Extent of peptide cross-linking in the peptidoglycan of Neisseria gonorrhoeae

Abstract

The extent of peptide cross-linking in peptidoglycan (PG) isolated from various strains of Neisseria gonorrhoeae was examined. Purified PG, specifically labeled in the peptide moiety with [(3)H]diaminopimelic acid (DAP) and labeled in the glycan with [(14)C]glucosamine and [(14)C]muramic acid, was digested completely with Chalaropsis B muramidase. Gel filtration of the digest on connected columns of Sephadex G-50 and G-25 revealed four well-defined peaks corresponding to soluble PG fragments and containing a constant ratio of (3)H to (14)C. On the basis of (i) K(D) values, (ii) amino acid composition, (iii) free amino group analysis of [(3)H]DAP residues, (iv) borohydride reduction, (v) the beta-elimination reaction, (vi) high-voltage electrophoresis, and (vii) paper chromatography in various solvents, the PG fragments were identified as un-cross-linked disaccharide peptide monomer, typical of chemotype I PG, and the corresponding peptide cross-linked dimers, trimers, and tetramers. The percent cross-linking of PG basically reflects the percentage of DAP residues that are involved in peptide cross-linking bonds. This value was estimated from the distribution of labeled fragments that resulted from the enzymatic digestion of PG and was confirmed by the analysis of free amino groups in [(3)H]DAP of intact PG. Although there were subtle, strain- and medium-dependent differences in percent cross-linking, these values varied only over a relatively narrow range (36 to 44%). The percent cross-linking of PG in the prototype strain, RD(5), grown in a standard gonococcal medium (LGCB(+)) was 41.0 +/- 2.0%. This is a relatively high degree of peptide cross-linking for a gram-negative bacterium. We also confirmed previous observations that the extent of PG cross-linking among isogenic gonococci was higher in strains, e.g., FA140 and FA136, carrying loci that govern increased resistance to multiple drugs.