The Elucidation of the Structure of Thermotoga maritima Peptidoglycan Reveals Two Novel Types of Cross-link

Abstract

Thermotoga maritima is a Gram-negative, hyperthermophilic bacterium whose peptidoglycan contains comparable amounts of L-and D-lysine. We have determined the fine structure of this cell-wall polymer. The muropeptides resulting from the digestion of peptidoglycan by mutanolysin were separated by high-performance liquid chromatography and identified by amino acid analysis after acid hydrolysis, dinitrophenylation, enzymatic determination of the configuration of the chiral amino acids, and mass spectrometry. The high-performance liquid chromatography profile contained four main peaks, two monomers, and two dimers, plus a few minor peaks corresponding to anhydro forms. Peptidoglycan (or murein) is a giant macromolecule whose main function is the protection of the cytoplasmic membrane against the internal osmotic pressure. It is composed of alternating residues of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) 2 cross-linked by short peptides (1). The composition of the peptide stem in nascent peptidoglycan is L-Ala 1 -␥-D-Glu 2 -X 3 -D-Ala 4 -D-Ala 5 , where X is most often meso-diaminopimelic acid (meso-A 2 pm) or L-lysine in Gram-negative and Gram-positive species, respectively (2, 3). In the mature macromolecule, the last D-Ala residue is removed. Cross-linking of the glycan chains generally occurs between the carboxyl group of D-Ala at position 4 of a donor peptide stem and the side-chain amino group of the diamino acid at position 3 of an acceptor peptide stem (433 cross-links). Cross-linking is either direct or through a short peptide bridge such as pentaglycine in Staphylococcus aureus (2, 3). The enzymes for the formation of the 433 cross-links are active-site serine DDtranspeptidases that belong to the penicillin-binding protein (PBP) family and are the essential targets of ␤-lactam antibiotics in pathogenic bacteria (4). Catalysis involves the cleavage of the D-Ala 4 -D-Ala 5 bond of a donor peptide stem and the formation of an amide bond between the carboxyl of D-Ala 4 and the side chain amine at the third position of an acceptor stem. Transpeptidases of the LD specificity are active-site cysteine enzymes that were shown to act as surrogates of the PBPs in mutants of Enterococcus faecium resistant to ␤-lactam antibiotics (5). They cleave the X 3 -D-Ala 4 bond of a donor stem peptide to form 333 cross-links. This alternate mode of crosslinking is usually marginal, although it has recently been shown to predominate in non-replicative "dormant" forms of Mycobacterium tuberculosis (6).Thermotoga maritima is a Gram-negative, extremely thermophilic bacterium isolated from geothermally heated sea floors by Huber et al. (7). A morphological characteristic is the presence of an outer sheath-like envelope called "toga." Although the organism has received considerable attention for its biotechnological potential, studies about its peptidoglycan are scarce (8 -11), and in particular the fine structure of the macromolecule is still unknown. In their initial work, Huber et al. (7) showed that the comp...

Primordial‐like enzymes from bacteria with reduced genomes

“…Members of all three taxa are characterized by having reduced genomes. The fine structure of the Thermotoga maritima peptidoglycan has been determined biochemically, and it is known to contain d ‐alanine (Boniface et al ., ). The well‐characterized member of the Pelagibacterales is Pelagibacter ubique , the genome sequence of which confirms that it synthesizes peptidoglycan (Giovannoni et al ., ).…”

Optimization of conditions for the glycosyltransferase activity of penicillin‐binding protein 1a from Thermotoga maritima

“…It must be noted that the substrates used in this study were either l ‐lysine‐containing lipid II, dansylated on the lysine, or radiolabeled meso‐diaminopimelic acid (meso‐A 2 pm)–lipid II. It has been found recently that the T. maritima peptidoglycan contains unusual stem peptides, which include d ‐lysine in nonconventional arrangements, as well as unusual cross‐links [44]. Future studies will investigate the substrate specificity of the TPase reaction of Tm‐1a*.…”

Elucidation of the d‐lysine biosynthetic pathway in the hyperthermophile Thermotoga maritima

“…Previously, d ‐Lys has been reported to exist in the peptide cross‐bridge only in B. rettgeri , implying that it is a rare component of peptidoglycan. Additionally, the degree of cross‐linking is lower in T. maritima peptidoglycan than in other Gram‐negative bacteria, and the average chain length is longer . It remains unclear whether these properties contribute to cellular thermostability, and this issue should be investigated in future studies.…”

“…d ‐Lys is a highly unusual component of peptidoglycan, and outside T. maritima it has only been reported in Butyribacterium rettgeri . MurE (UDP‐ N ‐acetylmuramoyl‐ l ‐alanyl‐ d ‐glutamate: meso ‐diaminopimelate ligase) from T. maritima links l ‐ and d ‐Lys to UDP‐MurNAc‐ l ‐Ala‐ d ‐Glu in vitro , and peptide stems containing l ‐ and d ‐Lys (e.g., MurNAc‐ l ‐Ala‐ d ‐Glu‐ l ‐Lys‐ d ‐Ala and MurNAc‐ l ‐Ala‐ d ‐Glu‐ d ‐Lys) are present in the fine structure of the peptidoglycan . However, the biosynthetic pathway of d ‐Lys in T. maritima remains unclear.…”