Teichuronic acid released from its phosphodiester linkage to peptidoglycan in the cell walls of Micrococcus luteus by mild acid treatment is resolved into a ladderlike series of bands by electrophoresis on polyacrylamide gels in the presence of borate. Each band of the ladder differs from its nearest neighbor by one disaccharide repeat unit, ----4)-2-acetamido-2-deoxy-beta-D-mannopyranuronosyl-(1----6)- alpha-D-glucopyranosyl-(1-. Acid-fragmented teichuronic acid, after conversion to the phenylamine derivative, was fractionated by preparative-scale molecular sieve column chromatography, which produced a series of elution peaks. Fast-atom-bombardment mass spectrometry of the smallest member of the series determined its molecular weight and established its identity as the phenylamine derivative of one disaccharide repeat unit of teichuronic acid. Homologous fractions of the same series were used to index the ladder of bands obtained by polyacrylamide gel electrophoresis from samples containing a more extensive distribution of polymer lengths. Nearly native teichuronic acid consists of polymers with a broad range of molecular sizes ranging from 20 to 55 disaccharide units. The most abundant species are those which have 25 to 40 repeat units. Prolonged treatment of teichuronic acid with the acid conditions used to release it from peptidoglycan causes gradual fragmentation of the teichuronic acid.
Teichuronic acid-peptidoglycan complex isolated from Micrococcus luteus cells by lysozyme digestion in osmotically stabilized medium was treated with mild acid to cleave the linkage joining teichuronic acid to peptidoglycan. This labile linkage was shown to be the phosphodiester which joins N-acetylglucosamine, the residue located at the reducing end of the teichuronic acid, through its anomeric hydroxyl group to a 6-phosphomuramic acid, a residue of the glycan strand of peptidoglycan. 31P nuclear magnetic resonance spectroscopy of the lysozyme digest of cell walls demonstrated the presence of a phosphodiester which was converted to a phosphomonoester by the conditions which released teichuronic acid from cell walls. Reduction of acid-liberated reducing end groups by NaB3H4 followed by complete acid hydrolysis yielded [3H] glucosaminitol from the true reducing end residue of teichuronic acid and [3H]glucitol from the sites of fragmentation of teichuronic acid. The amount of N-acetylglucosamine detected was approximately stoichiometric with the amount of phosphate in the complex. Partial fragmentation of teichuronic acid provides an explanation of the previous erroneous identification of the reducing end residue.Micrococcus luteus cell walls consist of a peptidoglycan matrix to which teichuronic acid is covalently attached (2,8,12,13,29). Although the structure of the disaccharide repeat unit of teichuronic acid has been determined by chemical procedures (9) and confirmed by 13C nuclear magnetic resonance (NMR) spectroscopy (15), the precise nature of the linkage of teichuronic acid to peptidoglycan has remained in dispute. A small amount of phosphate is present in M. luteus cell walls (2), and much of it can be recovered from strong acid hydrolysates as muramic acid-6-phosphate (24). Glucosamine-6-phosphate has also been detected (18,27). A phosphodiester has been suggested as part of the covalent link between peptidoglycan and teichuronic acid in M. luteus (10,25,26) just as it serves to link teichoic acids and other cell wall polymers to peptidoglycan in other microorganisms (4,16,17,19,23).Nasir-ud-Din and Jeanloz (25) claimed that glucose is the reducing end residue of teichuronic acid which links to peptidoglycan through the phosphodiester. Later Nasirud-Din et al. (26) reported experimental results which purported to show that glucose is the reducing end residue of teichuronic acid. Evidence presented by Hase and Matsushima (10) indicated instead that the reducing end residue is N-acetylglucosamine. Our studies of in vitro teichuronic acid biosynthesis (14, 31) showed that biosynthesis depends on the initial step which incorporated N-acetylglucosamine-1-phosphate into a carrier lipid-activated intermediate. Furthermore, the N-acetylglucosamine residue remains in product teichuronic acid at the reducing end, suggesting that teichuronic acid in native cell walls has an N-acetylglucosamine residue at its potentially reducing terminal residue.We report here that the true reducing end residue of teichuronic ...
The most direct and informative probe of ribosome structure is electron microscopy (EM), of negatively stained and antibody-labelled specimens. Ribosomes from Escherichia coli have been the most intensively studied, although those from other prokaryotes and eukaryotes have also been imaged [e.g., 3, 4, 5],The amount of structural information obtained from electron images can be significantly increased using multivariate statistical analysis and classification techniques [e.g., 6]. Noisy electron micrographs of single particles are sorted according to their principal features, and average images formed with an enhanced signal-to-noise ratio and better reproducible resolution. The class averages represent projections from which a three-dimensional reconstruction can be computed.Thermus aguaticus is a species of extremely thermophilic bacteria isolated from environments such as hot springs. This species possesses macromolecular enzyme complexes with a great thermostability which must be reflected somehow instructural differences. In this study, we investigate the structures of ribosomes and ribosomal subunits from this bacterium.
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