The secret life of plant-beneficial rhizosphere bacteria: insects as alternative hosts.

Root-colonizing bacteria have been intensively investigated for their intimate relationship with plants and their manifold plant-beneficial activities. They can inhibit growth and activity of pathogens or induce defence responses. In recent years, evidence has emerged that several plant-beneficial rhizosphere bacteria do not only associate with plants but also with insects. Their relationships with insects range from pathogenic to mutualistic and some rhizobacteria can use insects as vectors for dispersal to new host plants. Thus, the interactions of these bacteria with their environment are even more complex than previously thought and can extend far beyond the rhizosphere. The discovery of this secret life of rhizobacteria represents an exciting new field of research that should link the fields of plant-microbe and insect-microbe interactions. In this review, we provide examples of plant-beneficial rhizosphere bacteria that use insects as alternative hosts, and of potentially rhizosphere-competent insect symbionts. We discuss the bacterial traits that may enable a host-switch between plants and insects and further set the multi-host lifestyle of rhizobacteria into an evolutionary and ecological context. Finally, we identify important open research questions and discuss perspectives on the use of these rhizobacteria in agriculture.

Phylogenetically closely related pseudomonads isolated from arthropods exhibit differential insect-killing abilities and genetic variations in insecticidal factors.

Strains belonging to the Pseudomonas protegens and Pseudomonas chlororaphis species are able to control soilborne plant pathogens and to kill pest insects by producing virulence factors such as toxins, chitinases, antimicrobials or two-partner secretion systems. Most insecticidal Pseudomonas described so far were isolated from roots or soil. It is unknown whether these bacteria naturally occur in arthropods and how they interact with them. Therefore, we isolated P. protegens and P. chlororaphis from various healthy insects and myriapods, roots and soil collected in an agricultural field and a neighbouring grassland. The isolates were compared for insect killing, pathogen suppression and host colonization abilities. Our results indicate that neither the origin of isolation nor the phylogenetic position mirror the degree of insecticidal activity. Pseudomonas protegens strains appeared homogeneous regarding phylogeny, biocontrol and insecticidal capabilities, whereas P. chlororaphis strains were phylogenetically and phenotypically more heterogenous. A phenotypic and genomic analysis of five closely related P. chlororaphis isolates displaying varying levels of insecticidal activity revealed variations in genes encoding insecticidal factors that may account for the reduced insecticidal activity of certain isolates. Our findings point towards an adaption to insects within closely related pseudomonads and contribute to understand the ecology of insecticidal Pseudomonas.

Root-colonizing bacteria enhance the levels of (E)-ß-caryophyllene produced by maize roots in response to rootworm feeding.

When larvae of rootworms feed on maize roots they induce the emission of the sesquiterpene (E)-ß-caryophyllene (EßC). EßC is attractive to entomopathogenic nematodes, which parasitize and rapidly kill the larvae, thereby protecting the roots from further damage. Certain root-colonizing bacteria of the genus Pseudomonas also benefit plants by promoting growth, suppressing pathogens or inducing systemic resistance (ISR), and some strains also have insecticidal activity. It remains unknown how these bacteria influence the emissions of root volatiles. In this study, we evaluated how colonization by the growth-promoting and insecticidal bacteria Pseudomonas protegens CHA0 and Pseudomonas chlororaphis PCL1391 affects the production of EßC upon feeding by larvae of the banded cucumber beetle, Diabrotica balteata Le Conte (Coleoptera: Chrysomelidae). Using chemical analysis and gene expression measurements, we found that EßC production and the expression of the EßC synthase gene (tps23) were enhanced in Pseudomonas protegens CHA0-colonized roots after 72 h of D. balteata feeding. Undamaged roots colonized by Pseudomonas spp. showed no measurable increase in EßC production, but a slight increase in tps23 expression. Pseudomonas colonization did not affect root biomass, but larvae that fed on roots colonized by P. protegens CHA0 tended to gain more weight than larvae that fed on roots colonized by P. chlororaphis PCL1391. Larvae mortality on Pseudomonas spp. colonized roots was slightly, but not significantly higher than on non-colonized control roots. The observed enhanced production of EßC upon Pseudomonas protegens CHA0 colonization may enhance the roots' attractiveness to entomopathogenic nematodes, but this remains to be tested.

Correction to: Root-colonizing bacteria enhance the levels of (E)-ß-caryophyllene produced by maize roots in response to rootworm feeding.

Unfortunately, family name of author "Xavier Chiriboga M" was incorrectly identified in the original publication and the same is corrected here. The original article has been corrected.

Specific surface glycan decorations enable antimicrobial peptide resistance in plant-beneficial pseudomonads with insect-pathogenic properties.

Some plant-beneficial pseudomonads can invade and kill pest insects in addition to their ability to protect plants from phytopathogens. We explored the genetic basis of O-polysaccharide (O-PS, O-antigen) biosynthesis in the representative insecticidal strains Pseudomonas protegens CHA0 and Pseudomonas chlororaphis PCL1391 and investigated its role in insect pathogenicity. Both strains produce two distinct forms of O-PS, but differ in the organization of their O-PS biosynthesis clusters. Biosynthesis of the dominant O-PS in both strains depends on a gene cluster similar to the O-specific antigen (OSA) cluster of Pseudomonas aeruginosa. In CHA0 and other P. protegens strains, the OSA cluster is extensively reduced and new clusters were acquired, resulting in high diversity of O-PS structures, possibly reflecting adaptation to different hosts. CHA0 mutants lacking the short OSA form of O-PS were significantly impaired in insect virulence in Galleria injection and Plutella feeding assays. CHA0, PCL1391, and other insecticidal pseudomonads exhibited high resistance to antimicrobial peptides, including cecropins that are central to insect immune defense. Resistance of both model strains depended on the dominant OSA-type O-PS. Our results suggest that O-antigen is essential for successful insect infection and illustrate, for the first time, its importance in resistance of Pseudomonas to antimicrobial peptides.

Domain shuffling in a sensor protein contributed to the evolution of insect pathogenicity in plant-beneficial Pseudomonas protegens.

Pseudomonas protegens is a biocontrol rhizobacterium with a plant-beneficial and an insect pathogenic lifestyle, but it is not understood how the organism switches between the two states. Here, we focus on understanding the function and possible evolution of a molecular sensor that enables P. protegens to detect the insect environment and produce a potent insecticidal toxin specifically during insect infection but not on roots. By using quantitative single cell microscopy and mutant analysis, we provide evidence that the sensor histidine kinase FitF is a key regulator of insecticidal toxin production. Our experimental data and bioinformatic analyses indicate that FitF shares a sensing domain with DctB, a histidine kinase regulating carbon uptake in Proteobacteria. This suggested that FitF has acquired its specificity through domain shuffling from a common ancestor. We constructed a chimeric DctB-FitF protein and showed that it is indeed functional in regulating toxin expression in P. protegens. The shuffling event and subsequent adaptive modifications of the recruited sensor domain were critical for the microorganism to express its potent insect toxin in the observed host-specific manner. Inhibition of the FitF sensor during root colonization could explain the mechanism by which P. protegens differentiates between the plant and insect host. Our study establishes FitF of P. protegens as a prime model for molecular evolution of sensor proteins and bacterial pathogenicity.

Evolutionary patchwork of an insecticidal toxin shared between plant-associated pseudomonads and the insect pathogens Photorhabdus and Xenorhabdus.

BACKGROUND: Root-colonizing fluorescent pseudomonads are known for their excellent abilities to protect plants against soil-borne fungal pathogens. Some of these bacteria produce an insecticidal toxin (Fit) suggesting that they may exploit insect hosts as a secondary niche. However, the ecological relevance of insect toxicity and the mechanisms driving the evolution of toxin production remain puzzling. RESULTS: Screening a large collection of plant-associated pseudomonads for insecticidal activity and presence of the Fit toxin revealed that Fit is highly indicative of insecticidal activity and predicts that Pseudomonas protegens and P. chlororaphis are exclusive Fit producers. A comparative evolutionary analysis of Fit toxin-producing Pseudomonas including the insect-pathogenic bacteria Photorhabdus and Xenorhadus, which produce the Fit related Mcf toxin, showed that fit genes are part of a dynamic genomic region with substantial presence/absence polymorphism and local variation in GC base composition. The patchy distribution and phylogenetic incongruence of fit genes indicate that the Fit cluster evolved via horizontal transfer, followed by functional integration of vertically transmitted genes, generating a unique Pseudomonas-specific insect toxin cluster. CONCLUSIONS: Our findings suggest that multiple independent evolutionary events led to formation of at least three versions of the Mcf/Fit toxin highlighting the dynamic nature of insect toxin evolution.

Expanding the Pseudomonas diversity of the wheat rhizosphere: four novel species antagonizing fungal phytopathogens and with plant-beneficial properties.

Plant-beneficial Pseudomonas bacteria hold the potential to be used as inoculants in agriculture to promote plant growth and health through various mechanisms. The discovery of new strains tailored to specific agricultural needs remains an open area of research. In this study, we report the isolation and characterization of four novel Pseudomonas species associated with the wheat rhizosphere. Comparative genomic analysis with all available Pseudomonas type strains revealed species-level differences, substantiated by both digital DNA-DNA hybridization and average nucleotide identity, underscoring their status as novel species. This was further validated by the phenotypic differences observed when compared to their closest relatives. Three of the novel species belong to the P. fluorescens species complex, with two representing a novel lineage in the Pseudomonas phylogeny. Functional genome annotation revealed the presence of specific features contributing to rhizosphere colonization, including flagella and components for biofilm formation. The novel species have the genetic potential to solubilize nutrients by acidifying the environment, releasing alkaline phosphatases and their metabolism of nitrogen species, indicating potential as biofertilizers. Additionally, the novel species possess traits that may facilitate direct promotion of plant growth through the modulation of the plant hormone balance, including the ACC deaminase enzyme and auxin metabolism. The presence of biosynthetic clusters for toxins such as hydrogen cyanide and non-ribosomal peptides suggests their ability to compete with other microorganisms, including plant pathogens. Direct inoculation of wheat roots significantly enhanced plant growth, with two strains doubling shoot biomass. Three of the strains effectively antagonized fungal phytopathogens (Thielaviopsis basicola, Fusarium oxysporum, and Botrytis cinerea), demonstrating their potential as biocontrol agents. Based on the observed genetic and phenotypic differences from closely related species, we propose the following names for the four novel species: Pseudomonas grandcourensis sp. nov., type strain DGS24T ( = DSM 117501T = CECT 31011T), Pseudomonas purpurea sp. nov., type strain DGS26T ( = DSM 117502T = CECT 31012T), Pseudomonas helvetica sp. nov., type strain DGS28T ( = DSM 117503T = CECT 31013T) and Pseudomonas aestiva sp. nov., type strain DGS32T ( = DSM 117504T = CECT 31014T).

Fragmented micro-growth habitats present opportunities for alternative competitive outcomes.

Bacteria in nature often thrive in fragmented environments, like soil pores, plant roots or plant leaves, leading to smaller isolated habitats, shared with fewer species. This spatial fragmentation can significantly influence bacterial interactions, affecting overall community diversity. To investigate this, we contrast paired bacterial growth in tiny picoliter droplets (1-3 cells per 35 pL up to 3-8 cells per species in 268 pL) with larger, uniform liquid cultures (about 2 million cells per 140 µl). We test four interaction scenarios using different bacterial strains: substrate competition, substrate independence, growth inhibition, and cell killing. In fragmented environments, interaction outcomes are more variable and sometimes even reverse compared to larger uniform cultures. Both experiments and simulations show that these differences stem mostly from variation in initial cell population growth phenotypes and their sizes. These effects are most significant with the smallest starting cell populations and lessen as population size increases. Simulations suggest that slower-growing species might survive competition by increasing growth variability. Our findings reveal how microhabitat fragmentation promotes diverse bacterial interaction outcomes, contributing to greater species diversity under competitive conditions.

Entomopathogenic pseudomonads can share an insect host with entomopathogenic nematodes and their mutualistic bacteria.

A promising strategy to overcome limitations in biological control of insect pests is the combined application of entomopathogenic pseudomonads (EPPs) and nematodes (EPNs) associated with mutualistic bacteria (NABs). Yet, little is known about interspecies interactions such as competition, coexistence, or even cooperation between these entomopathogens when they infect the same insect host. We investigated the dynamics of bacteria-bacteria interactions between the EPP Pseudomonas protegens CHA0 and the NAB Xenorhabdus bovienii SM5 isolated from the EPN Steinernema feltiae RS5. Bacterial populations were assessed over time in experimental systems of increasing complexity. In vitro, SM5 was outcompeted when CHA0 reached a certain cell density, resulting in the collapse of the SM5 population. In contrast, both bacteria were able to coexist upon haemolymph-injection into Galleria mellonella larvae, as found for three further EPP-NAB combinations. Finally, both bacteria were administered by natural infection routes i.e. orally for CHA0 and nematode-vectored for SM5 resulting in the addition of RS5 to the system. This did not alter bacterial coexistence nor did the presence of the EPP affect nematode reproductive success or progeny virulence. CHA0 benefited from RS5, probably by exploiting access routes formed by the nematodes penetrating the larval gut epithelium. Our results indicate that EPPs are able to share an insect host with EPNs and their mutualistic bacteria without major negative effects on the reproduction of any of the three entomopathogens or the fitness of the nematodes. This suggests that their combination is a promising strategy for biological insect pest control.

Natural plant disease suppressiveness in soils extends to insect pest control.

BACKGROUND: Since the 1980s, soils in a 22-km2 area near Lake Neuchâtel in Switzerland have been recognized for their innate ability to suppress the black root rot plant disease caused by the fungal pathogen Thielaviopsis basicola. However, the efficacy of natural disease suppressive soils against insect pests has not been studied. RESULTS: We demonstrate that natural soil suppressiveness also protects plants from the leaf-feeding pest insect Oulema melanopus. Plants grown in the most suppressive soil have a reduced stress response to Oulema feeding, reflected by dampened levels of herbivore defense-related phytohormones and benzoxazinoids. Enhanced salicylate levels in insect-free plants indicate defense-priming operating in this soil. The rhizosphere microbiome of suppressive soils contained a higher proportion of plant-beneficial bacteria, coinciding with their microbiome networks being highly tolerant to the destabilizing impact of insect exposure observed in the rhizosphere of plants grown in the conducive soils. We suggest that presence of plant-beneficial bacteria in the suppressive soils along with priming, conferred plant resistance to the insect pest, manifesting also in the onset of insect microbiome dysbiosis by the displacement of the insect endosymbionts. CONCLUSIONS: Our results show that an intricate soil-plant-insect feedback, relying on a stress tolerant microbiome network with the presence of plant-beneficial bacteria and plant priming, extends natural soil suppressiveness from soilborne diseases to insect pests. Video Abstract.

Control and host-dependent activation of insect toxin expression in a root-associated biocontrol pseudomonad.

Pseudomonas fluorescens CHA0 is a root-associated biocontrol agent that suppresses soil-borne fungal diseases of crops. Remarkably, the pseudomonad is also endowed with systemic and oral activity against pest insects which depends on the production of the insecticidal Fit toxin. The toxin gene (fitD) is part of a virulence cassette encoding three regulators (FitF, FitG, FitH) and a type I secretion system (FitABC-E). Immunoassays with a toxin-specific antibody and transcriptional analyses involving fitG and fitH deletion and overexpression mutants identified LysR family regulator FitG and response regulator FitH as activator and repressor, respectively, of Fit toxin and transporter expression. To visualize and quantify toxin expression in single live cells by fluorescence microscopy, we developed reporters which in lieu of the native toxin protein express a fusion of the Fit toxin with red fluorescent mCherry. In a wild-type background, expression of the mCherry-tagged Fit toxin was activated at high levels in insect hosts, i.e. when needed, yet not on plant roots or in batch culture. By contrast, a derepressed fitH mutant expressed the toxin in all conditions. P. fluorescens hence can actively induce insect toxin production in response to the host environment, and FitH and FitG are key regulators in this mechanism.

Oral insecticidal activity of plant-associated pseudomonads.

Biocontrol pseudomonads are most known to protect plants from fungal diseases and to increase plant yield, while intriguing aspects on insecticidal activity have been discovered only recently. Here, we demonstrate that Fit toxin producing pseudomonads, in contrast to a naturally Fit-deficient strain, exhibit potent oral activity against larvae of Spodoptera littoralis, Heliothis virescens and Plutella xylostella, all major insect pests of agricultural crops. Spraying plant leaves with suspensions containing only 1000 Pseudomonas cells per ml was sufficient to kill 70-80% of Spodoptera and Heliothis larvae. Monitoring survival kinetics and bacterial titres in parallel, we demonstrate that Pseudomonas fluorescens CHA0 and Pseudomonas chlororaphis PCL1391, two bacteria harbouring the Fit gene cluster colonize and kill insects via oral infection. Using Fit mutants of CHA0 and PCL1391, we show that production of the Fit toxin contributes substantially to oral insecticidal activity. Furthermore, the global regulator GacA is required for full insecticidal activity. Our findings demonstrate the lethal oral activity of two root-colonizing pseudomonads so far known as potent antagonists of fungal plant pathogens. This adds insecticidal activity to the existing biocontrol repertoire of these bacteria and opens new perspectives for applications in crop pest control and in research on their ecological behaviour.

Evolutionary and ecological role of extracellular contractile injection systems: from threat to weapon.

Contractile injection systems (CISs) are phage tail-related structures that are encoded in many bacterial genomes. These devices encompass the cell-based type VI secretion systems (T6SSs) as well as extracellular CISs (eCISs). The eCISs comprise the R-tailocins produced by various bacterial species as well as related phage tail-like structures such as the antifeeding prophages (Afps) of Serratia entomophila, the Photorhabdus virulence cassettes (PVCs), and the metamorphosis-associated contractile structures (MACs) of Pseudoalteromonas luteoviolacea. These contractile structures are released into the extracellular environment upon suicidal lysis of the producer cell and play important roles in bacterial ecology and evolution. In this review, we specifically portray the eCISs with a focus on the R-tailocins, sketch the history of their discovery and provide insights into their evolution within the bacterial host, their structures and how they are assembled and released. We then highlight ecological and evolutionary roles of eCISs and conceptualize how they can influence and shape bacterial communities. Finally, we point to their potential for biotechnological applications in medicine and agriculture.

Biological hydrogen cyanide emission globally impacts the physiology of both HCN-emitting and HCN-perceiving Pseudomonas.

IMPORTANCE: Bacteria communicate by exchanging chemical signals, some of which are volatile and can remotely reach other organisms. HCN was one of the first volatiles discovered to severely impact exposed organisms by inhibiting their respiration. Using HCN-deficient mutants in two Pseudomonas strains, we demonstrate that HCN's impact goes beyond the sole inhibition of respiration and affects both emitting and receiving bacteria in a global way, modulating their motility, biofilm formation, and production of antimicrobial compounds. Our data suggest that bacteria could use HCN not only to control their own cellular functions, but also to remotely influence the behavior of other bacteria sharing the same environment. Since HCN emission occurs in both clinically and environmentally relevant Pseudomonas, these findings are important to better understand or even modulate the expression of bacterial traits involved in both virulence of opportunistic pathogens and in biocontrol efficacy of plant-beneficial strains.

Relation of pest insect-killing and soilborne pathogen-inhibition abilities to species diversification in environmental Pseudomonas protegens.

Strains belonging to the Pseudomonas protegens phylogenomic subgroup have long been known for their beneficial association with plant roots, notably antagonising soilborne phytopathogens. Interestingly, they can also infect and kill pest insects, emphasising their interest as biocontrol agents. In the present study, we used all available Pseudomonas genomes to reassess the phylogeny of this subgroup. Clustering analysis revealed the presence of 12 distinct species, many of which were previously unknown. The differences between these species also extend to the phenotypic level. Most of the species were able to antagonise two soilborne phytopathogens, Fusarium graminearum and Pythium ultimum, and to kill the plant pest insect Pieris brassicae in feeding and systemic infection assays. However, four strains failed to do so, likely as a consequence of adaptation to particular niches. The absence of the insecticidal Fit toxin explained the non-pathogenic behaviour of the four strains towards Pieris brassicae. Further analyses of the Fit toxin genomic island evidence that the loss of this toxin is related to non-insecticidal niche specialisation. This work expands the knowledge on the growing Pseudomonas protegens subgroup and suggests that loss of phytopathogen inhibition and pest insect killing abilities in some of these bacteria may be linked to species diversification processes involving adaptation to particular niches. Our work sheds light on the important ecological consequences of gain and loss dynamics for functions involved in pathogenic host interactions of environmental bacteria.

Molecular and evolutionary basis of O-antigenic polysaccharide-driven phage sensitivity in environmental pseudomonads.

IMPORTANCE: The application of plant-beneficial microorganisms to protect crop plants is a promising alternative to the usage of chemicals. However, biocontrol research often faces difficulties in implementing this approach due to the inconsistency of the bacterial inoculant to establish itself within the root microbiome. Beneficial bacterial inoculants can be decimated by the presence of their natural predators, notably bacteriophages (also called phages). Thus, it is important to gain knowledge regarding the mechanisms behind phage-bacteria interactions to overcome this challenge. Here, we evidence that the major long O-antigenic polysaccharide (O-PS, O-antigen) of the widely used model plant-beneficial bacterium Pseudomonas protegens CHA0 is the receptor of its natural predator, the phage ΦGP100. We examined the distribution of the gene cluster directing the synthesis of this O-PS and identified signatures of horizontal gene acquisitions. Altogether, our study highlights the importance of bacterial cell surface structure variation in the complex interplay between phages and their Pseudomonas hosts.

Changes in structure and assembly of a species-rich soil natural community with contrasting nutrient availability upon establishment of a plant-beneficial Pseudomonas in the wheat rhizosphere.

BACKGROUND: Plant-beneficial bacterial inoculants are of great interest in agriculture as they have the potential to promote plant growth and health. However, the inoculation of the rhizosphere microbiome often results in a suboptimal or transient colonization, which is due to a variety of factors that influence the fate of the inoculant. To better understand the fate of plant-beneficial inoculants in complex rhizosphere microbiomes, composed by hundreds of genotypes and multifactorial selection mechanisms, controlled studies with high-complexity soil microbiomes are needed. RESULTS: We analysed early compositional changes in a taxa-rich natural soil bacterial community under both exponential nutrient-rich and stationary nutrient-limited growth conditions (i.e. growing and stable communities, respectively) following inoculation with the plant-beneficial bacterium Pseudomonas protegens in a bulk soil or a wheat rhizosphere environment. P. protegens successfully established under all conditions tested and was more abundant in the rhizosphere of the stable community. Nutrient availability was a major factor driving microbiome composition and structure as well as the underlying assembly processes. While access to nutrients resulted in communities assembled mainly by homogeneous selection, stochastic processes dominated under the nutrient-deprived conditions. We also observed an increased rhizosphere selection effect under nutrient-limited conditions, resulting in a higher number of amplicon sequence variants (ASVs) whose relative abundance was enriched. The inoculation with P. protegens produced discrete changes, some of which involved other Pseudomonas. Direct competition between Pseudomonas strains partially failed to replicate the observed differences in the microbiome and pointed to a more complex interaction network. CONCLUSIONS: The results of this study show that nutrient availability is a major driving force of microbiome composition, structure and diversity in both the bulk soil and the wheat rhizosphere and determines the assembly processes that govern early microbiome development. The successful establishment of the inoculant was facilitated by the wheat rhizosphere and produced discrete changes among other members of the microbiome. Direct competition between Pseudomonas strains only partially explained the microbiome changes, indicating that indirect interactions or spatial distribution in the rhizosphere or soil interface may be crucial for the survival of certain bacteria. Video Abstract.

The conserved iol gene cluster in Pseudomonas is involved in rhizosphere competence.

The Pseudomonas genus has shown great potential as a sustainable solution to support agriculture through its plant-growth-promoting and biocontrol activities. However, their efficacy as bioinoculants is limited by unpredictable colonization in natural conditions. Our study identifies the iol locus, a gene cluster in Pseudomonas involved in inositol catabolism, as a feature enriched among superior root colonizers in natural soil. Further characterization revealed that the iol locus increases competitiveness, potentially caused by an observed induction of swimming motility and the production of fluorescent siderophore in response to inositol, a plant-derived compound. Public data analyses indicate that the iol locus is broadly conserved in the Pseudomonas genus and linked to diverse host-microbe interactions. Together, our findings suggest the iol locus as a potential target for developing more effective bioinoculants for sustainable agriculture.

From microbiome composition to functional engineering, one step at a time.

SUMMARYCommunities of microorganisms (microbiota) are present in all habitats on Earth and are relevant for agriculture, health, and climate. Deciphering the mechanisms that determine microbiota dynamics and functioning within the context of their respective environments or hosts (the microbiomes) is crucially important. However, the sheer taxonomic, metabolic, functional, and spatial complexity of most microbiomes poses substantial challenges to advancing our knowledge of these mechanisms. While nucleic acid sequencing technologies can chart microbiota composition with high precision, we mostly lack information about the functional roles and interactions of each strain present in a given microbiome. This limits our ability to predict microbiome function in natural habitats and, in the case of dysfunction or dysbiosis, to redirect microbiomes onto stable paths. Here, we will discuss a systematic approach (dubbed the N+1/N-1 concept) to enable step-by-step dissection of microbiome assembly and functioning, as well as intervention procedures to introduce or eliminate one particular microbial strain at a time. The N+1/N-1 concept is informed by natural invasion events and selects culturable, genetically accessible microbes with well-annotated genomes to chart their proliferation or decline within defined synthetic and/or complex natural microbiota. This approach enables harnessing classical microbiological and diversity approaches, as well as omics tools and mathematical modeling to decipher the mechanisms underlying N+1/N-1 microbiota outcomes. Application of this concept further provides stepping stones and benchmarks for microbiome structure and function analyses and more complex microbiome intervention strategies.