Chemical Composition of Cell Wall Peptidoglycan From Clostridium Saccharoperbutylacetonicum Studied With Phage Endolysins and Gas Chromatography

Abstract

The enzymic digest of cell wall peptidoglycan from Clostridium saccharoperbutylacetonicum by phage HM 7 endolysin (N-acetylmuramyl-Lalanine amidase) was separated into two constituents on ion-exchange chromatography. One was a polysaccharide, which contained N-acetylglucosamine and N-acetylmuramic acid in the molar ratio of 1.00: 0.78. This polysaccharide was digested by phage HM 3 endolysin (N-acetylmuramidase), and the digested product was a saccharide composed of N-acetylglucosamine and N-acetylmuramic acid. The other was a peptide composed of glutamic acid, alanine, and diaminopimelic acid in the molar ratio of 1.00: 2.09: 1.05. Amino acid sequence of the isolated peptide was determined by Edman degradation method, and optical configuration of the component amino acids was confirmed by gas chromatography using their N-trifiuoroacetyl menthyl esters. These analyses indicated that the isolated peptide was composed of a tetrapeptide subunits of NH2-terminal-L-AIa-D-Glu-Dpm-D-Ala. A reasonable structure for the cell wall peptidoglycan was also proposed.Bacterial cell wall peptidoglycan is a major constituent of the cell wall and maintains the cell rigidity and shape. It is typically composed of an alternating polymer of i3-1,4-linked N-acetylglucosamine and N-acetylmuramic acid. Each muramic acid residue bears a short peptide chain consisting of D-glutamic acid, L-and D-alanine, and either meso-diaminopimelic acid or L-lysine. Little is known, however, about the study of cell wall peptidoglycan of genus Clostridium except for C. botulinum (1, 2) and C. welchii (3). In our previous paper (4), we presented hypothesis for the structure of the cell wall peptidoglycan from C. saccharoper-65

Analysis of muramic acid in holocene microbial environments by gas chromatography, electron impact, and fast atom bombardment mass spectrometry

“…The peptidoglycan has been described as composed of a matrix consisting of N-acetylglucosamine and N-acetylmuramic acid linked by ß-1,4 linkages and pentapeptide side chains comprising D-and L-alanine, D-glutamic acid, and either L-lysine or mesodiaminopimelic acid (Ogata et al, 1975;Rose, 1976). Recent developments in analytical biochemistry have made it possible to estimate the bacterial biomass in different environments by measuring the specific biochemical constituents of bacteria, such as Downloaded by [University of California, San Diego] at 12:36 29 June 2016 adenosine triphosphate (Karl, 1979), total lipid fatty acids (White et al, 1979), and muramic acid (Millar and Casida, 1970;Moriarty, 1977;Findlay et al, 1983).…”

“…These include paper chromatography (Yamada and Komagata, 1970), thin layer chromatography (Boone and Pine, 1968), ion exchange chromatography (Vasstrand et al, 1980), enzymatic analysis (Ogata et al, 1975), and gas chromatography. Several derivatization techniques have been applied to the gas Chromatographie procedure.…”

The analysis of actinomycete wall amino acids by gas chromatography

“…Paper chromatography and TLC have been used primarily to provide a rapid qualitative assessment of the wall amino acid composition and to detect diagnostic diamino acids [4,5]. An established technique, applied only rarely to bacterial amino acids [6,7], is the conversion of amino acids into volatile derivatives and their analysis by gas chromatography (GC) [8]. This approach is of potential value since it provides reliable quantitative data on wall amino acids without the use of an amino acid analyser.…”

Quantitative Chemical Analyses and Antigenic Properties of Peptidoglycans from Clostridium botulinum and Other Clostridia

“…The chemical constituents of the cell walls of various species of the genus Clostridium were qualitatively determined by Cummins and colleague [2,3] and these results were used for classification of this genus [19]. Although there are a few studies on the quantitative chemical analyses of peptidoglycans of some clostridia [12,13,15], there are no reports on various types of Clostridium botulinum, except type A [21]. Our previous studies [24] on the cell walls of C. botulinum type A have shown that the peptidoglycan is of the directly cross-linked diaminopimelic acid (DAP)-containing type in which the cross-linkage of peptide moiety extends from the r-amino group of DAP of one peptide to the carboxyl group of D-alanine of another adjacent peptide subunit.…”