Heiko T. Kiesewalter

Bacillus subtilis is ubiquitously and broadly distributed in various environments but is mostly isolated from soil. Given that B. subtilis is known as a plant growth‐promoting rhizobacterium in agriculture, we aimed to describe the natural distribution of this species and uncover how biotic and abiotic factors affect its distribution. When comparing different soils, we discovered that B. subtilis group species are most abundant in grasslands but can rarely be isolated from forest soil, even if the soil sample sites are situated in proximity. Differential analysis revealed that spore‐forming bacteria exhibited enrichments in the grassland, suggesting niche overlap or synergistic interactions leading to the proliferation of certain Bacillus species in grassland environments. Network analysis further revealed that Bacillus and other Bacillota established a densely interconnected hub module in the grassland, characterised by positive associations indicating co‐occurrence, a pattern not observed in the forest soil. Speculating that this difference was driven by abiotic factors, we combined amplicon sequencing with physico‐chemical analysis of soil samples and found multiple chemical variables, mainly pH, to affect microbial composition. Our study pinpoints the factors that influence B. subtilis abundance in natural soils and, therefore, offers insights for designing B. subtilis ‐based biocontrol products in agricultural settings.

Bacillus subtilis is ubiquitously and broadly distributed in various environments but mostly isolated from soil. Given that species of B. subtilis are known as key plant growth-promoting rhizobacteria in agriculture, we here aimed to describe the natural distribution of this species and uncover how biotic and abiotic factors affect its distribution. When comparing different soils, we discovered that B. subtilis is most abundant in grasslands, but can rarely be isolated from forest soil, even if the sample sites for the two types of soil are situated in proximity. Differential analysis revealed that spore-forming bacteria exhibited enrichments in the grassland, suggesting niche overlap or synergistic interactions leading to the proliferation of certain Bacillus species in grassland environments. Network analysis further revealed that Bacillus and other Bacillota established a densely interconnected hub module in the grassland soil samples, characterized by positive associations indicating co-occurrence, a pattern not observed in the forest soil. Speculating that this difference was driven by abiotic factors, we next combined amplicon sequencing with physio-chemical analysis of soil samples, and found multiple chemical variables, mainly pH, to affect microbial composition. Our study pinpoints the factors that influence B. subtilis abundance in natural soils and, therefore, offers insights for designing B. subtilis -based biocontrol products in agricultural settings.

The majority of ecological, industrial and medical impacts of bacteria result from diverse communities containing multiple species. This diversity presents a significant challenge as co-cultivation of multiple bacterial species frequently leads to species being outcompeted and, with this, the possibility to manipulate, evolve and improve bacterial communities is lost. Ecological theory predicts that a solution to this problem will be to grow species in structured environments, which reduces the likelihood of competitive exclusion. Here, we explored the ability of cultivation in a structured environment to facilitate coexistence, evolution, and adaptation in an industrially important community:Lactococcus lactisandLeuconostoc mesenteroidesfrequently used as dairy starter cultures. As commonly occurs, passaging of these two species together in a liquid culture model led to the loss of one species in 6 of 20 lineages (30%). By contrast, when we co-cultured the two species as biofilms on beads, a stable coexistence was observed in all lineages studied for over 100 generations. Moreover, we show that the co-culture drove evolution of new high-yield variants, which compared to the ancestor grew more slowly, yielded more cells and had enhanced capability of biofilm formation. Importantly, we also show that these high-yield biofilm strains did not evolve when each species was passaged in monoculture in the biofilm model. Therefore, both co-culture and the biofilm model were conditional for these high-yield strains to evolve. Our study underlines the power of ecological thinking—namely, the importance of structured environments for coexistence—to facilitate cultivation, evolution, and adaptation of industrially important bacterial communities.

Secondary or specialized metabolites with antimicrobial activities define the biocontrol properties of microorganisms. Members of the Bacillus genus produce a plethora of secondary metabolites, of which nonribosomally produced lipopeptides in particular display strong antifungal activity.

Secondary metabolites provide Bacillus subtilis with increased competitiveness towards other microorganisms. In particular, nonribosomal peptides (NRPs) have an enormous antimicrobial potential by causing cell lysis, perforation of fungal membranes, enzyme inhibition, or disruption of bacterial protein synthesis. This knowledge was primarily acquired in vitro when B. subtilis was competing with other microbial monocultures. However, our understanding of the true ecological role of these small molecules is limited. In this study, we have established soil-derived semisynthetic mock communities containing 13 main genera and supplemented them with B. subtilis P5_B1 WT, the NRP-deficient strain sfp, or single-NRP mutants incapable of producing surfactin, plipastatin, or bacillaene. Through 16S amplicon sequencing, it was revealed that the invasion of NRP-producing B. subtilis strains had no major impact on the bacterial communities. Still, the abundance of the two genera Lysinibacillus and Viridibacillus was reduced. Interestingly, this effect was diminished in communities supplemented with the NRP-deficient strain. Growth profiling of Lysinibacillus fusiformis M5 exposed to either spent media of the B. subtilis strains or pure surfactin indicated the sensitivity of this strain towards the biosurfactant surfactin. Our study provides a more in-depth insight into the influence of B. subtilis NRPs on semisynthetic bacterial communities and helps to understand their ecological role.

Secondary metabolites provide Bacillus subtilis with increased competitiveness towards other microorganisms. In particular nonribosomal peptides (NRPs) have an enormous antimicrobial potential by causing cell lysis, perforation of fungal membranes, enzyme inhibition or disruption of bacterial protein synthesis. This knowledge was primarily acquired in vitro when B. subtilis was competing with other microbial monocultures. However, our understanding of the true ecological role of these small molecules is limited. In this study, we have established soil-derived semi-synthetic mock communities containing 13 main genera and supplemented them with B. subtilis P5_B1 WT, its NRP deficient strain sfp or single NRP mutants incapable of producing surfactin, plipastatin or bacillaene. 16S amplicon sequencing revealed that the invasion of NRP-producing B. subtilis strains had no major impact on the bacterial communities. Still, the abundances of the two genera Lysinibacillus and Viridibacillus were reduced. Interestingly, this effect was diminished in communities supplemented with the NRPs deficient strain. Growth profiling of Lysinibacillus fusiformis M5 exposed to either spent media of the B. subtilis strains or pure surfactin indicates the sensitivity of this strain towards the biosurfactant surfactin. Our study provides a more in-depth insight into the influence of B. subtilis NRPs on semi-synthetic bacterial communities and helps to understand their ecological role.

Bacillus subtilis produces a wide range of secondary metabolites providing diverse plant-growth-promoting and biocontrol abilities. These secondary metabolites include non-ribosomal peptides (NRPs) with strong antimicrobial properties, causing either cell lysis, pore formation in fungal membranes, inhibition of certain enzymes, or bacterial protein synthesis. However, the natural products of B. subtilis are mostly studied either in laboratory strains or in individual isolates and therefore, a comparative overview of B. subtilis secondary metabolites is missing. In this study, we have isolated 23 B. subtilis strains from eleven sampling sites, compared the fungal inhibition profiles of wild types and their NRPs mutants, followed the production of targeted lipopeptides, and determined the complete genomes of 13 soil isolates. We discovered that non-ribosomal peptide production varied among B. subtilis strains co-isolated from the same soil samples. In vitro antagonism assays revealed that biocontrol properties depend on the targeted plant pathogenic fungus and the tested B. subtilis isolate. While plipastatin alone is sufficient to inhibit Fusarium sp., a combination of plipastatin and surfactin is required to hinder the growth of Botrytis cinerea . Detailed genomic analysis revealed that altered NRP production profiles in certain isolates is due to missing core genes, nonsense mutation, or potentially altered gene regulation. Our study combines microbiological antagonism assays with chemical NRPs detection and biosynthetic gene cluster predictions in diverse B. subtilis soil isolates to provide a broader overview of the secondary metabolite chemodiversity of B. subtilis . IMPORTANCE: Secondary or specialized metabolites with antimicrobial activities define the biocontrol properties of microorganisms. Members of the Bacillus genus produce a plethora of secondary metabolites, of which non-ribosomally produced lipopeptides in particular display strong antifungal activity. To facilitate prediction of the biocontrol potential of new Bacillus subtilis isolates, we have explored the in vitro antifungal inhibitory profiles of recent B. subtilis isolates, combined with analytical natural product chemistry, mutational analysis, and detailed genome analysis of biosynthetic gene clusters. Such a comparative analysis helped to explain why selected B. subtilis isolates lack production of certain secondary metabolites.

Bacillus subtilis is a plant-benefiting soil-dwelling Gram-positive bacterium with secondary metabolite production potential. Here, we report the complete genome sequences of 13 B. subtilis strains isolated from different soil samples in Germany and Denmark.