy; (3) acetylene reduction to ethylene by the cells, and inhibition of this reaction by bromoethanesulfonic acid (BES), a methanogen inhibitor. High-resolution 15N-NMR analysis of ethanol extracts of these organisms and cross-polarization magic-angle sample spinning analysis of the solid debris from these extracts are compared to labeled material from Methanococcus thermolithotrophicus, a methanogen previously determined to fix dinitrogen.

31P-NMR studies of the oral pathogen Streptococcus mutans: observation of lipoteichoic acid

We have used 31P-nuclear magnetic resonance spectroscopy to identify phosphorus-containing compounds in whole cells of two serotype c strains of the oral pathogen Streptococcus mutans. The major resonance, centered at 0 ppm in whole cells, was attributed to lipoteichoic acid on the basis of its chemical shift, insensitivity to pH changes, cellular localization and a comparison with spectra obtained with purified lipoteichoic acid from S. mutans. The linewidths of resonances observed for intact cells and purified lipoteichoic acid were moderately narrowed by increasing the ionic strength, and substantially broadened in the presence of the lectin concanavalin A. Experiments with purified lipoteichoic acid suggest that this compound in whole cells is complexed with divalent cations such as Mg2+. Intracellular pools of other phosphorus-containing metabolites were found to be low when compared to the lipoteichoic acid concentration in both starved and glycolyzing cells.