Re-Thinking the Roles of Pathogens and Mutualists: Exploring the Continuum of Symbiosis in the Context of Microbial Ecology and Evolution

Systems of classification are important for guiding research activities and providing a common platform for discussion and investigation. One such system is assigning microbial taxa to the roles of mutualists and pathogens. Yet, there are often challenges and even inconsistencies in reports of research findings when microbial taxa display behaviors outside of these two static conditions (e.g. commensal). Over the last two decades, there has been some effort to highlight a continuum of symbiosis, wherein certain microbial taxa may exhibit mutualistic or pathogenic traits depending on environmental contexts, life stages, and plant host associations. However, gaps remain in understanding how to apply the continuum approach to host-microbe pairs across a range of environmental and ecological factors. This commentary presents an alternative framework for evaluating the continuum of symbiosis using dominant archetypes that define symbiotic ranges. We focus particularly on fungi and bacteria, though we recognize that archaea and other microeukaryotes play important roles in host-microbe interactions that may be described by this approach. This framework is centered in eco-evolutionary theory and aims to enhance communication among researchers, as well as prioritize holistic consideration of the factors shaping microbial life strategies. We discuss the influence of plant-mediated factors, habitat constraints, co-evolutionary forces, and the genetic contributions which shape different microbial lifestyles. Looking to the future, using a continuum of symbiosis paradigm will enable greater flexibility in defining the roles of target microbes and facilitate a more holistic view of the complex and dynamic relationship between microbes and plants.

Pseudomonas Lipopeptide-Mediated Biocontrol: Chemotaxonomy and Biological Activity

Pseudomonas lipopeptides (Ps-LPs) play crucial roles in bacterial physiology, host–microbe interactions and plant disease control. Beneficial LP producers have mainly been isolated from the rhizosphere, phyllosphere and from bulk soils. Despite their wide geographic distribution and host range, emerging evidence suggests that LP-producing pseudomonads and their corresponding molecules display tight specificity and follow a phylogenetic distribution. About a decade ago, biocontrol LPs were mainly reported from the P. fluorescens group, but this has drastically advanced due to increased LP diversity research. On the one hand, the presence of a close-knit relationship between Pseudomonas taxonomy and the molecule produced may provide a startup toolbox for the delineation of unknown LPs into existing (or novel) LP groups. Furthermore, a taxonomy–molecule match may facilitate decisions regarding antimicrobial activity profiling and subsequent agricultural relevance of such LPs. In this review, we highlight and discuss the production of beneficial Ps-LPs by strains situated within unique taxonomic groups and the lineage-specificity and coevolution of this relationship. We also chronicle the antimicrobial activity demonstrated by these biomolecules in limited plant systems compared with multiple in vitro assays. Our review further stresses the need to systematically elucidate the roles of diverse Ps-LP groups in direct plant–pathogen interactions and in the enhancement of plant innate immunity.

Accurate and robust inference of microbial growth dynamics from metagenomic sequencing reveals personalized growth rates

Patterns of sequencing coverage along a bacterial genome---summarized by a peak-to-trough ratio (PTR)---have been shown to accurately reflect microbial growth rates, revealing a new facet of microbial dynamics and host-microbe interactions. Here, we introduce CoPTR (Compute PTR): a tool for computing PTRs from complete reference genomes and assemblies. Using simulations and data from growth experiments in simple and complex communities, we show that CoPTR is more accurate than the current state-of-the-art, while also providing more PTR estimates overall. We further develop theory formalizing a biological interpretation for PTRs. Using a reference database of 2935 species, we applied CoPTR to a case-control study of 1304 metagenomic samples from 106 individuals with inflammatory bowel disease. We show that growth rates are personalized, are only loosely correlated with relative abundances, and are associated with disease status. We conclude by demonstrating how PTRs can be combined with relative abundances and metabolomics to investigate their effect on the microbiome.

Gut mélange à trois: fluctuating selection modulated by microbiota, host immune system, and antibiotics

Iron is critical in host-microbe interactions, and its availability is under tight regulation in the mammalian gut. Antibiotics and inflammation are known to perturb iron availability in the gut, which could subsequently alter host-microbe interactions. Here, we show that an adaptive allele of iscR, encoding a major regulator of iron homeostasis of Escherichia coli, is under fluctuating selection in the mouse gut. In vivo competitions in immune-competent, immune-compromised, and germ-free mice reveal that the selective pressure on an iscR mutant E. coli is modulated by the presence of antibiotics, other members of the microbiota, and the immune system. In vitro assays show that iron availability is an important mediator of the iscR allele fitness benefits or costs. We identify Lipocalin-2, a host's innate immune system protein that prevents bacterial iron acquisition, as a major host mechanism underlying fluctuating selection of the iscR allele. Our results provide a remarkable example of strong fluctuating selection acting on bacterial iron regulation in the mammalian gut.

Considerations for the Analysis of Bacterial Membrane Vesicles: Methods of Vesicle Production and Quantification Can Influence Biological and Experimental Outcomes

Recent years have seen a surge in interest in the roles of BMVs in host-microbe interactions and interbacterial communication. As a result of such rapid growth in the field, there is a lack of uniformity in BMV enumeration.

A broad-spectrum cloning vector that exists as both an integrated element and a free plasmid in Chlamydia trachomatis

Plasmid transformation of chlamydiae has created new opportunities to investigate host–microbe interactions during chlamydial infections; however, there are still limitations. Plasmid transformation requires a replicon derived from the native Chlamydia plasmid, and these transformations are species-specific. We explored the utility of a broad host-range plasmid, pBBR1MCS-4, to transform chlamydiae, with a goal of simplifying the transformation process. The plasmid was modified to contain chromosomal DNA from C. trachomatis to facilitate homologous recombination. Sequences flanking incA were cloned into the pBBR1MCS-4 vector along with the GFP:CAT cassette from the pSW2-GFP chlamydial shuttle vector. The final plasmid construct, pBVR2, was successfully transformed into C. trachomatis strain L2-434. Chlamydial transformants were analyzed by immunofluorescence microscopy and positive clones were sequentially purified using limiting dilution. PCR and PacBio-based whole genome sequencing were used to determine if the plasmid was maintained within the chromosome or as an episome. PacBio sequencing of the cloned transformants revealed allelic exchange events between the chromosome and plasmid pBVR2 that replaced chromosomal incA with the plasmid GFP:CAT cassette. The data also showed evidence of full integration of the plasmid into the bacterial chromosome. While some plasmids were fully integrated, some were maintained as episomes and could be purified and retransformed into E. coli. Thus, the plasmid can be successfully transformed into chlamydia without a chlamydial origin of replication and can exist in multiple states within a transformed population.

Structural analysis of the sulfatase AmAS from Akkermansia muciniphila

Akkermansia muciniphila, an anaerobic Gram-negative bacterium, is a major intestinal commensal bacterium that can modulate the host immune response. It colonizes the mucosal layer and produces nutrients for the gut mucosa and other commensal bacteria. It is believed that mucin desulfation is the rate-limiting step in the mucin-degradation process, and bacterial sulfatases that carry out mucin desulfation have been well studied. However, little is known about the structural characteristics of A. muciniphila sulfatases. Here, the crystal structure of the premature form of the A. muciniphila sulfatase AmAS was determined. Structural analysis combined with docking experiments defined the critical active-site residues that are responsible for catalysis. The loop regions I–V were proposed to be essential for substrate binding. Structure-based sequence alignment and structural superposition allow further elucidation of how different subclasses of formylglycine-dependent sulfatases (FGly sulfatases) adopt the same catalytic mechanism but exhibit diverse substrate specificities. These results advance the understanding of the substrate-recognition mechanisms of A. muciniphila FGly-type sulfatases. Structural variations around the active sites account for the different substrate-binding properties. These results will enhance the understanding of the roles of bacterial sulfatases in the metabolism of glycans and host–microbe interactions in the human gut environment.

Novel metabolic interactions and environmental conditions mediate the boreal peatmoss-cyanobacteria mutualism

Interactions between Sphagnum (peat moss) and cyanobacteria play critical roles in terrestrial carbon and nitrogen cycling processes. Knowledge of the metabolites exchanged, the physiological processes involved, and the environmental conditions allowing the formation of symbiosis is important for a better understanding of the mechanisms underlying these interactions. In this study, we used a cross-feeding approach with spatially resolved metabolite profiling and metatranscriptomics to characterize the symbiosis between Sphagnum and Nostoc cyanobacteria. A pH gradient study revealed that the Sphagnum–Nostoc symbiosis was driven by pH, with mutualism occurring only at low pH. Metabolic cross-feeding studies along with spatially resolved matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) identified trehalose as the main carbohydrate source released by Sphagnum, which were depleted by Nostoc along with sulfur-containing choline-O-sulfate, taurine and sulfoacetate. In exchange, Nostoc increased exudation of purines and amino acids. Metatranscriptome analysis indicated that Sphagnum host defense was downregulated when in direct contact with the Nostoc symbiont, but not as a result of chemical contact alone. The observations in this study elucidated environmental, metabolic, and physiological underpinnings of the widespread plant–cyanobacterial symbioses with important implications for predicting carbon and nitrogen cycling in peatland ecosystems as well as the basis of general host-microbe interactions.