File S1 - Pseudomonas aeruginosa Promotes Escherichia coli Biofilm Formation in Nutrient-Limited Medium

Figures S1–S7. Figure S1. Mean population density of E. coli and P. aeruginosa grown in R2A medium over a 3-day period at 37°C. Both populations remained steady for the duration of the experiment, with P. aeruginosa exhibiting a higher population concentration. Figure S2. Mean population density of E. coli and P. aeruginosa grown in R2A medium over a 3-day period at 24°C. Both populations remained relatively steady for the duration of the experiment, with P. aeruginosa exhibiting a higher population concentration. Figure S3. Co-development of 3-day old E. coli and P. aeruginosa biofilms under varying R2A medium concentrations in a microfluidic flow cell. Column: A) 3-day old co-inoculated P. aeruginosa – E. coli biofilms with under 4x and 8x R2A concentrations (grid unit is 23.8 µm). B) Horizontal section near the base of the biofilm and vertical sections of the biofilm shown in panel A (scale bar = 40 µm). C) Biomass fraction vs. biofilm height graph. Biofilms were counter-stained by SYTO 62. E. coli appears red and P. aeruginosa-GFP appears green. Figure S4. Average biofilm biomass vs. R2A concentration for co-development of 3-day old E. coli and P. aeruginosa biofilms. Figure S5. Co-development of 3-day old E. coli and P. aeruginosa biofilms under a controlled flow gradient in a planar flow cell and in R2A medium. Results are presented for two local fluid velocities, 0.96 mm/s (top row) and 1.69 mm/s (bottom row). Column: A) 3-day old co-inoculated P. aeruginosa – E. coli biofilms (grid unit is 23.8 µm). B) Horizontal section near the base of the biofilm and vertical sections of the biofilm shown in panel A (scale bar = 20 µm). C) Distribution of P. aerugiosa and E. coli biomass as function of height, indicating that E. coli was the dominant species throughout a majority of the biofilm. E. coli appears red and P. aeruginosa-GFP appears green. Figure S6. Biofilm biomass on day 3 for the co-development of P. aeruginosa and E. coli biofilms subjected to different local velocities in R2A medium. Figure S7. Colonization of E. coli BW25113 biofilms by P. aeruginosa in R2A medium. E. coli appears red and P. aeruginosa-GFP appears green or yellow. A) After 3-days of mono-species growth, E. coli formed sparse biofilms composed of small, isolated cell clusters (grid unit is 23.8 µm). B) Mixed P. aeruginosa and E. coli biofilm on day 6, after 3 days of mono-species E. coli growth plus 3 additional days of multi-species growth after inoculation of P. aeruginosa. (grid unit in B is 23.8 µm). Following introduction of P. aeruginosa, E. coli grew prolifically and adopted a configuration similar to that observed in other mixed-species experiments. C) Horizontal section near the base of the biofilm and vertical sections of the biofilm shown in panel B (scale bar = 40 µm). (DOCX)

补充图S1~S7。
补充图S1：37℃下于R2A培养基中培养3天的大肠杆菌（E. coli）与铜绿假单胞菌（P. aeruginosa）的平均种群密度。实验全程两种菌群的种群密度均保持稳定，且铜绿假单胞菌的种群浓度更高。
补充图S2：24℃下于R2A培养基中培养3天的大肠杆菌与铜绿假单胞菌的平均种群密度。实验全程两种菌群的种群密度均相对稳定，且铜绿假单胞菌的种群浓度更高。
补充图S3：微流体流动池内不同R2A培养基浓度下，3日龄大肠杆菌与铜绿假单胞菌生物膜的共发育情况。列示内容如下：A）4倍、8倍R2A浓度下共接种的大肠杆菌-铜绿假单胞菌3日龄生物膜（网格单位为23.8 μm）。B）A组生物膜底部附近的水平切面与该生物膜的垂直切面（比例尺=40 μm）。C）生物量占比随生物膜高度变化的图表。生物膜经SYTO 62复染，大肠杆菌呈红色，绿色荧光标记的铜绿假单胞菌（P. aeruginosa-GFP）呈绿色。
补充图S4：3日龄大肠杆菌与铜绿假单胞菌共发育生物膜的平均生物膜生物量随R2A培养基浓度的变化关系。
补充图S5：平面流动池内、R2A培养基中，受控流动梯度下3日龄大肠杆菌与铜绿假单胞菌生物膜的共发育情况。结果展示了两种局部流体流速：0.96 mm/s（上排）与1.69 mm/s（下排）。列示内容如下：A）3日龄共接种的大肠杆菌-铜绿假单胞菌生物膜（网格单位为23.8 μm）。B）A组生物膜底部附近的水平切面与该生物膜的垂直切面（比例尺=20 μm）。C）大肠杆菌与铜绿假单胞菌生物量随高度的分布情况，结果显示大肠杆菌在生物膜绝大多数区域均为优势菌种。大肠杆菌呈红色，绿色荧光标记的铜绿假单胞菌呈绿色。
补充图S6：R2A培养基中施加不同局部流速时，大肠杆菌与铜绿假单胞菌共发育生物膜在第3日的生物膜生物量。
补充图S7：R2A培养基中铜绿假单胞菌定植于大肠杆菌BW25113生物膜的情况。大肠杆菌呈红色，绿色荧光标记的铜绿假单胞菌呈绿色或黄色。A）单菌种培养3天后，大肠杆菌形成由小型孤立细胞簇组成的稀疏生物膜（网格单位为23.8 μm）。B）接种铜绿假单胞菌后，先经3天单菌种大肠杆菌培养，再进行3天多菌种共培养，第6日的大肠杆菌-铜绿假单胞菌混合生物膜（B组网格单位为23.8 μm）。引入铜绿假单胞菌后，大肠杆菌大量增殖，其群落结构与其他多菌种实验中观察到的结果相似。C）B组生物膜底部附近的水平切面与该生物膜的垂直切面（比例尺=40 μm）。（DOCX）