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

www.nature.com/scientificreports OPEN Received: 30 May 2017 Accepted: 15 November 2017 Published: xx xx xxxx A meta-proteomics approach to study the interspecies interactions affecting microbial biofilm development in a model community Jakob Herschend1, Zacharias B. V. Damholt2, Andrea M. Marquard3, Birte Svensson2, Søren J. Sørensen1, Per Hägglund 2,4 & Mette Burmølle 1 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. Microbial biofilms are dynamic communities, where the architecture and community function is shaped by inter-species interactions, resulting in pH, oxygen and nutrient gradients. In natural and man-made environments, biofilms often host a plethora of different species, making it difficult to predict how these communities develop. This knowledge is however indispensable as microbial biofilms are highly associated with chronic infections1, colonization of catheters and implants2, crop pathogenicity3–5 and contamination of food products with spoilage and potential pathogenic strains6,7. Additionally, implementation of microbial communities for bioremediation8, waste-water treatment9, plant growth promotion3–5, and bio-energy10 provides environmentally friendly and low-cost substitutes to current technologies. To better harness beneficial biofilms and prevent harmful ones, there is a need to unravel the development and functionality of complex microbial communities. Importantly, knowledge of community development and functionality cannot be inferred by studying the single species constituents, as inter-species interactions severely alter the lifestyle of the community members11. In a previous study, seven different bacterial species were isolated from Danish agricultural soil and screened in combinations of four for synergistic biofilm formation12. In more than half of the assembled communities 1 Section of Microbiology, Department of Biology, University of Copenhagen, Copenhagen, Denmark. 2Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, Denmark. 3Section for Immunology and Vaccinology, National Veterinary Institute, Technical University of Denmark, Lyngby, Denmark. 4Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark. Jakob Herschend and Zacharias B. V. Damholt contributed equally to this work. Correspondence and requests for materials should be addressed to M.B. (email: ) SCIeNTIfIC REPOrtS | 7: 16483 | DOI:10.1038/s41598-017-16633-6 1 www.nature.com/scientificreports/ Figure 1. Overview of experimental design. Biofilm was cultivated in a drip flow reactor for 48 hrs and single and four-species biofilm was scraped off the glass slide. Proteins were extracted sonication, digested with trypsin and the resulting peptides were separated into three fractions using SDB-RPS filter tips. Mass spectrometric analysis was performed on a Q-Exactive and data was analyzed using MaxQuant with label free quantification. inter-species interactions facilitated synergistic biofilm formation, compared to the best performing single species. One combination, composed of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus, showed a 4–5 fold increase in biofilm formation during co-cultivation. All species in the consortium were indispensable for the synergy and all increased in cell counts, compared to the single species biofilm13. A subsequent study indicated amino acid complementation as a potential driver behind the synergistic biofilm formation, as Xanthomonas uniquely altered its metabolism of different amino acids when co-cultured in the four-species consortium, compared to both mono and dual species biofilm14. Another study showed that the community synergy was not limited to batch cultivation, but could also be identified in continuous flow systems such as a drip flow reactor. The study also showed that the synergy was linked to a unique spatial localization of the four individual species in the four species biofilm15. Meta-proteomics is an emerging technology within microbiology and several studies have successfully used the technique to describe microbial communities growing as model biofilms; including acid mine drainage16,17 and oral infection model biofilms18,19. The functional characterization of biofilm community by meta-proteomics can provide valuable information about the mechanisms driving community development and structure20, e.g. by providing information on how participating members cooperate and compete for nutrients and other resources21,22, and how metabolic activities are distributed between community members. For example a study applying meta-proteomics showed that elevated temperatures caused a community and genus level response in an acid mine drainage biofilm, as elevated temperatures increased the abundance of proteins involved in amino acid metabolism from members of the Leptospirillum group II 5-way, UBA genotypes and group III17. One of the challenges of studying complex microbial communities with current omics technologies is obtaining sufficient sampling depth and taxonomic resolution between related organisms to describe the community interactions and metabolic activity on a species and preferably strain level. This becomes even more problematic when community functionality is influenced by the low abundant community members15,23,24. In the present study, we perform meta-proteomics profiling of a microbial consortium with the aim of elaborating on the inter-species interactions aff (...truncated)