mbo382-sup-0001-movieS1.movvideo/mov14615KMovie 1. Cell aggregate of the ΔEmbRS mutant: a polymeric material surrounds the cells in the aggregate and serves as a physical barrier against the entry of the free cells in the polymeric aggregate. Transmitted light image was acquired after 2 h crystal violet stain. The movie represents 2 min of acquisition of the same culture.
mbo382-sup-0002-movieS2.movvideo/mov10593KMovie 2. Biofilm formation by the ΔEmbRS mutant. Formation of the bacterial veil around the toothpick scaffold. Cells start growing in the whole plate; then the shrinking of the biofilm occurs and stops when the veil comes in contact with the top of the toothpicks, generating an organized polygonal network (Fig. 3).

Figure S1. Autoaggregation ability of the wild type and the ΔEmbRS null mutant. Cells were grown photosynthetically. The percentage of autoaggregation is expressed as: [(OD total- OD in suspension) ×100/OD total].

Figure S2. Crystal violet staining of ΔEmbRS cells form aggregate. The polymeric material delimited by * can be visualized in the movie M1.

Figure S3. Successive images of the biofilm formation around the toothpick scaffold. For more details see movie M2.

Figure S4. Examples of EmbRS-Tn5 mutants isolated in this study. (A) Mutants have recovered a partial or quasi-smooth phenotype compared to the parental ΔEmbRS null mutant on plates. (B) Photosynthetic liquid cultures of WT and ΔEmbRS compared with ΔEmbRS-GltA::Tn5, ΔEmbRS-Stik::Tn5 and to ΔEmbRS-BmfS::Tn5, transposon mutants.

Figure S5. (A) Organization of the genes encoding the BmfSR two-component system and the predicted domains within the histidine sensor kinase BmfS and the transcriptional regulator BmfR (Smart at EMBL). (B) Sequence alignment between EmbR and BmfR transcriptional regulators.

Figure S6. Putative regulatory model of the biofilm formation and autoaggregation in Rubrivivax gelatinosus based on the genetic and molecular data in this study. EmbRS functions as a negative two-component system that control genes (cpsG, wcbM…) involved in the synthesis of exopolysaccharides (extracellular matrix). BmfSR is a second two-component system also involved in biofilm formation. However, BmfSR should activate the expression of some genes involved in the synthesis of exopolysaccharides. The model includes also BdcA, a putative diguanylate cyclase, that may control exopolysaccharides and biofilm formation in R. gelatinosus.

Figure S7. Biofilm formation and sinking of the wild-type strain occur in the late stationary growth phase probably as a result of nutrient-limited environment. This event is accelerated in the ΔEmbRS mutant.

Figure S8. Putative role of EmbRS in the evolution of photosynthesis in Rubrivivax gelatinosus. Acquisition of embRS either concomitantly to the PGC (scenario 1) or after the PGC acquisition (scenario 2) may have allowed cells to migrate to the euphotic zone and to set up photosynthesis.

mbo382-sup-0005-TableS1.pdfapplication/PDF101KTable S1. Strains and plasmids.
mbo382-sup-0006-TableS2.pdfapplication/PDF12KTable S2. Primers.

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