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Microbial biofilms are omnipresent in nature and relevant to a broad spectrum of industries ranging from bioremediation and food production to biomedical applications. To date little is understood about how multi-species biofilm communities develop and function on a molecular level, due to the complexity of these biological systems. Here we apply a meta-proteomics approach to investigate the mechanisms influencing biofilm formation in a model consortium of four bacterial soil isolates;  Stenotrophomonas rhizophila ,  Xanthomonas retroflexus ,  Microbacterium oxydans  and  Paenibacillus amylolyticus . Protein abundances in community and single species biofilms were compared to describe occurring inter-species interactions and the resulting changes in active metabolic pathways. To obtain full taxonomic resolution between closely related species and empower correct protein quantification, we developed a novel pipeline for generating reduced reference proteomes for spectral database searches. Meta-proteomics profiling indicated that community development is dependent on cooperative interactions between community members facilitating cross-feeding on specific amino acids. Opposite regulation patterns of fermentation and nitrogen pathways in  Paenibacillus amylolyticus  and  Xanthomonas retroflexus  may, however, indicate that competition for limited resources also affects community development. Overall our results demonstrate the multitude of pathways involved in biofilm formation in mixed communities.

A meta-proteomics approach to study the interspecies interactions affecting microbial biofilm development in a model community