Figure 3. Robust towers arise from transiently restricted NTHI in a mixed culture.

The reporter strains [86-028NP/pGM1.1 (production of GFP) and 86-028NP/pKM1.1 (production of mCherry)] were cultured for 24 hours in the presence or absence of heme-iron to generate “replete” and “restricted” populations. Restricted 86-028NP/pGM1.1 was co-cultured with equivalent numbers of replete 86-028NP/pKM1.1 for 48 hours in 2 µg heme mL⁻¹ (A) or 20 µg heme mL⁻¹ (C) in a glass chamber slide. Restricted 86-028NP/pKM1.1 was co-cultured with equivalent numbers of replete 86-028NP/pGM1.1 for 48 hours in 2 µg heme mL⁻¹ (B) in a glass chamber slide. Optical sections were rendered to form the top down and orthogonal views. Biofilms formed from co-culture of restricted 86-028NP/pKM1.1 with replete 86-028NP/pGM1.1 were subjected to disruption to enumerate the total numbers of each reporter strain present following 48 hours of biofilm growth (D). Restricted 86-028NP/pGM1.1 was co-cultured with 1000 fold more 86-028NP/pKM1.1 replete for heme-iron for 48 hours in 2 µg heme mL⁻¹ (E) in a glass chamber slide. Restricted 86-028NP/pKM1.1 was co-cultured with 1000 times more 86-028NP/pGM1.1 replete for heme-iron for 48 hours in 2 µg heme mL⁻¹ (F) in a glass chamber slide. Orthogonal views of 3D rendered image are depicted (E, F). Heights of the biofilm are indicated in each panel. Statistical analysis was performed using a two-tailed t-test.