Self-Organization in a Synthetic Consortium Figure 2. The consortium adopts a specific, layered structure which is associated with a growth advantage. (A) After 80 hours, the blue population remains primarily near the substrate while the yellow population forms clumps attached to the blue population, as shown in this cross- sectional projection taken at 1/3 the total height of the biofilm. (B) After 80 hours of growth, the yellow population begins to exhibit a consistently larger biomass median (yellow lines, throughout figure) than the blue population (blue lines, throughout figure), revealing that the yellow population grows further from the substrate while the blue population remains close to the substrate. (The biomass median indicates the average distance from the substrate at which cells of a given population are found.) Gray bars, plotted against the right-hand axis, indicate total biomass accumulation for the entire consortium at each time-point throughout the figure (errors throughout figure are s.d.). (C) Maximum total biomass accumulated by the downstream biofilm is double that in the initial biofilm (compare gray bars in [B] and [C]). Additionally, the downstream biofilm assumes the layered structure more quickly: the biomass medians reveal structure after 24 hours of growth. (D) When aggregates are disrupted prior to transfer, leaving all else constant, this treated effluent can form biofilms, but they never exhibit the layered structure or growth advantage. (E) The layered structure is recovered when the sorted blue-and-yellow aggregate fraction forms downstream biofilms. In fact, the consortium starts with this structure, exhibiting it by 24 hours after inoculation. Further, maximum total biomass accumulation is more than double the highest amount observed in the predecessor (illustrated in [D]), suggesting recovery of the growth advantage. (F) The single-cell fraction consists of more than 99% yellow cells, and thus neither the blue population nor the layered structure nor any growth advantage is evident in the downstream biofilm it forms. This biofilm accumulates less biomass than the biofilm formed by the aggregate fraction (compare to grey bars in [E]). (G) Here, effluent is taken from the treated biofilms, which are less productive and do not exhibit structure (illustrated in [D]). Although this effluent is left untreated, it forms initially dense, monomorphic, and primarily yellow downstream biofilms that do not exhibit layered structure. These biofilms consistently lose biomass. doi:10.1371/journal.pone.0016791.g002 We have used an engineered symbiotic microbial consortium to explore spatial self-organization and its benefits to a microbial community. Here, two symbiotic populations of E. coli grow to form a defined, layered structure which provides a growth advantage to both. This engineered consortium allowed us to observe the critical roles of self-organization, layering, and aggregation in the growth, movement, and ability of a microbial consortium to colonize new environments. The persistence assured by aggregates allows evolution and adaptation of interacting microbial communities despite environmental disruptions. It may eventually be possible to use engineered consortia like this one to determine how relationships between interacting, co-evolving populations are enhanced and preserved by particular physical structures. Materials and Methods Strains To construct the knockout strains of E. coli MG1655, we used recombination with the lambda red recombinase plasmid pKD46, as outlined in [33]. To compromise metabolism we deleted dapD to make E. coli MG1655DDapD [27]. Biofilm formation was compromised by deleting the csgAB and csgDEFG operons and the DwcaL–wza gene locus in strain AAEC191 to create E. coli MG1655Dfim, DwcaL–wza, DcsgC-csgG. Further details are found in Supporting Information S1. Plasmids The engineered plasmid in the blue population encodes constitutive production of RhlR from the p(LacIq) promoter with strong ribosome binding site RBSII, and RhlR-dependent expression of DapD from the qsc119 promoter with the weak ribosome binding site RBSH. DapD was expressed with an LVA degradation tag (DapD-LVA). The engineered plasmid in the yellow population encodes very strong constitutive expression of RhlI from the strong promoter p(JM2300) coupled with RBSII. More information is available in Supporting Information S1. Growth conditions Throughout all experiments, cultures and biofilms were grown at 30uC in M9-AADO (Amino Acid Drop Out) medium without lysine, containing 50 mg ml21 kanamycin and 20 mg ml21 tetracycline to maintain the engineered and the marker plasmids, respectively [34]. M9-AADO (per liter). 200 mL 5xM9, 100 mL 10x AADO Solution without lysine, 2 mM MgSO4, 0.5% glycerol, 0.01% thymine. 5x M9 (per liter). 18 g anhydrous Na2HPO4, 15g KH2PO4, 5g NH4Cl, 2.5g NaCl. 10x Amino Acid Drop Out Solution (without Lysine, per liter). 300 mg l-isoleucine; 1500 mg l-valine; 200 mg l-adenine hemisulfate salt; 200 mg l-arginine HCl; 200 mg l-histidine HCl monohydrate; 1000 mg l-leucine; 200 mg l-methionine; 500 mg l- phenylalanine; 2000 mg l-threonine; 200 mg l-tryptophan; 300 mg l-tyrosine; 200 mg l-uracil. Biofilm Experiments The biofilm flow apparatus was described in detail previously [35], and additional details are found in Supporting information S6. To begin initial biofilms, separate overnight cultures of blue and yellow populations were shaken, in M9-AADO medium with antibiotics as detailed above, to saturation. Cultures of the blue population were supplemented with 10 mM C4HSL (Sigma, O9945). Cultures were centrifuged at 4000 RPM for 8 minutes, cells were resuspended in 1 mL 0.9% NaCl solution containing the same antibiotics, then diluted into 0.9% NaCl solution with the antibiotics to an OD600 of 0.07, which corresponds to approxi- mately 46107 cells mL21. 1 mL of a 50/50 mixture of blue and yellow cells was inoculated into each flow lane for experimental replicates. Control biofilms were started with a 50/50 mixture of blue or yellow cells and 0.9% NaCl solution. Prior to inoculation each 164640 mm lane of each flow chamber (Stovall Life Sciences, ACFL0001) was incubated for at least 90 minutes at 37uC with 200 mL of a solution of 10 mg mL21 bovine ribonuclease B (Sigma, R7884) suspended in 0.02 M bicarbonate buffer. Each lane was then quenched with 200 mL of 0.2% bovine serum albumin (Sigma, A4503). Flow of M9-AADO with antibiotics through the flow chambers was initiated for five minutes prior to inoculation. After inoculation, flow chambers were incubated glass- coverslip-down for 4 minutes, and then flow was reinstated for 4 minutes prior to returning the flow chambers to the upright position. The flow rate of medium through each lane was approximately 230 mL min21, and flow cells were incubated at 30uC62uC throughout the length of each experiment. Medium reservoirs were replaced every 12 hours to ensure freshness of the antibiotics. Treated and Untreated Biofilms To begin both treated and untreated downstream biofilms, effluents from three separate replicates (in separate lanes) of the type of biofilm to be propagated were mixed. This mixture was divided into treated and untreated cases. In the untreated case, OD600 was adjusted to 0.07 as necessary, and 1 mL was inoculated into each fresh flow lane. In the treated case, the effluent was vortexed for 5 minutes and then passed through a PLoS ONE | www.plosone.org 5 February 2011 | Volume 6 | Issue 2 | e16791