Membrane-wall interrelationship in Gaffkya homari: sulfhydryl sensitivity and heat lability of nascent peptidoglycan incorporation into walls.

Membrane-walls from Gaffkya homari require a specific interrelationship between membrane and wall that functions in the incorporation of nascent peptidoglycan into the preexisting peptidoglycan of the wall. Two different methods were used to inhibit selectively this incorporation process: (i) sensitivity to sulfhydryl reagents and (ii) heat inactivation. Of the sulfhydryl reagents tested, 2.2 mM iodoacetamide inhibited the synthesis of wall peptidoglycan 50%, whereas greater than 100 mM was required to inhibit the synthesis of sodium dodecyl sulfate (SDS)-soluble peptidoglycan. Heat treatment at 37 degrees C (t 1/2 = 5.7 min) inhibited wall peptidoglycan synthesis without affecting SDS-soluble peptidoglycan synthesis. Inhibition of LD-carboxypeptidase by iodoacetamide and heat gave 50% inhibition and t 1/2 values similar to those observed for the incorporation process. Thus, it is suggested that the LD-carboxypeptidase may be one of the enzymes responsible for the sulfhydryl sensitivity and heat lability and that this enzyme may play a role in the relationship between membrane and wall in G. homari.     

Characterization of a novel linkage unit between ribitol teichoic acid and peptidoglycan in Listeria monocytogenes cell walls

The structure of the linkage unit between ribitol teichoic acid and peptidoglycan in the cell walls of Listeria monocytogenes EGD was studied. A teichoic-acid--glycopeptide preparation isolated from lysozyme digests of the cell walls of this strain contained mannosamine, glycerol, glucose and muramic acid 6-phosphate in an approximate molar ratio of 1:1:2:1, together with large amounts of glucosamine and other components of teichoic acid and glycopeptides. A teichoic-acid-linked sugar preparation, obtained by heating the cell walls at pH 2.5, also contained glucosamine, mannosamine, glycerol and glucose in an approximate molar ratio of 25:1:1:2. Part of the glucosamine residues were shown to be involved in the linkage unit. Thus, on mild alkaline hydrolysis, the teichoic-acid-linked sugar preparation gave a disaccharide characterized as N-acetylmannosaminyl(beta 1----4)-N-acetylglucosamine [ManNAc(beta 1----4)GlcNAc] in addition to the ribitol teichoic acid moiety, whereas the teichoic-acid - glycopeptide was separated into disaccharide-linked glycopeptide and the ribitol teichoic acid moiety by the same procedure. Furthermore, Smith degradation of the cell walls gave a characteristic fragment, EtO2-P-Glc(beta 1----3)Glc(beta 1----1/3)Gro-P-ManNAc(beta 1----4)GlcNAc (where EtO2 = 1,2-ethylenediol and Gro = glycerol). The results lead to the conclusion that in the cell walls of this organism, the ribitol teichoic acid chain is linked to peptidoglycan through a novel linkage unit, Glc(beta 1----3)Glc(beta 1----1/3)Gro-P-(3/4)ManNAc-(beta 1----4)GlcNAc.     

Biosynthesis of peptidoglycan in Staphylococcus aureus: incorporation of the Nepsilon-Ala-Lys moiety into the peptide subunit of nascent peptidoglycan.

UDP-MurNAc-Ala-DGlu-Lys(Nepsilon-Ala)-DAla-DAla was isolated from extracts of Staphylococcus aureus Copenhagen. This nucleotide accumulated in media deficient in glycine. To establish its role in peptidoglycan biosynthesis, the nucleotide-hexapeptide was compared with UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla in the reaction catalyzed by phospho-MurNAc-pentapeptide translocase and in the membrane-catalyzed nascent peptidoglycan-synthetizing system. In the exchange reaction catalyzed by the translocase, the Rmax and Rmax/Km are 1.79 muM/min and 4.47 X 10(-2)/min, respectively, for UDP-MurNAc-pentapeptide and 1.81 muM/min and 4.46 X 10(-2)/min, respectively, for UDP-Mur-NAc-hexapeptide. In the synthesis of nascent peptidoglycan, the Vmax is 1.8 muM/min X 10(-2) for both the nucleotide-hexapeptide and -pentapeptide. The Vmax/Km is 5.6 X 10(-4) and 4.3 X 10(-4)/min for the nucleotide-pentapeptide and -hexapeptide, respectively. Schleifer, Hammes, and Kandler (Adv. Microb. Physiol. in press) observed that growth of S. aureus Copenhagen on a glycine-poor medium results in a peptidoglycan structure in which 20% of the lysine residues are substituted at the epsilon-amino group by L-alanine residues that do not participate in interpeptide bridge information. The in vitro studies demonstrate that UDP-MurNAc-Ala-DGlu-Lys(Nepsilon-Ala)-DAla-DAla is a possible precursor of the Nepsilon-Ala-Lys moiety.     

Tunicamycin-induced growth and inhibition of glucosamine incorporation into cell walls of rice coleoptiles

Tunicamycin, 0.25 to 2.5 μ M , promotes elongation of rice coleoptile sections after a 2 h lag. Tunicamycin decreased the minimum stress-relaxation time of the cell wall, T₀; and the wall loosening is recognized as the cause of this growth promotion. Bacitracin did not have significant effects on growth or T₀ except for inhibition of elongation at high concentration. Coleoptile sections were incubated with [¹⁴C]-glucosamine, and the synthetic pathway of the hexosamine-containing cell wall component was examined by a pulse-chase experiment. This component seems to be synthesized in the particulate fraction and secreted mainly into the hemicellulose I fraction. Tunicamycin strongly inhibited glucosamine incorporation into the particulate fraction and stopped the labeling of the cell wall. At 2.5 μ M , tunicamycin had no effect on incorporation of mannose, leucine or proline. These results suggest that the hexosamine-containing wall component is a kind of asparagine-linked glycoprotein, and that this component plays a principal role in formation of the cell wall network and in growth regulation.     

A common linkage saccharide unit between teichoic acids and peptidoglycan in cell walls of Bacillus coagulans

Teichoic acid-glycopeptide complexes were isolated from lysozyme digests of the cell walls of Bacillus coagulans AHU 1631, AHU 1634, and AHU 1638, and the structure of the teichoic acid moieties and their linkage regions was studied. On treatment with hydrogen fluoride, each of the complexes gave a hexosamine-containing disaccharide, which was identified to be glucosyl(beta 1----4)N-acetylglucosamine, in addition to dephosphorylated repeating units of the teichoic acids, namely, galactosyl(alpha 1----2)glycerol and either galactosyl(alpha 1----2)[glucosyl(alpha 1----1/3)]glycerol (AHU 1638) or galactosyl(alpha 1----2)[glucosyl(beta 1----1/3)]glycerol (AHU 1631 and AHU 1634). From the results of Smith degradation, methylation analysis, and partial acid hydrolysis, the teichoic acids from these strains seem to have the same backbone chains composed of galactosyl(alpha 1----2)glycerol phosphate units joined by phosphodiester bonds at C-6 of the galactose residues. The presence of the disaccharide, glucosyl(beta 1----4)N-acetylglucosamine, in the linkage regions between teichoic acids and peptidoglycan was confirmed by the isolation of a disaccharide-linked glycopeptide fragment from each complex after treatment with mild alkali and of a teichoic acid-linked saccharide from each cell wall preparation after treatment with mild acid. Thus, it is concluded that despite structural differences in the glycosidic branches, the teichoic acids in the cell walls of the three strains are linked to peptidoglycan through a common linkage saccharide, glucosyl (beta 1----4) N-acetylglucosamine.     

Biosynthesis of peptidoglycan in Pseudomonas aeruginosa. 1. The incorporation of peptidoglycan into the cell wall.

Ether-treated cells of Pseudomonas aeruginosa catalyze the formation of crosslinked peptidoglycan from the two nucleotide precursors uridinediphospho-N-acetylglucosamine and uridinediphospho-N-acetylmuramyl-L-alanyl-D-gamma-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine. The main enzymatic reactions of biosynthesis were similar to those found in Escherichia coli. Part of the reaction products were soluble in 4% sodium dodecylsulfate whereas the other part was covalently bound to the preexisting cell wall peptidoglycan sacculus. The incorporation into cell wall is carried out by a transpeptidation reaction in which the nascent peptidoglycan functions mainly as the donor and the preexisting one as acceptor. The detergent-soluble peptidoglycan is composed of partially crosslinked peptidoglycan strands as well as low-molecular-weight peptidoglycan fragments. Pulse-chase biosynthesis experiments show that the detergent-soluble peptidoglycan is an intermediate that eventually becomes covalently bound to the wall. The DD-carboxypeptidase activity of P. aeruginosa is membrane-bound and does not hydrolyse C-terminal D-alanine residues from the L-lysine-containing nucleotide-precursor analogue. An LD-carboxypeptidase was also detected in P. aeruginosa.     

Structural features of cell wall teichoic acid and peptidoglycan of Actinomadura cremea INA 292

The teichoic acid from the cell wall of Actinomadura cremea INA 292 has an unusual structure, being a poly(galactosylglycerol phosphate) chain with glycerol phosphate groups. Monomeric units of 1-O, beta-D-galactopyranosylglycerol monophosphate are joined in the polymer by phosphodiester links involving the glycerol C3 and the galactose C6 atoms. Approximately every second galactosyl substituent has a glycerol phosphate residue at its C3 atom. The teichoic acid structure was established by chemical analysis and 13C-NMR spectroscopy. There also is a peptidoglycan belonging to the A1 gamma type: as well as meso-2,6-diaminopimelic acid it contains small amounts of the LL form and glycine.     

N-acetylmannosaminyl(1----4)N-acetylglucosamine, a linkage unit between glycerol teichoic acid and peptidoglycan in cell walls of several Bacillus strains.

The structure of teichoic acid-glycopeptide complexes isolated from lysozyme digests of cell walls of Bacillus subtilis (four strains) and Bacillus licheniformis (one strain) was studied to obtain information on the structural relationship between glycerol teichoic acids and their linkage saccharides. Each preparation of the complexes contained equimolar amounts of muramic acid 6-phosphate and mannosamine in addition to glycopeptide components and glycerol teichoic acid components characteristic of the strain. Upon treatment with 47% hydrogen fluoride, these preparations gave, in common, a hexosamine-containing disaccharide, which was identified as N- acetylmannosaminyl (1----4) N-acetylglucosamine, along with large amounts of glycosylglycerols presumed to be the dephosphorylated repeating units of teichoic acid chains. The glycosylglycerol obtained from each bacterial strain was identified as follows: B. subtilis AHU 1392, glucosyl alpha (1----2)glycerol; B. subtilis AHU 1235, glucosyl beta(1----2) glycerol; B. subtilis AHU 1035 and AHU 1037, glucosyl alpha (1----6)galactosyl alpha (1----1 or 3)glycerol; B. licheniformis AHU 1371, galactosyl alpha (1----2)glycerol. By means of Smith degradation, the galactose residues in the teichoic acid-glycopeptide complexes from B. subtilis AHU 1035 and AHU 1037 and B. licheniformis AHU 1371 were shown to be involved in the backbone chains of the teichoic acid moieties. Thus, the glycerol teichoic acids in the cell walls of five bacterial strains seem to be joined to peptidoglycan through a common linkage disaccharide, N- acetylmannosaminyl (1----4)N-acetylglucosamine, irrespective of the structural diversity in the glycosidic branches and backbone chains.     

A teichoic acid of Nocardioides albus VKM Ac-805T cell walls

A teichoic acid of Nocardioides albus VKM Ac-805T cell walls, a typical species of the genus Nocardioides, contains a poly(glycosylglycerol phosphate). The repeating unit of the polymer has the structure: [figure]. These units are in phosphodiester linkage at C-3 of glycerol and C-3 of beta-D-galactopyranose. beta-D-Galactopyranosyl residues are substituted at C-4 by beta-D-glucopyranose carrying a 4,6-pyruvate ketal group in S-configuration. The presence of pyruvic acid in the majority of repeating units increases the anionic properties of the polymer in comparison with most other common teichoic acids. This is the first report of the occurrence of a beta-D-galactofuranosyl residue in teichoic acids; it probably acts as a terminator of an extending chain of the polymer. The ratio of beta-D-galactopyranosyl to beta-D-galactofuranosyl units is 7:1. The polymer structure was determined by NMR spectroscopy. This type of teichoic acid structure has not been reported previously.     

Bacterial cell walls and membranes. Discovery of the teichoic acids

Teichoic acids are major wall components of most Gram-positive bacteria. Their discovery followed that of their nucleotide precursors. Lipoteichoic acids associated with the cell membrane were discovered at the same time. Events leading to these discoveries and the probable function of teichoic acids in cation control are described.     

Influence of phosphate supply on teichoic acid and teichuronic acid content of Bacillus subtilis cell walls

Bacillus subtilis 168 was grown in chemostat culture in fully defined media containing a constant concentration of magnesium and concentrations of phosphate that varied from those giving phosphate-limited growth to those in which phosphate was present in excess and magnesium was limiting. Phosphate-limited bacteria were deficient in wall teichoic acid and contained less than half as much cellular phosphate as did bacteria grown in excess of phosphate. Approximately 70% of the additional phosphate in the latter bacteria was present as wall teichoic acid, indicating that the ability of the bacteria to discontinue teichoic acid synthesis when grown under phosphate limitation permits a substantial increase in their growth yield. Since not all of the additional phosphate is present as wall teichoic acid other cellular phosphates may also be present in reduced amounts in the phosphate-limited bacteria. The content of phosphate groups in walls of magnesium-limited bacteria was similar to the content of uronic acid groups in walls of phosphate-limited bacteria, and walls of bacteria grown in media of intermediate composition contained intermediate proportions of the two anionic polymers. Phage SP50, used as a marker for the presence of teichoic acid, bound densely to nearly all of the bacteria in samples containing down to 22% of the maximum content of teichoic acid. Apparently, therefore, nearly all of these bacteria contain teichoic acid, and the population does not consist of a mixture of individuals having exclusively one kind of anionic polymer. Bacteria containing less than 22% of the maximum content of teichoic bound in a nonuniform manner, and possible explanations for this are discussed.     

Efficient and quantitative incorporation of diaminopimelic acid into the peptidoglycan of Salmonella typhimurium

Abstract Diaminopimelic acid is incorporated into the peptidoglycan of Salmonella typhimurium in an efficient and quantitative manner. The amount of DAP incorporated is similar to the number of molecules estimated to exist in the Salmonella cell wall. In contrast, strains of E. coli , including those most used for studies of cell wall synthesis, are much less efficient in the incorporation of diaminopimelic acid. The lysine-requiring strains of E. coli appear to excrete diaminopimelic acid related material during growth and this accounts, in part, for the inefficient incorporation of radioactive diaminopimelic acid into Escherichia strains. In addition, the Escherichia strains are much less permeable to DAP than Salmonella strains. Cysteine and cystine inhibit the incorporation of DAP into the cell and this result suggests that Salmonella uses the cystine uptake system to allow DAP into the cell.     

In vitro system for the synthesis of teichoic acid linked to peptidoglycan.

A crude cell wall preparation from Staphylococcus aureus H prepared by the method of Mirelman and Sharon (1972) was shown to catalyze the synthesis of polyribitol phosphate linked to the cell wall peptidoglycan. The reaction used cytidine diphosphate (CDP)-ribitol as a substrate and in addition required the presence of CDP-glycerol, uridine diphosphate (UDP)-N-acetyl-D-glucosamine, and adenosine triphosphate. Incubation of radioactive CDP-glycerol with the crude cell wall preparation resulted in the transfer of glycerol phosphate residues to the cell wall; this reaction was greatly stimulated by the presence of UDP-N-acetylglucosamine. These data suggest that polyribitol phosphate is linked to the cell wall peptidoglycan by an oligomer contaning N-acetyl-D-glucosamine and glycerol phosphate.     

In vitro synthesis of the unit that links teichoic acid to peptidoglycan.

The role of cytidine diphosphate (CDP)-glycerol in gram-positive bacteria whose walls lack poly(glycerol phosphate) was investigated. Membrane preparations from Staphylococcus aureus H, Bacillus subtilis W23, and Micrococcus sp. 2102 catalyzed the incorporation of glycerol phosphate residues from radioactive CDP-glycerol into a water-soluble polymer. In toluenized cells of Micrococcus sp. 2102, some of this product became linked to the wall. In each case, maximum incorporation of glycerol phosphate residues required the presence of the nucleotide precursors of wall teichoic acid and of uridine diphosphate-N-acetylglucosamine. In membrane preparations capable of synthesizing peptidoglycan, vancomycin caused a decrease in the incorporation of isotope from CDP-glycerol into polymer. Synthesis of the poly (glycerol phosphate) unit thus depended at an early stage on the concomitant synthesis of wall teichoic acid and later on the synthesis of peptidoglycan. It is concluded that CDP-glycerol is the biosynthetic precursor of the tri(glycerol phosphate) linkage unit between teichoic acid and peptidoglycan that has recently been characterized in S. aureus H.