High Spatial Resolution Imaging Mass Spectrometry Reveals Chemical Heterogeneity Across Bacterial Microcolonies.

Microbes interact with the world around them at the chemical level. However, directly examining the chemical exchange between microbes and microbes and their environment, at ecological scales, i.e., the scale of a single bacterial cell or small groups of cells, remains a key challenge. Here we address this obstacle by presenting a methodology that enables matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) of bacterial microcolonies. By combining optimized sample preparation with subatmospheric pressure MALDI, we demonstrate that chemical output from groups of as few as ∼50 cells can be visualized with MALDI-IMS. Application of this methodology to Bacillus subtilis and Streptomyces coelicolor revealed heterogeneity in chemical output across microcolonies and asymmetrical metabolite production when cells grew within physiological gradients produced by Medicago sativa roots. Taken together, these results indicate that MALDI-IMS can readily visualize metabolites made by very small assemblages of bacterial cells and that even these small groups of cells can differentially produce metabolites in response to local chemical gradients.

Chemical signaling involved in plant-microbe interactions.

Microorganisms are found everywhere, and they are closely associated with plants. Because the establishment of any plant-microbe association involves chemical communication, understanding crosstalk processes is fundamental to defining the type of relationship. Although several metabolites from plants and microbes have been fully characterized, their roles in the chemical interplay between these partners are not well understood in most cases, and they require further investigation. In this review, we describe different plant-microbe associations from colonization to microbial establishment processes in plants along with future prospects, including agricultural benefits.

microbeMASST: a taxonomically informed mass spectrometry search tool for microbial metabolomics data.

microbeMASST, a taxonomically informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbe-derived metabolites and relative producers without a priori knowledge will vastly enhance the understanding of microorganisms' role in ecology and human health.

Multiple lineages of Streptomyces produce antimicrobials within passalid beetle galleries across eastern North America.

Some insects form symbioses in which actinomycetes provide defense against pathogens by making antimicrobials. The range of chemical strategies employed across these associations, and how these strategies relate to insect lifestyle, remains underexplored. We assessed subsocial passalid beetles of the species Odontotaenius disjunctus, and their frass (fecal material), which is an important food resource within their galleries, as a model insect/actinomycete system. Through chemical and phylogenetic analyses, we found that O. disjunctus frass collected across eastern North America harbored multiple lineages of Streptomyces and diverse antimicrobials. Metabolites detected in frass displayed synergistic and antagonistic inhibition of a fungal entomopathogen, Metarhizium anisopliae, and multiple streptomycete isolates inhibited this pathogen when co-cultivated directly in frass. These findings support a model in which the lifestyle of O. disjunctus accommodates multiple Streptomyces lineages in their frass, resulting in a rich repertoire of antimicrobials that likely insulates their galleries against pathogenic invasion.

Cooperation, Competition, and Specialized Metabolism in a Simplified Root Nodule Microbiome.

Microbiomes associated with various plant structures often contain members with the potential to make specialized metabolites, e.g., molecules with antibacterial, antifungal, or siderophore activities. However, when and where microbes associated with plants produce specialized metabolites, and the potential role of these molecules in mediating intramicrobiome interactions, is not well understood. Root nodules of legume plants are organs devoted to hosting symbiotic bacteria that fix atmospheric nitrogen and have recently been shown to harbor a relatively simple accessory microbiome containing members with the ability to produce specialized metabolites in vitro On the basis of these observations, we sought to develop a model nodule microbiome system for evaluating specialized microbial metabolism in planta Starting with an inoculum derived from field-grown Medicago sativa nodules, serial passaging through gnotobiotic nodules yielded a simplified accessory community composed of four members: Brevibacillus brevis, Paenibacillus sp., Pantoea agglomerans, and Pseudomonas sp. Some members of this community exhibited clear cooperation in planta, while others were antagonistic and capable of disrupting cooperation between other partners. Using matrix-assisted laser desorption ionization-imaging mass spectrometry, we found that metabolites associated with individual taxa had unique distributions, indicating that some members of the nodule community were spatially segregated. Finally, we identified two families of molecules produced by B. brevisin planta as the antibacterial tyrocidines and a novel set of gramicidin-type molecules, which we term the britacidins. Collectively, these results indicate that in addition to nitrogen fixation, legume root nodules are likely also sites of active antimicrobial production.

Inducible Antibacterial Activity in the Bacillales by Triphenyl Tetrazolium Chloride.

The world is in the midst of an antimicrobial resistance crisis, driving a need to discover novel antibiotic substances. Using chemical cues as inducers to unveil a microorganism's full metabolic potential is considered a successful strategy. To this end, we investigated an inducible antagonistic behavior in multiple isolates of the order Bacillales, where large inhibition zones were produced against Ralstonia solanacearum only when grown in the presence of the indicator triphenyl tetrazolium chloride (TTC). This bioactivity was produced in a TTC-dose dependent manner. Escherichia coli and Staphylococcus sp. isolates were also inhibited by Bacillus sp. strains in TTC presence, to a lesser extent. Knockout mutants and transcriptomic analysis of B. subtilis NCIB 3610 cells revealed that genes from the L-histidine biosynthetic pathway, the purine, pyrimidine de novo synthesis and salvage and interconversion routes, were significantly upregulated. Chemical space studied through metabolomic analysis, showed increased presence of nitrogenous compounds in extracts from induced bacteria. The metabolites orotic acid and L-phenylalaninamide were tested against R. solanacearum, E. coli, Staphylococcus sp. and B. subtilis, and exhibited activity against pathogens only in the presence of TTC, suggesting a biotransformation of nitrogenous compounds in Bacillus sp. cells as the plausible cause of the inducible antagonistic behavior.