Production and interconversion of 1,2-propanediol and hydroxyacetone by Escherichia coli.

The anaerobic metabolism of marginally lethal levels of [13C]formaldehyde by Escherichia coli (K12, MU352, CRB, and CR63) was followed in vivo by 13C NMR. The products include 1,2-propanediol. Under aeration, the 1,2-propanediol is converted to hydroxyacetone. The hydroxyacetone is reconverted to 1,2-propanediol when aeration is stopped. The process can be cycled by varying the rate of aeration.

Nuclear magnetic resonance spectroscopy reveals the metabolic origins of proline excreted by an Escherichia coli derivative during growth on [13C]acetate.

We have used 13C nuclear magnetic resonance to monitor acetate metabolism in a proline-overproducing strain of Escherichia coli growing on 13C-labeled acetate. The conversion of 13C-labeled acetate to proline by actively dividing cells was followed in vivo, and the site specificity of the incorporation of the acetate carbons in the proline was determined from spectra of butanol extracts of the growth media. The degree of incorporation of deuterium from partially deuterated water into various sites on the proline was monitored from the beta-deuterium-shifted signals in the 13C spectra. The spectra provide information on the origin of the carbons and the protons during proline biosynthesis.

Formaldehyde metabolism by Escherichia coli. Detection by in vivo 13C NMR spectroscopy of S-(hydroxymethyl)glutathione as a transient intracellular intermediate.

In vivo 13C NMR has been used to detect the transient formation of S-(hydroxymethyl)glutathione (GSCH2OH) from glutathione and [13C]formaldehyde in Escherichia coli. Two-dimensional 1H-13C shift correlation was used to locate the chemical shift of the formaldehyde-derived protons of the adduct. The adduct GSCH2OH is formed by chemical reaction in the first few minutes after cells are challenged with formaldehyde and remains within the cell until consumed by metabolism.

Formaldehyde metabolism by Escherichia coli. Carbon and solvent deuterium incorporation into glycerol, 1,2-propanediol, and 1,3-propanediol.

Escherichia coli were grown on 14.3% uniformly 13C-labeled glucose as the sole carbon source and challenged anaerobically with 90% 13C-labeled formaldehyde. The major multiply labeled metabolites were identified by 13C NMR spectroscopy to be glycerol and 1,2-propanediol, and a minor metabolite was shown to be 1,3-propanediol. In each case, formaldehyde is incorporated only into the C1 position. A novel form of 13C NMR isotope dilution analysis of the major products reveals that all the 1,2-diol C1 is formaldehyde derived but that about 40% of the glycerol C1 is derived from bacterial sources. Glycerokinase converted the metabolite [1-13C]glycerol to equal amounts of [3-13C]glycerol 3-phosphate and [1-13C]glycerol 3-phosphate, demonstrating that the metabolite is racemic. When [13C]formaldehyde incubation was carried out in H2O/D2O mixtures, deuterium incorporation was detected by beta- and gamma-isotope shifts. The 1,3-diol is deuterium labeled only at C2 and only once, while the 1,2-diol and glycerol are each labeled independently at both C2 and C3; C3 is multiply labeled. Deuterium incorporation levels are different for each metabolite, indicating that the biosynthetic pathways probably diverge early.

Formaldehyde metabolism by Escherichia coli. In vivo carbon, deuterium, and two-dimensional NMR observations of multiple detoxifying pathways.

13C NMR has been used to demonstrate the metabolism of dilute solutions of labeled formaldehyde by Escherichia coli to methanol, formate, carbon dioxide, and several other unidentified metabolites which contain labeled CH2 groups. Aeration of bacterial suspensions within the spectrometer dramatically increased the rate of oxidation to formate and carbon dioxide. Deoxygenation with nitrogen gas virtually abolished all metabolism, as did the exposure of bacteria to very high formaldehyde concentrations. Deuterium NMR of whole cells in deuterium-depleted water further demonstrated the conversion of formaldehyde-d2 to methanol-d2, ruling out a formaldehyde dismutase as an important species. Two-dimensional proton-carbon chemical shift correlation was used to reveal the chemical shifts of the protons attached to 13C labels in metabolites. The results indicate that formaldehyde is efficiently detoxified by the bacterial cell through a route or routes which do not appear to involve tetrahydrofolate. This detoxification may be in competition with the lethal antibacterial processes associated with formaldehyde.

A compound representing the D-glycerate terminus of the methylglucose-containing polysaccharide of Mycobacterium smegmatis.

In order to study the structure of the methylglucose-containing polysaccharide (MGP) of Mycobacterium smegmatis by NMR spectroscopy, we have prepared the model compound O-alpha-D-glucopyranosyl-(1 leads to 2)-D-glyceric acid. This compound, which represents the aglycon-containing terminus of MGP, was made from leucorse [O-alpha-D-glucopyranosyl-(1 leads to 5)-D-fructopyranose] by successive treatment with sodium borohydride, lead tetraacetate, and hypobromite. The structure of O-alpha-D-glucopyranosy.-(1 leads to 2)-D-glyceric acid was confirmed by chemical and enzymic methods. 13C and 1H NMR spectra of this compound, together with spectra of several disaccharides, were obtained for future reference in the polysaccharide study. The nine resonances in the 13C spectrum were assigned by comparison with the spectrum of methyl alpha-D-glucopyranoside. Analysis of the 1H NMR spectrum showed that the two methylene protons on C-3 of the glycerate moiety were less equivalent in the sodium salt than in the acid. This may be attributable to hydrogen bonding between the carboxylate and the hydrogen atom of the glycerate 3-hydroxyl group.