FIG. 4.

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 mM d-glucose) (panels 2), RMg− plus 30 mM l-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.