Contribution of a Thickened Cell Wall and Its Glutamine Nonamidated Component to the Vancomycin Resistance Expressed by Staphylococcus aureus Mu50

Metabolic pathways for the synthesis of cell-wall peptidoglycan. Two major biosynthetic pathways supply UDP-GlcNAc, the cardinal precursor metabolite for cell-wall peptidoglycan. Two enzymes involved in the cell-wall synthesis pathways require glutamine as the NH₄ ⁺ donor for the amidation reaction catalyzed by the enzymes: GlmS catalyzes the conversion of Fru-6-P to GlcN-6-P, and a putative enzyme catalyzes the amidation of iso-glutamate of the murein monomer precursor (in the form of lipid I or II). GS helps the activities of these enzymes by supplying glutamine by assimilating ammonia into glutamate.

Rate of cell-wall synthesis and GS and GlmS activities of Mu50. (A) The rates of ¹⁴C-labeled d-glucose incorporation into the cell wall among the test strains were compared. The time course of incorporation of ¹⁴C-labeledd-glucose into the cell walls in RMg− was evaluated. Symbols: closed triangle, Mu50; small closed circle, Mu3; large open triangle, H1; small open triangle, Mu50ω; large closed circle, N315; open circle, FDA209P. (B) GlmS and GS activities in Mu50 and other test strains. Bacteria in the logarithmic growth phase were harvested for measurement of the enzyme activities as described in Materials and Methods. GS was tested for both its biosynthetic (glutamine synthetase) and its transferase (glutamyltransferase) activities. ND, not done. (C) Effect of l-glutamine on rate of glucose incorporation. The rates of incorporation of ¹⁴C-labeled d-glucose into the cell walls of test strains in medium containing various concentrations of l-glutamine were compared. The bacterial culture in BHI medium was harvested at an OD₅₇₈ of 0.7, washed twice with RMg−, and incubated in RMg− with various concentration of l-glutamine 30 min before the addition of radioactive glucose. The values shown are those obtained at 20 min in the incorporation assay. Note that in the case of Mu50, more than twice the amount of glucose was incorporated in the presence of 10 mM glutamine.

Correlation between the cell-wall thickness, cell-wall structure, and TRG in the presence of vancomycin in Mu50. Mu50 was grown in BHI medium to an OD₆₀₀ of 0.7, washed twice with RMg− and then incubated at 37°C for 2 h with shaking in either one of the following media: RMg− (panels 1), RM (RMg− plus 30 mMd-glucose) (panels 2), RMg− plus 30 mMl-glutamine (panels 3), RMg− plus 30 mM GlcNAc (panels 4), and RM plus 30 mM l-glutamine (panels 5). The cells were analyzed for cell-wall thickness by transmission electron microscopy (A), cell-wall composition by HPLC (B), and the level of resistance by observing the cell growth in BHI medium containing 30 μg of vancomycin (solid line in panel C) per ml. The vancomycin concentration in the medium was determined by bioassay, as described in Materials and Methods, by using the sterile filtrates of the culture at various times after the initiation of culture (broken line in panel C). Magnification in panel A, ×22,500. M9/M4 and D/M in panel B indicate the ratio of the peak area of the glutamine-nonamidated monomer (M9 peak) versus that of the amidated monomer (M4 peak) and the ratio of the total area of dimer peaks versus that of monomer peaks (8), respectively. The numbers used to label the panels and lines in panels A, B, and C correspond to one another. The values given under each picture in panel A are the means and SDs of the cell wall thickness (in nanometers). In panel C, the symbols for the broken lines (vancomycin concentration) correspond to the numerals for the solid lines (cell growth), as follows: ▵, 1; ○, 2; ●, 3; □, 4; ◊, 5.