Resistance towards and biotransformation of a Pseudomonas-produced secondary metabolite during community invasion.

The role of antagonistic secondary metabolites produced by Pseudomonas protegens in suppression of soil-borne phytopathogens has been clearly documented. However, their contribution to the ability of P. protegens to establish in soil and rhizosphere microbiomes remains less clear. Here, we use a four-species synthetic community (SynCom) in which individual members are sensitive towards key P. protegens antimicrobial metabolites (DAPG, pyoluteorin, and orfamide A) to determine how antibiotic production contributes to P. protegens community invasion and to identify community traits that counteract the antimicrobial effects. We show that P. protegens readily invades and alters the SynCom composition over time, and that P. protegens establishment requires production of DAPG and pyoluteorin. An orfamide A-deficient mutant of P. protegens invades the community as efficiently as wildtype, and both cause similar perturbations to community composition. Here, we identify the microbial interactions underlying the absence of an orfamide A mediated impact on the otherwise antibiotic-sensitive SynCom member, and show that the cyclic lipopeptide is inactivated and degraded by the combined action of Rhodococcus globerulus D757 and Stenotrophomonas indicatrix D763. Altogether, the demonstration that the synthetic community constrains P. protegens invasion by detoxifying its antibiotics may provide a mechanistic explanation to inconsistencies in biocontrol effectiveness in situ.

Enhanced surface colonisation and competition during bacterial adaptation to a fungus.

Bacterial-fungal interactions influence microbial community performance of most ecosystems and elicit specific microbial behaviours, including stimulating specialised metabolite production. Here, we use a co-culture experimental evolution approach to investigate bacterial adaptation to the presence of a fungus, using a simple model of bacterial-fungal interactions encompassing the bacterium Bacillus subtilis and the fungus Aspergillus niger. We find in one evolving population that B. subtilis was selected for enhanced production of the lipopeptide surfactin and accelerated surface spreading ability, leading to inhibition of fungal expansion and acidification of the environment. These phenotypes were explained by specific mutations in the DegS-DegU two-component system. In the presence of surfactin, fungal hyphae exhibited bulging cells with delocalised secretory vesicles possibly provoking an RlmA-dependent cell wall stress. Thus, our results indicate that the presence of the fungus selects for increased surfactin production, which inhibits fungal growth and facilitates the competitive success of the bacterium.

Competition for iron shapes metabolic antagonism between Bacillus subtilis and Pseudomonas marginalis.

Siderophores have long been implicated in sociomicrobiology as determinants of bacterial interrelations. For plant-associated genera, like Bacillus and Pseudomonas, siderophores are well known for their biocontrol functions. Here, we explored the functional role of the Bacillus subtilis siderophore bacillibactin (BB) in an antagonistic interaction with Pseudomonas marginalis. The presence of BB strongly influenced the outcome of the interaction in an iron-dependent manner. The BB producer B. subtilis restricts colony spreading of P. marginalis by repressing the transcription of histidine kinase-encoding gene gacS, thereby abolishing production of secondary metabolites such as pyoverdine and viscosin. By contrast, lack of BB restricted B. subtilis colony growth. To explore the specificity of the antagonism, we cocultured B. subtilis with a collection of fluorescent Pseudomonas spp. and found that the Bacillus-Pseudomonas interaction is conserved, expanding our understanding of the interplay between two of the most well-studied genera of soil bacteria.

Establishment of a transparent soil system to study Bacillus subtilis chemical ecology.

Bacterial secondary metabolites are structurally diverse molecules that drive microbial interaction by altering growth, cell differentiation, and signaling. Bacillus subtilis, a Gram-positive soil-dwelling bacterium, produces a wealth of secondary metabolites, among them, lipopeptides have been vastly studied by their antimicrobial, antitumor, and surfactant activities. However, the natural functions of secondary metabolites in the lifestyles of the producing organism remain less explored under natural conditions, i.e. in soil. Here, we describe a hydrogel-based transparent soil system to investigate B. subtilis chemical ecology under controllable soil-like conditions. The transparent soil matrix allows the growth of B. subtilis and other isolates gnotobiotically and under nutrient-controlled conditions. Additionally, we show that transparent soil allows the detection of lipopeptides production and dynamics by HPLC-MS, and MALDI-MS imaging, along with fluorescence imaging of 3-dimensional bacterial assemblages. We anticipate that this affordable and highly controllable system will promote bacterial chemical ecology research and help to elucidate microbial interactions driven by secondary metabolites.

Complete Genome Sequences of Four Soil-Derived Isolates for Studying Synthetic Bacterial Community Assembly.

Here, we report the complete genome sequences of four bacterial soil isolates, Chryseobacterium sp., Stenotrophomonas indicatrix, Pedobacter sp., and Rhodococcus globerulus. These isolates can be used for studying microbial interactions and community assembly in vitro.

Phages carry interbacterial weapons encoded by biosynthetic gene clusters.

Bacteria produce diverse specialized metabolites that mediate ecological interactions and serve as a rich source of industrially relevant natural products. Biosynthetic pathways for these metabolites are encoded by organized groups of genes called biosynthetic gene clusters (BGCs). Understanding the natural function and distribution of BGCs provides insight into the mechanisms through which microorganisms interact and compete. Further, understanding BGCs is extremely important for biocontrol and the mining of new bioactivities. Here, we investigated phage-encoded BGCs (pBGCs), challenging the relationship between phage origin and BGC structure and function. The results demonstrated that pBGCs are rare, and they predominantly reside within temperate phages infecting commensal or pathogenic bacterial hosts. Further, the vast majority of pBGCs were found to encode for bacteriocins. Using the soil- and gut-associated bacterium Bacillus subtilis, we experimentally demonstrated how a temperate phage equips a bacterium with a fully functional BGC, providing a clear competitive fitness advantage over the ancestor. Moreover, we demonstrated a similar transfer of the same phage in prophage form. Finally, using genetic and genomic comparisons, a strong association between pBGC type and phage host range was revealed. These findings suggest that bacteriocins are encoded in temperate phages of a few commensal bacterial genera. In these cases, lysogenic conversion provides an evolutionary benefit to the infected host and, hence, to the phage itself. This study is an important step toward understanding the natural role of bacterial compounds encoded by BGCs, the mechanisms driving their horizontal transfer, and the sometimes mutualistic relationship between bacteria and temperate phages.

Genomic and Chemical Diversity of Bacillus subtilis Secondary Metabolites against Plant Pathogenic Fungi.

Bacillus subtilis produces a wide range of secondary metabolites providing diverse plant growth-promoting and biocontrol abilities. These secondary metabolites include nonribosomal peptides 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 secondary metabolites from various environmental B. subtilis strains is missing. In this study, we isolated 23 B. subtilis strains from 11 sampling sites, compared the fungal inhibition profiles of wild types and their nonribosomal peptide mutants, followed the production of targeted lipopeptides, and determined the complete genomes of 13 soil isolates. We discovered that nonribosomal peptide production varied among B. subtilis strains coisolated 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 spp., a combination of plipastatin and surfactin is required to hinder growth of Botrytis cinerea Detailed genomic analysis revealed that altered nonribosomal peptide production profiles in specific isolates are due to missing core genes, nonsense mutation, or potentially altered gene regulation. Our study combines microbiological antagonism assays with chemical nonribosomal peptide 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 nonribosomally produced lipopeptides in particular display strong antifungal activity. To facilitate the 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 the production of certain secondary metabolites.

Secondary metabolites of Bacillus subtilis impact the assembly of soil-derived semisynthetic bacterial communities.

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.

Metal ions weaken the hydrophobicity and antibiotic resistance of Bacillus subtilis NCIB 3610 biofilms.

Surface superhydrophobicity makes bacterial biofilms very difficult to fight, and it is a combination of their matrix composition and complex surface roughness which synergistically protects these biomaterials from wetting. Although trying to eradicate biofilms with aqueous (antibiotic) solutions is common practice, this can be a futile approach if the biofilms have superhydrophobic properties. To date, there are not many options available to reduce the liquid repellency of biofilms or to prevent this material property from developing. Here, we present a solution to this challenge. We demonstrate how the addition of metal ions such as copper and zinc during or after biofilm formation can render the surface of otherwise superhydrophobic B. subtilis NCIB 3610 biofilms completely wettable. As a result of this procedure, these smoother, hydrophilic biofilms are more susceptible to aqueous antibiotics solutions. Our strategy proposes a scalable and widely applicable step in a multi-faceted approach to eradicate biofilms.

Complete Genome Sequences of 13 Bacillus subtilis Soil Isolates for Studying Secondary Metabolite Diversity.

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.