Exploring the Impact of Microcystin on Interspecies Interactions between Microcystis aeruginosa and Associated Heterotrophic Bacteria

Bacteria inhabit diverse of environments, where multitudes of different species coexist and form complex microbiomes. Within these microbiome communities a delicate interplay of beneficial and harmful interactions occurs among their microbial members. These interactions frequently appear in the form of nutrient exchange, but certain metabolites also function as chemical signals or modulators of microbial metabolism. Particularly relevant examples of such microbial interactions are found between phototrophic and heterotrophic microorganisms. The cyanobacterium Microcystis is a prominent representative of phototrophic bacteria, that forms complex colonies in which a diverse and specific microbiome, the so-called phycosphere, is enriched. Microcystis exhibits remarkable genotypic and phenotypic plasticity and produces a variety of specialized metabolites encoded by biosynthesis gene clusters (BGCs). These BGCs are only sporadically present in the genome, leading to a ubiquitous coexistence of producing and non-producing strains. Among these specialized metabolites, the hepatotoxin microcystin (MC) is of particular ecological and toxicological relevance. While the effects of MC on eukaryotes are well-studied, its function in the prokaryotic, microbial community context is only partially understood. An initial screening of the composition of single Microcystis colony phycospheres revealed that MC-producing colonies display reduced bacterial richness compared to non-producing ones, with specific taxa preferentially associating with either genotype. To further explore these relationships, synthetic community (SynCom) experiments were performed comparing the MC-producing strain M. aeruginosa PCC 7806 (WT) with its non-producing ΔmcyB mutant in co-cultivation with a defined consortium of heterotrophic bacteria. Over twenty-eight days, distinct temporal dynamics between the Microcystis strains and its heterotrophic partners were observed, including ‘tug-of-war’-like fluctuations of cyanobacterial and heterotrophic bacterial relative abundances followed by a stabilization phase towards the end of the experiment. Notably, the Sphingomonas sp. UP3 strain became dominant in co-cultivation with the M. aeruginosa WT, while Flavobacterium sp. UP2 was enriched in co-cultivation with the MC-deficient ΔmcyB mutant. The Agrobacterium sp. UP1 strain, however, reached high abundances in co-cultivation with both genotypes. Metabolic pathway completeness estimation, based on whole genome sequence analysis, revealed complementary metabolic capabilities between the heterotrophic bacteria and Microcystis, indicating nutrient exchange (cross-feeding) as a central interaction mechanism. In addition, substrate utilization assays demonstrated an enhanced degradation of polymeric structures (e.g. Tween-40, Tween-80) in the Sphingomonas sp. UP3 strain, particularly the presence of M. aeruginosa WT exudates, while Agrobacterium sp. UP1 exhibited broad substrate utilization regardless of the origin of the exudates. Transcriptomic analyses further revealed that Microcystis responds in an MC-dependent manner to the presence of heterotrophic bacteria. The MC-producing strain showed reduced expression of nitrate transporters, while simultaneously exhibiting increased expression of transporters for the import of cofactors and the export of amino acids, proteins, and lipopolysaccharides (LPS), compared to the non-producing mutant. Additionally, marked alterations in the expression of genes related to the photosynthetic apparatus were observed, accompanied by a reallocation of metabolic resources from chlorophyll a to cobalamin biosynthesis. These findings suggest that MC influences resource allocation, cell surface composition, and interspecific nutrient exchange of Microcystis. Complementary experiments with the Planctomycete Planctopirus limnophila revealed a potentially harmful interaction characterized by reduced growth of the ΔmcyB mutant, increased extracellular MC levels, and altered cell surface morphologies, particularly in M. aeruginosa WT, which suggests a possible role for MC in the defense against bacterial predators. This work provides new insights into the ecological functions of MC. It shows that MC shapes the phycosphere composition in a selective manner, modulates cross-feeding interactions, and possibly enhances the resilience of producing strains under competitive or stressful conditions. The results show that the multifaceted functions of MC extend way beyond its toxic effects and highlight its impact as a chemical mediator in the assembly and dynamics of microbial communities.