13C nuclear magnetic resonance studies on selectively labeled bacterial biofilms

Bacterial biofilms of Pseudomonas aeruginosa selectively labeled by introduction of 2-¹³C-glycerol was studied by solid-state and high-resolution nuclear magnetic resonance. The ¹³C nuclei were mainly integrated into mannuronate and guluronate, the two monomer units forming the bacterial alginate. The signal for the C5 position of the mannuronate, which was easily identified and well separated from other peaks, was analyzed for molecular mobility. The result indicated a high degree of motional freedom within the molecular network of the alginate. Despite the fact that the alginate was part of a solid aqueous gel phase, the reorientation mechanism of the monomer units came close to isotropic tumbling. Solid-state spectra of biofilms labeled in the described manner may serve as a valuable tool for noninvasive analyses of molecular mobility of the alginate component under various influences, thereby revealing important structural information. In addition, the effect of a monovalent electrolyte (LiCl) on the molecular mobility of alginate fragments in an aqueous solution was studied by determining the spin–lattice relaxation times, line widths and line shapes under variations of the ion concentration. The presence of ions accelerated overall motions but left rapid local motions virtually unaffected. Journal of Industrial Microbiology & Biotechnology (2001) 26, 62–69. Received 26 January 2000/ Accepted in revised form 30 August 2000     

13C nuclear magnetic resonance studies of egg phosphatidylcholine.

Spin lattice relaxation times (T1) and apparent spin-spin relaxation times (T2) derived from linewidth have been used to investigate model membranes composed of egg yolk phosphatidylcholine. T1 measurements appear to be largely dominated by segmental motion and as a consequence are not very sensitive to small changes in membrane structure. On the contrary, apparent T2 times are shown to be sensitive to such changes in the membrane and are thus suggested as a useful tool for further investigation of membrane structure.     

13C nuclear magnetic resonance studies of the biosynthesis by Microbacterium ammoniaphilum of L-glutamate selectively enriched with carbon-13

13C NMR of isotopically enriched metabolites has been used to study the metabolism of Microbacterium ammoniaphilum, a bacterium which excretes large quantities of L-glutamic acid into the medium. Biosynthesis from 90% [1-13C]glucose results in relatively high specificity of the label, with [2,4-13C2]glutamate as the major product. The predominant biosynthetic pathway for synthesis of glutamate from glucose was determined to be the Embden Meyerhof glycolytic pathway followed by P-enolpyruvate carboxylase and the first third of the Krebs cycle. Different metabolic pathways are associated with different correlations in the enrichment of the carbons, reflected in the spectrum as different 13C-13C scalar multiplet intensities. Hence, intensity and 13C-13C multiplet analysis allows quantitation of the pathways involved. Although blockage of the Krebs cycle at the alpha-ketoglutarate dehydrogenase step is the basis for the accumulation of glutamate, significant Krebs cycle activity was found in glucose grown cells, and extensive Krebs cycle activity in cells metabolizing [1-13C]acetate. In addition to the observation of the expected metabolites, the disaccharide alpha, alpha-trehalose and alpha, beta-glucosylamine were identified from the 13C NMR spectra.     

Natural abundance 13C nuclear magnetic resonance studies of live cestodes

1. Natural abundance carbon-13 nmr spectra of several intact cestodes have been obtained. 2. All spectra show peaks assignable to triglycerides and the N(CH3)3 carbons of the choline moiety. 3. The olefinic region of the 13C nmr spectra indicated that the cestode larvae Mesocestoides corti and Echinococcus multilocularis have a larger concentration of polyunsaturated fatty acids than Hymenolepis adults. 4. Mobile fragments of glycogen were detected in all species studied, but its apparent concentration in individual cestodes was highly variable.     

Carbon-13 nuclear magnetic resonance studies of cobalamins

The carbon-13 nuclear magnetic resonance spectra of aquocobalamin, adenosylcobalamin, methylcobalamin, and (carboxymethyl)cobalamin have been interpreted. The assignments were made by a comparison of the spectra with that of cyanocobalamin, by a study of the pH dependence of the chemical shifts, by an analysis of the effect of the axial ligands on the carbon atoms of the corrin ring, and by a study of the specific line broadening effect of the paramagnetic ions Mn2+ and Gd3+. The chemical shift changes that accompany the "base-on"----"base-off" conversion of the organocobalamins demonstrate that the conformation of the "western" half of the corrin ring and the conformations of the a, b, c, d, f, and g side chains are relatively constant. In contrast, the conformations of the "eastern" half of the corrin ring and the e propionamide side chain are highly variable.     

13C nuclear magnetic resonance studies of anaerobic glycolysis inTrypanosoma brucei spp.

Anaerobic glycolysis inTrypanosoma brucei spp. has been studied by¹³C NMR at 50 and 75.5 MHz. The uptake of [U-¹³C]glucose by cell suspensions ofT. b. brucei was monitored by time-course spectroscopy, and while no anomeric specificity was found, the end -products of glycolysis were confirmed as glycerol and pyruvate together with alanine and dihydroxypropionat e. The intermediacy of L-glycerol-3-phosphate was also ascertained. The incorporation of C-I of [1-¹³C]glucose and of C-6 of [6-¹³C]glucose into glycerol and pyruvate inT. b. gambiense was quantified by measurement of the longitudinal relaxation times of the species involved. An incorporation to the extent of 66% of each substrate into equimolar amounts of glycerol and pyruvate indicate that Keq for the triosephosphate-isomerase-mediated reaction approaches unity.     

Control of porphyrin biosynthesis in Rhodopseudomonas spheroides and Propionibacterium shermanii. A direct 13C nuclear-magnetic-resonance spectroscopy study.

The facultative anaerobes Rhodopseudomonas spheroides and Propionibacterium shermanii were grown under anaerobic and aerobic conditions. The effect of light was studied with the photosynthetic R. spheroides, and the adaptation of both species to dark anaerobic life was monitored by direct observation of 5-amino[5-13C]laevulinic acid metabolism by using 13C nuclear-magnetic-resonance spectroscopy.     

Temperature-dependent 13C nuclear magnetic resonance studies of human serum low density lipoproteins.

The natural abundance 13C nuclear magnetic resonance (NMR) spectrum of human serum low density lipoproteins (LDL) shows significant temperature-dependent changes. These temperature-dependent spectra have been used to monitor changes in the organization of cholesterol esters within the LDL particle. Comparison with 13C NMR spectra of both cholesterol linoleate and an aqueous codispersion of cholesterol linoleate and egg phosphatidylcholine suggests that at low temperatures (10 degrees C), the cholesterol esters in LDL are organized in a smectic-like, liquid-crystalline arrangement. At temperatures above the order-disorder transition exhibited by the cholesterol esters of LDL, the cholesterol esters appear to be partially melted but still are motionally restricted compared with liquid cholesterol esters.     

13C Fourier transform nuclear magnetic resonance studies of fractionated Candida utilis membranes.

13C Fourier transform nuclear magnetic resonance has been used to study the lipid structure and dynamics of fractionated Candida utilis cell membranes. Measurements of the spin-lattice relaxation times indicate the existence of mobility gradients in the direction of increased mobility from the glycerol backbone toward the terminal methyl group of the fatty acid and toward the choline methyls. The temperature dependence of the relaxation times gives activation energies of approximately 4-6 kcal/mol for the rotations about various carbon-carbon bonds which determine the relaxation rates. In general, comparison with data which have been reported for artificial membrane systems indicates that the contributions of protein-lipid interactions to the T1 gradient are of negligible importance in the yeast membrane system. A dynamical model for the motion about bonds near unsaturated bonds which determined the relaxation of the unsaturated carbons is also proposed. Measurements of chemical shifts with temperature also exhibit a correlation with chain position. On the basis of these data a correlation of deltaE, the energy difference between gauche and anti conformations for gamma carboms, with chain position is inferred. In addition, an estimate of 1.2 kcal/mol can be obtained for deltaE for carbons near the end of the fatty acid chain. This value indicates that intermolecular interactions contribute substantially to deltaE since a value of approximately 0.5 kcal/mol can be ascribed to intramolecular interactions.     

13C nuclear magnetic resonance and circular dichroism studies of the tuftsin conformation in water

13C-NMR and circular dichroic (CD) spectra of tuftsin and its analogues are discussed in connection with our hypothesis that the beta-turn is the biologically active conformation of tuftsin. The changes in CD spectra evoked by an increase in pH are interpreted as a demonstration of the increasing amount of beta-turn conformers in solution. Configurational changes in successive residues of tuftsin showed that residues 2 and 3 of the peptide chain are important for the tuftsin conformation.     

Propionate precursors in the biosynthesis of daunomycin and adriamycin: A 13C nuclear magnetic resonance study

A ¹³C-NMR study of the biosynthesis of daunomycin adriamycin from propionate[1-¹³C] has been carried out in cultures of Streptomyces peucetius var. caesius. Results give direct support for the postulate that a propionate ‘starter’ is involved in the biosynthesis of both metabolites.