Experimental design

Our experiment comprised six treatments, each consisting of 10 independent replicate microcosm communities: (1) a control treatment with all species present and containing B. subtilis WT; (2) removal of the apex predator Didinium; (3) removal of B. subtilis; (4) all species present with replacement of B. subtilis WT with the ΔsinI mutant, reducing biofilm production; (5) all species present with replacement of B. subtilis WT with the ΔsinR mutant, increasing biofilm production; (6) all species present with replacement of B. subtilis WT with the ΔphoA mutant, as a procedural control.

Community assembly

Culture methods followed closely those of in refs 48, 49. Microcosms consisted of loosely capped 200-ml glass bottles with 15-g glass microbeads providing habitat structure. Each microcosm received 100 ml medium consisting of one protist pellet (Carolina Biological Supply, Burlington, NC, USA) per 1-l spring water and two wheat seeds to provide a slow-release nutrient source. All media were sterilized before use. Microcosms were maintained at 22 °C and under a 12:12-h light:dark cycle. Nutrients in the microcosms were replenished with weekly replacement of 7 ml of the microcosm volume with sterile medium and one additional sterile wheat seed. Paramecium and Colpidium species, generalist consumers with similar interaction strengths with each of the inoculated bacteria species (Fig. 1 and Supplementary Fig. 1; (refs 50, 51, 52, 53, 54, 55, 56, 57); MANOVA, Pillai’s Trace=0.05, F 2,8 =2.5, P =0.14 (see Primary consumer experiment subsection below)), were obtained from Carolina Biological Supply (Burlington, NC, USA). Didinium were obtained from Sciento (Manchester, UK). Sources and strains of all bacterial strains used are listed in Supplementary Table 2.

Overnight cultures of strains NCIB3610 (WT), LSB369 (sinI), LSB370 (sinR), LSB377 (phoA) and S. marcescens ATCC 29632 grown in TY medium (Luria–Bertani (LB) broth supplemented with 10 mM MgSO 4 and 100 μM MnSO 4 after autoclaving58 plus 100 μg ml−1 spectinomycin when appropriate), were diluted into fresh TY medium at OD 600 (optical density) ∼0.03 and grown at 37 °C until late exponential phase (OD 600 ∼1.0), at which time 1 ml of each bacterial culture was inoculated into 100-ml microcosm medium, according to the experimental set-up. B. megaterium (ATCC 19213) was also added but failed to establish in any microcosms.

Microcosms were then left for 48 h at 37 °C to facilitate growth of the bacteria and ensure sufficient numbers before the addition of the bacterivorous protists. Microcosms were inoculated with six pipette drops of stock cultures of protozoans: P. caudatum, P. aurelia and Colpidium (∼50–70 individuals of each species) and allowed to settle for 1 week at 22 °C. Predators (Didinium) were added (∼10 individuals) to appropriate microcosms 7 days after the addition of the bacterivores. The consumer protist cultures used for this experiment were laboratory cultures and therefore, while not inoculated with any bacterial populations, not entirely sterile; therefore, cultures were mixed thoroughly before addition of consumers to ensure that any bacteria present in the media had an equal chance of colonizing each microcosm. Both Klebsiella and Aeromonas colonized all microcosm units in this manner.

The point of addition of the predators is considered as Day 0 of the experiment with measurements taking place on Day 14. Samples were also taken on Day 7 to check for persistence of species and the presence of contaminants. As bacterial sampling is destructive, requiring vortexing of microcosms to strip biofilm and reduce within-mesocosm variability, only data from Day 14 are used in analyses. Pilot experiments, using the mutant- and predator-removed treatments, found that this period was sufficient for all organisms in the community to reach equilibrium densities, and a consistent difference between the treatments and control show that the effect was not transient (Supplementary Fig. 2). Protists were sampled by gently swirling the microcosms to homogenize contents and to suspend the protists, and up to 1-ml sample was examined using stereo (Olympus SZX9) and compound (Olympus BX60) microscopes. Rare species were counted in the entire sample and more numerous species were counted in appropriately diluted subsamples. Bacterial density was measured through direct colony counts on plates (of nutrient agar for all species and LB supplemented with spectinomycin for easy quantification of sinI, sinR and phoA strains) from appropriately diluted samples.

Bacterial mutant strain construction

Chromosomal deletions were first created in the 168 background. Strains LSB362, LSB363 and LSB368 were generated using an adaptation of long flanking homology PCR. The protocol is modified from the published procedure58. In brief, the spc gene (encoding for spectinomycin resistance) was amplified from plasmid template pDG1726 (ref. 59) using primers spc fwd/spc rev (Supplementary Table 3). Two primer pairs were designed to amplify ∼750-bp DNA fragments flanking the region to be deleted at its 5′ and 3′ ends using Phusion Polymerase. These fragments contained ∼25-bp homologous to the spc cassette. Primer pairs sinI up fwd/sinI up rev (spc) and sinI do fwd (spc)/sinI do rev were used for strain LSB362; sinI up fwd/sinR up rev (spc) and sinR do fwd (spc)/sinR do rev were used for strain LSB363; phoA up fwd/phoA up rev (spc) and phoA do fwd (spc)/phoA do rev were used for strain LSB368. Overall, 150–200 ng of the flanking fragments and 250–300 ng of the resistance cassette were joined together using the Expand Long Template PCR System (Roche) and the specific up fwd and down rev primers. The resulting PCR product was used to transform B. subtilis 168. Transformants were screened by direct colony PCR, using the up-forward primer with a reverse primer annealing inside the spc resistance cassette.

Mutations in the NCIB3610 background (strains LSB369, LSB370 and LSB377) were created by SPP1-mediated transduction from strains LSB362, LSB363 and LSB368, respectively, as described previously60. Transductants were screened by direct colony PCR, using the up-forward primer with a reverse primer annealing inside the spc resistance cassette. The resulting strains (LSB369, LSB370 and LSB377) were verified using diagnostic PCR and DNA sequencing.

Pellicle (biofilm)/colony morphology assays

Strains 168, NCIB3610, LSB369 (ΔsinI::spc), LSB370 (ΔsinR::spc) and LSB377 (ΔphoA::spc) were cultivated in LB medium61 until mid-exponential growth (OD 600 ∼0.5) at which time 10 μl of each culture was inoculated into 10 ml of MSgg medium62 in six-well plates, which were then incubated at room temperature (pellicle assay), or 5 μl was spotted on a 1.5% Agar MSgg plate that was initially incubated overnight at 37 oC and later kept at room temperature (colony morphology).

Primary consumer experiment

We established a subsidiary experiment to test whether the individual effects of the three primary consumer species used in our experiment on bacterial community structure varied either among each other or among communities containing the different B. subtilis phenotypes (WT, ΔsinI and ΔsinR). The experiments were performed in 64-well plates in 2-ml cells. Each cell contained sterile protist medium (identical to that used in the microcosms) and 100 μl of Klebsiella, Serratia and Aeromonas strains. To this was added 100 μl of one of the three B. subtilis phenotypes used in the experiment. Plates were incubated for 24 h, after which time five individuals of either P. caudatum, P. aurelia or Colpidium were added to cells. Each treatment was replicated three times. Control plates with no consumers were also included for comparison and to enable quantification of interaction strengths between each primary consumer species and their bacterial prey. After 2 days, the bacteria were enumerated on nutrient agar from appropriately diluted subsamples.

Statistical analyses

We used MANOVA to test whether our treatments significantly affected the structure of the microcosm communities. All abundances were log-transformed and mean-standardized (expressed in units of s.d.) before analysis. To assess the extent of change in community composition among treatments, we used a permutation test (with 104 permutations) to test for differences in the location of community centroids between each treatment and the control. We then examined differences in the relative magnitudes of these changes in community composition by bootstrapping, using 104 samples of the data taken with replacement. We tested for correlations in species abundances with the axis of biofilm formation phenotype using Spearman’s rank correlation tests. Total interaction strengths between each bacterial species and each of the three primary consumers used in our experiment were quantified as the natural logarithm of the ratio of untransformed densities of each bacterial species in each microcosm containing each primary consumer species in isolation to their mean untransformed density in control microcosms without primary consumers63.