Illuminating microbial mat assembly: Cyanobacteria and Chloroflexota cooperate to structure light-responsive biofilms

Microbial mats are stratified communities often dominated by unicellular and filamentous phototrophs within an exopolymer matrix. It is challenging to quantify the dynamic responses of community members in situ as they experience steep gradients and rapid fluctuations of light. To address this, we developed a binary consortium using two representative isolates from hot spring mats: the unicellular oxygenic phototrophic cyanobacterium Synechococcus OS-B (Syn OS-B) and the filamentous anoxygenic phototroph Chloroflexus MS-CIW-1 (Chfl MS-1). We quantified the motility of individual cells and entire colonies and demonstrated that Chfl MS-1 formed bundles of filaments that moved in all directions with no directional bias to light. Syn OS- B was slightly less motile but exhibited positive phototaxis. This binary consortium displayed cooperative behavior by moving further than either species alone and formed ordered arrays where both species aligned with the light source. No cooperative motility was observed when a non-motile pilB mutant of Syn OS-B was used instead of Syn OS-B. The binary consortium also produced more adherent biofilm than individual species, consistent with the close interspecies association revealed by electron microscopy. We propose that cyanobacteria and Chloroflexota cooperate in forming natural microbial mats, by colonizing new niches and building robust biofilms. SignificanceMicrobial mats are dense, layered communities with ancient origins and widespread occurrence, but how they assemble is not well understood. To investigate how microbial motility, physical interactions, and responses to light affect mat assembly, we developed a binary consortium from representative hot spring mat isolates. Individually, the Cyanobacteria and Chloroflexota isolates displayed significant differences in motility and biofilm formation. When combined, the consortium exhibited enhanced motility towards light and formed more robust biofilms. This model consortium approach complements in situ studies by directly testing the role of motility and physical cooperation in shaping microbial mats, and could inform biofilm applications in industrial settings.