FIG 3.

RodA and PBP1b promote cell growth by inserting nascent PG material at the poles. Overnight cultures of the indicated strains from Fig. 1 grown in BHIS medium at 30°C were diluted 1:1,000 in BHIS and grown for 3 h at 30°C. The cells were loaded into a CellASIC microfluidic device and grown for 30 min in BHIS medium at 30°C. Following this equilibration period, the cells were imaged every 5 min using phase-contrast and fluorescence optics. Cells were pulse labeled with the fluorescent d-amino acid TADA (39) for 3 min at the 6-min mark. The label was then progressively washed away by the flow of fresh medium lacking label. Every 6th frame in the time-lapse series is shown. The length of unlabeled cell wall was measured after the TADA pulse (t = 20 min) and at the end of the time lapse (t = 120 min) using Oufti (N > 150 cells); the unlabeled portion of each cell was defined as that with a TADA signal <20% of the maximum TADA signal for that cell. The mean and standard error of the rate of polar elongation were calculated by taking the mean difference in the length of unlabeled cell wall at the initial and final time points divided by the time elapsed.