Niche availability and competitive loss by facilitation control proliferation of bacterial strains intended for soil microbiome interventions.

Microbiome engineering - the targeted manipulation of microbial communities - is considered a promising strategy to restore ecosystems, but experimental support and mechanistic understanding are required. Here, we show that bacterial inoculants for soil microbiome engineering may fail to establish because they inadvertently facilitate growth of native resident microbiomes. By generating soil microcosms in presence or absence of standardized soil resident communities, we show how different nutrient availabilities limit outgrowth of focal bacterial inoculants (three Pseudomonads), and how this might be improved by adding an artificial, inoculant-selective nutrient niche. Through random paired interaction assays in agarose micro-beads, we demonstrate that, in addition to direct competition, inoculants lose competitiveness by facilitating growth of resident soil bacteria. Metatranscriptomics experiments with toluene as selective nutrient niche for the inoculant Pseudomonas veronii indicate that this facilitation is due to loss and uptake of excreted metabolites by resident taxa. Generation of selective nutrient niches for inoculants may help to favor their proliferation for the duration of their intended action while limiting their competitive loss.

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.

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.

Fitness-Conditional Genes for Soil Adaptation in the Bioaugmentation Agent Pseudomonas veronii 1YdBTEX2.

Strain inoculation (bioaugmentation) is a potentially useful technology to provide microbiomes with new functionalities. However, there is limited understanding of the genetic factors contributing to successful establishment of inoculants. This work aimed to characterize the genes implicated in proliferation of the monoaromatic compound-degrading Pseudomonas veronii 1YdBTEX2 in nonsterile polluted soils. We generated two independent mutant libraries by random minitransposon-delivered marker insertion followed by deep sequencing (Tn-seq) with a total of 5.0 × 105 unique insertions. Libraries were grown in multiple successive cycles for up to 50 generations either in batch liquid medium or in two types of soil microcosms with different resident microbial content (sand or silt) in the presence of toluene. Analysis of gene insertion abundances at different time points (passed generations of metapopulation growth), in comparison to proportions at start and to in silico generated randomized insertion distributions, allowed to define ~800 essential genes common to both libraries and ~2,700 genes with conditional fitness effects in either liquid or soil (195 of which resulted in fitness gain). Conditional fitness genes largely overlapped among all growth conditions but affected approximately twice as many functions in liquid than in soil. This indicates soil to be a more promiscuous environment for mutant growth, probably because of additional nutrient availability. Commonly depleted genes covered a wide range of biological functions and metabolic pathways, such as inorganic ion transport, fatty acid metabolism, amino acid biosynthesis, or nucleotide and cofactor metabolism. Only sparse gene sets were uncovered whose insertion caused fitness decrease exclusive for soils, which were different between silt and sand. Despite detectable higher resident bacteria and potential protist predatory counts in silt, we were, therefore, unable to detect any immediately obvious candidate genes affecting P. veronii biological competitiveness. In contrast to liquid growth conditions, mutants inactivating flagella biosynthesis and motility consistently gained strong fitness advantage in soils and displayed higher growth rates than wild type. In conclusion, although many gene functions were found to be important for growth in soils, most of these are not specific as they affect growth in liquid minimal medium more in general. This indicates that P. veronii does not need major metabolic reprogramming for proliferation in soil with accessible carbon and generally favorable growth conditions. IMPORTANCE Restoring damaged microbiomes is still a formidable challenge. Classical widely adopted approaches consist of augmenting communities with pure or mixed cultures in the hope that these display their intended selected properties under in situ conditions. Ecological theory, however, dictates that introduction of a nonresident microbe is unlikely to lead to its successful proliferation in a foreign system such as a soil microbiome. In an effort to study this systematically, we used random transposon insertion scanning to identify genes and possibly, metabolic subsystems, that are crucial for growth and survival of a bacterial inoculant (Pseudomonas veronii) for targeted degradation of monoaromatic compounds in contaminated nonsterile soils. Our results indicate that although many gene functions are important for proliferation in soil, they are general factors for growth and not exclusive for soil. In other words, P. veronii is a generalist that is not a priori hindered by the soil for its proliferation and would make a good bioaugmentation candidate.

Reproducible Propagation of Species-Rich Soil Bacterial Communities Suggests Robust Underlying Deterministic Principles of Community Formation.

Microbiomes are typically characterized by high species diversity but it is poorly understood how such system-level complexity can be generated and propagated. Here, we used soil microcosms as a model to study development of bacterial communities as a function of their starting complexity and environmental boundary conditions. Despite inherent stochastic variation in manipulating species-rich communities, both laboratory-mixed medium complexity (21 soil bacterial isolates in equal proportions) and high-diversity natural top-soil communities followed highly reproducible succession paths, maintaining 16S rRNA gene amplicon signatures prominent for known soil communities in general. Development trajectories and compositional states were different for communities propagated in soil microcosms than in liquid suspension. Compositional states were maintained over multiple renewed growth cycles but could be diverged by short-term pollutant exposure. The different but robust trajectories demonstrated that deterministic taxa-inherent characteristics underlie reproducible development and self-organized complexity of soil microbiomes within their environmental boundary conditions. Our findings also have direct implications for potential strategies to achieve controlled restoration of desertified land. IMPORTANCE There is now a great awareness of the high diversity of most environmental ("free-living") and host-associated microbiomes, but exactly how diverse microbial communities form and maintain is still highly debated. A variety of theories have been put forward, but testing them has been problematic because most studies have been based on synthetic communities that fail to accurately mimic the natural composition (i.e., the species used are typically not found together in the same environment), the diversity (usually too low to be representative), or the environmental system itself (using designs with single carbon sources or solely mixed liquid cultures). In this study, we show how species-diverse soil bacterial communities can reproducibly be generated, propagated, and maintained, either from individual isolates (21 soil bacterial strains) or from natural microbial mixtures washed from top-soil. The high replicate consistency we achieve both in terms of species compositions and developmental trajectories demonstrates the strong inherent deterministic factors driving community formation from their species composition. Generating complex soil microbiomes may provide ways for restoration of damaged soils that are prevalent on our planet.

Bacterial surface properties influence the activity of the TAT-RasGAP317-326 antimicrobial peptide.

Antibiotic resistance is an increasing threat for public health, underscoring the need for new antibacterial agents. Antimicrobial peptides (AMPs) represent an alternative to classical antibiotics. TAT-RasGAP317-326 is a recently described AMP effective against a broad range of bacteria, but little is known about the conditions that may influence its activity. Using RNA-sequencing and screening of mutant libraries, we show that Escherichia coli and Pseudomonas aeruginosa respond to TAT-RasGAP317-326 by regulating metabolic and stress response pathways, possibly implicating two-component systems. Our results also indicate that bacterial surface properties, in particular integrity of the lipopolysaccharide layer, influence peptide binding and entry. Finally, we found differences between bacterial species with respect to their rate of resistance emergence against this peptide. Our findings provide the basis for future investigation on the mode of action of TAT-RasGAP317-326, which may help developing antimicrobial treatments based on this peptide.