Competitive growth analysis of WT and ΔnsiR4. Competitive growth analysis of WT and ΔnsiR4. (A) Experimental setup. Three independent, exponentially growing cultures of WT and ΔnsiR4 were diluted to an OD750 of 0.1 and mixed 1:1. (B) Growth performance during long-term cultivation. Cultures were rediluted every 3 or 4 d to an OD750 of 0.1 and 25 µL of a 1:1,000 dilution were dropped on BG11 agar plates with and without 40 µg/mL kanamycin. Data are the mean ± SD of WT and ΔnsiR4, as well as three mixed WT/ΔnsiR4 cocultures. Due to the lowered NO3− content (1 mM instead of 17.6 mM NO3−), a strong fluctuation in the cellular N status could be assumed, which was also indicated by the cultures switching between green or bleached appearance at lower (0.1–0.8) and higher (>0.8) ODs (see also C). (C) Whole cell absorption spectra of the WT culture indicating cellular bleaching, which was due to pigment degradation as response to initiated internal N limitation. (D) Number of generations that were observed over 50 d at average growth rates of µ = 0.751 ± 0.065. (E) Evolution of the ratio between kanamycin-resistant (ΔnsiR4) and total (WT + ΔnsiR4) colony forming units (CFUs) during consecutive cultivation and redilution. (F) Photographs of CFUs at the beginning of the experiment and after 33 and 48 generations (g). (G) Verification of the ΔnsiR4 mutant allele reduction (upper band) compared with the WT allele (lower band) by PCR. *To one of the WT/ΔnsiR4 cocultures once per day 100 µM NH4Cl (f.c.) was added to intensify the fluctuation in the N availability. However, no change in growth performance compared with the other two cocultures was observed. Thus, all three cocultures were averaged as shown in E. Stephan Klähn et al. PNAS 2015;112:45:E6243-E6252 ©2015 by National Academy of Sciences