SecMet-FISH: labeling, visualization, and enumeration of secondary metabolite producing microorganisms.

Our understanding of the role of secondary metabolites in microbial communities is challenged by intrinsic limitations of culturing bacteria under laboratory conditions and hence cultivation independent approaches are needed. Here, we present a protocol termed Secondary Metabolite FISH (SecMet-FISH), combining advantages of gene-targeted fluorescence in situ hybridization (geneFISH) with in-solution methods (in-solution FISH) to detect and quantify cells based on their genetic capacity to produce secondary metabolites. The approach capitalizes on the conserved nature of biosynthetic gene clusters (BGCs) encoding adenylation (AD) and ketosynthase (KS) domains, and thus selectively targets the genetic basis of non-ribosomal peptide and polyketide biosynthesis. The concept relies on the generation of amplicon pools using degenerate primers broadly targeting AD and KS domains followed by fluorescent labeling, detection, and quantification. Initially, we obtained AD and KS amplicons from Pseuodoalteromonas rubra, which allowed us to successfully label and visualize BGCs within P. rubra cells, demonstrating the feasibility of SecMet-FISH. Next, we adapted the protocol and optimized it for hybridization in both Gram-negative and Gram-positive bacterial cell suspensions, enabling high-throughput single cell analysis by flow cytometry. Ultimately, we used SecMet-FISH to successfully distinguish secondary metabolite producers from non-producers in a five-member synthetic community.

Small Spatial Scale Drivers of Secondary Metabolite Biosynthetic Diversity in Environmental Microbiomes.

In the search for novel drug candidates, diverse environmental microbiomes have been surveyed for their secondary metabolite biosynthesis potential, yet little is known about the biosynthetic diversity encoded by divergent microbiomes from different ecosystems, and the environmental parameters driving this diversity. Here, we used targeted amplicon sequencing of adenylation (AD) and ketosynthase (KS) domains along with 16S sequencing to delineate the unique biosynthetic potential of microbiomes from three separate habitats (soil, water, and sediments) exhibiting unique small spatial scale physicochemical gradients. The estimated richness of AD domains was highest in marine sediments with 656 ± 58 operational biosynthetic units (OBUs), while the KS domain richness was highest in soil microbiomes with 388 ± 67 OBUs. Microbiomes with rich and diverse bacterial communities displayed the highest PK potential across all ecosystems, and on a small spatial scale, pH and salinity were significantly, positively correlated to KS domain richness in soil and aquatic systems, respectively. Integrating our findings, we were able to predict the KS domain richness with a RMSE of 31 OBUs and a R2 of 0.91, and by the use of publicly available information on bacterial richness and diversity, we identified grassland biomes as being particularly promising sites for the discovery of novel polyketides. Furthermore, a focus on acidobacterial taxa is likely to be fruitful, as these were responsible for most of the variation in biosynthetic diversity. Overall, our results highlight the importance of sampling diverse environments with high taxonomic diversity in the pursuit for novel secondary metabolites. IMPORTANCE To counteract the antibiotic resistance crisis, novel anti-infective agents need to be discovered and brought to market. Microbial secondary metabolites have been important sources of inspiration for small-molecule therapeutics. However, the isolation of novel antibiotics is difficult, and the risk of rediscovery is high. With the overarching purpose of identifying promising microbiomes for discovery of novel bioactivity, we mapped out the most significant drivers of biosynthetic diversity across divergent microbiomes. We found the biosynthetic potential to be unique to individual ecosystems, and to depend on bacterial taxonomic diversity. Within systems, and on small spatial scales, pH and salinity correlated positively to the biosynthetic richness of the microbiomes, Acidobacteria representing the taxa most highly associated with biosynthetic diversity. Ultimately, understanding the key drivers of the biosynthesis potential of environmental microbiomes will allow us to focus bioprospecting efforts and facilitate the discovery of novel therapeutics.

The natural product biosynthesis potential of the microbiomes of Earth - Bioprospecting for novel anti-microbial agents in the meta-omics era.

As we stand on the brink of the post-antibiotic era, we are in dire need of novel antimicrobial compounds. Microorganisms produce a wealth of so-called secondary metabolites and have been our most prolific source of antibiotics so far. However, rediscovery of known antibiotics from well-studied cultured microorganisms, and the fact that the majority of microorganisms in the environment are out of reach by means of conventional cultivation techniques, have led to the exploration of the biosynthetic potential in natural microbial communities by novel approaches. In this mini review we discuss how sequence-based analyses have exposed an unprecedented wealth of potential for secondary metabolite production in soil, marine, and host-associated microbiomes, with a focus on the biosynthesis of non-ribosomal peptides and polyketides. Furthermore, we discuss how the complexity of natural microbiomes and the lack of standardized methodology has complicated comparisons across biomes. Yet, as even the most commonly sampled microbiomes hold promise of providing novel classes of natural products, we lastly discuss the development of approaches applied in the translation of the immense biosynthetic diversity of natural microbiomes to the procurement of novel antibiotics.

Roseobacter Group Probiotics Exhibit Differential Killing of Fish Pathogenic Tenacibaculum Species.

Fish-pathogenic bacteria of the Tenacibaculum genus are a serious emerging concern in modern aquaculture, causing tenacibaculosis in a broad selection of cultured finfish. Data describing their virulence mechanisms are scarce and few means, antibiotic treatment aside, are available to control their proliferation in aquaculture systems. We genome sequenced a collection of 19 putative Tenacibaculum isolates from outbreaks at two aquaculture facilities and tested their susceptibility to treatment with tropodithietic acid (TDA)-producing Roseobacter group probiotics. We found that local outbreaks of Tenacibaculum can involve heterogeneous assemblages of species and strains with the capacity to produce multiple different virulence factors related to host invasion and infection. The probiotic Phaeobacter piscinae S26 proved efficient in killing pathogenic Tenacibaculum species such as T. maritimum, T. soleae, and some T. discolor strains. However, the T. mesophilum and T. gallaicum species exhibit natural tolerance toward TDA and are hence not likely to be easily killed by TDA-producing probiotics. Tolerance toward TDA in Tenacibaculum is likely involving multiple inherent physiological features pertaining to electron and proton transport, iron sequestration, and potentially also drug efflux mechanisms, since genetic determinants encoding such features were significantly associated with TDA tolerance. Collectively, our results support the use of TDA producers to prevent tenacibaculosis; however, their efficacy is likely limited to some Tenacibaculum species. IMPORTANCE A productive and sustainable aquaculture sector is needed to meet the UN sustainable development goals and supply the growing world population with high-protein food sources. A sustainable way to prevent disease outbreaks in the industry is the application of probiotic bacteria that can antagonize fish pathogens in the aquaculture systems. TDA-producing Roseobacter group probiotics have proven efficient in killing important vibrio pathogens and protecting fish larvae against infection, and yet their efficacy against different fish pathogenic species of the Tenacibaculum genus has not been explored. Therefore, we tested the efficacy of such potential probiotics against a collection of different Tenacibaculum isolates and found the probiotic to efficiently kill a subset of relevant strains and species, supporting their use as sustainable disease control measure in aquaculture.