The biofilm matrix and its functional role in the ecology of bacterial communities

Project Information

BioMatrix

Grant agreement ID: 101002208

DOI

10.3030/101002208

EC signature date 11 March 2021

Start date 1 September 2021

End date 28 February 2027

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 978 602,00

EU contribution

€ 1 978 602,00

Coordinated by

KOBENHAVNS UNIVERSITET
Denmark

Periodic Reporting for period 3 - BioMatrix (The biofilm matrix and its functional role in the ecology of bacterial communities)

Reporting period: 2024-09-01 to 2026-02-28

Summary of the context and overall objectives of the project

Bacteria in nature live in biofilms, which are assemblages of bacteria embedded in a matrix, produced by the bacteria themselves. Biofilms are found everywhere: on plant roots, on decomposing organic material, in aquatic sediments and much more. Biofilms are useful in biotechnological applications, but they are also problematic, as they form at unwanted sites in industrial settings and can lead to chronic infections in humans. This is mainly because biofilm associated bacteria resist cleaning regimes, attach strongly to surfaces to resist removal and have enhanced tolerance towards various stressors including antibiotics. When different types of bacteria live in proximity in a biofilm, we often identify features of this microbial community, which neither of the bacteria has when forming biofilm individually; examples include induced biomass production, enhanced tolerance towards stress and inhibitors, and new metabolic capabilities. The interactions and mechanisms causing such “community properties” are largely unknown, but we hypothesize that the matrix play a fundamental role in shaping and facilitating these interactions.

The overall objective of BioMatrix is to evaluate the functional and ecological impact of the matrix of biofilms comprised of multiple types of bacteria. We aim at identifying matrix components that directly impact functionality and/or tolerance of biofilms in nature. New experimental models will be designed to better mimic the real life of biofilm bacteria in nature. BioMatrix will uncover fundamental mechanisms shaping bacterial communities and thereby close a primary knowledge gap in microbial ecology. Moreover, specific targets, facilitating efficient biofilm manipulation, are provided. This is useful in biotechnology for enhanced exploitation of bacterial community activity and in the development of novel and improved strategies for controlling and preventing unwanted and detrimental biofilm formation. Hopefully, such strategies will lead to better treatment of chronic biofilm infections and more efficient control of harmful biofilms in industry.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The experimental foundation of the research conducted in BioMatrix is a bacterial model community composed of four species originally isolated from soil: the Gram-negative Stenotrophomonas rhizophila and Xanthomonas retroflexus, and the Gram-positive Microbacterium oxydans and Paenibacillus amylolyticus. When combined, these four strains interact to produce more biofilm biomass, have higher tolerance to chemical inhibitors and predation, and gain new functionalities. BioMatrix explores the impact of the biofilm matrix components for facilitating these community intrinsic properties.
Initially, we characterized the biofilm matrix of single species and the community, using (i) specific fluorescent stains/compounds combined with microscopic and (ii) proteomic analysis of the matrix proteome. We have identified biofilm matrix structures uniquely present in either individual species or in the community, confirming the hypothesis that bacterial interactions shape biofilm matrix structure and composition. Based on this, and analysis of the bacterial genomes, we have identified genes potentially encoding matrix components. We are currently genetically engineering the strains, enabling the study of expression of these genes in mono vs multispecies biofilms, and over time. Moreover, we will delete selected genes from the genomes to assess the functional consequences imposed on the bacteria by lacking specific matrix components. To date, to such mutants have been constructed, one lacking the ability to express structural matrix proteins, amyloid fibres, and one that does not produce flagella.
For assessments of bacterial activities when forming biofilms individually, with partners and when lacking matrix components, several community intrinsic properties have been identified. We have shown that expression of some enzymes is strongly induced in the multispecies biofilm setting, including xylanase, catalysing degradation of the hemicellulose component xylan to the simple sugar xylose. We have also show that, in contrast to individual species, the community stimulates plant root development and moves on surfaces (also referred to as swarming). These and other community intrinsic properties will be assessed upon replacement of type strains with their matrix mutant counterparts.
We have developed methodology for 3D printing of artificial leaves, optimised for studies of biofilms in settings mimicking their natural environments and compatible with confocal microscopy.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

From our matrix characterizations, we have identified matrix components uniquely present in either mono or multispecies biofilms including specific hydrocarbons and proteins involved in bacterial motility (flagella components). This shows that the mono and multispecies biofilm matrices differ, which may underlie the community intrinsic properties observed. We are currently testing this hypothesis using our genetically engineered strains deficient in expression of specific matrix components.
We have identified novel community intrinsic properties, including plant stimulation and swarming motility that occurs only in the community setting. This is of applied interests, as plant promoting bacteria are traditionally applied as single strains. Our results show that bacterial effects on plants are restricted to the community context. In ecological perspectives, results identify a keystone species shaping community function but only in the presence of other strains. We are currently evaluating potential advantages of individual strains gained from participating in collective surface movement (swarming) and we expect that exciting novel findings will arise.
Methodology for 3D printing of biodegradable, artificial leaves was developed, with the overall aim to analyze biofilm communities in conditions mimicking their natural habitats. The leaves are printed in carbon sources representing those of leaves and can be manipulated. They are also compatible with microscopy, so we can study bacterial interactions in biofilms directly on the leaves. Combined with artificial, sterile soil and non-sterile soil, not only can we study biofilm formation and interactions of our model strains, but also their interactions with the indigenous soil community, including other microbes, predators etc., and the impact of specific matrix components.