ABSTRACT Bioaugmentation, the process of soil restoration by introducing microorganisms capable of degrading pollutants, is a promising and cost-effective strategy for environmental remediation. Aromatic hydrocarbons, such as benzene, toluene, ethylbenzene, and p-xylene (BTEX), are highly toxic environmental contaminants that could be transformed to less harmful products through the inoculation of certain organisms capable of BTEX degradation. However, a barrier to successful bioaugmentation is the inoculant’s failure to establish within the resident microbial community. In an effort to improve inoculant proliferation, we have investigated phosphite as a phosphorus source for selective nutrient supply. Phosphite is an inaccessible form of phosphorus to organisms that lack the capacity for phosphite oxidation to phosphate. We introduced a phosphite dehydrogenase-coding gene (ptxD) into the genome of the toluene-degrading bacterium Pseudomonas veronii 1YdBTEX2 to couple phosphite metabolism and aromatic hydrocarbon clearance. When inoculated in either soil matrix or liquid soil extract, P. veronii proliferates in a phosphite- and toluene-dependent manner in both growing and stable synthetic soil microbial communities, although the selective effects of phosphite and toluene were not additive in a carbon-limited context. Once toluene is metabolized, P. veronii abundance decays, and the microbial community recovers diversity and abundance resembling the uninoculated controls. Additional members of the microbial community were also enriched in the presence of phosphite, and genomic analysis suggests that these microorganisms utilize an alkaline phosphatase, phoV, for phosphite assimilation. IMPORTANCE Bioaugmentation is a promising solution to soil contamination, but its practical application is limited due to poor inoculant establishment in the native soil community. This can often be attributed to low nutrient availability and resource competition with native microorganisms. We proposed the use of phosphite as a selective nutrient source to support the growth of a toluene-degrading bacterium, Pseudomonas veronii, in a model soil system. We engineered a strain of this organism that was capable of using phosphite as a phosphorus source and saw that phosphite application enhanced the abundance of the inoculant sixfold within a synthetic soil community. In this study, we present the first investigation of a phosphite selection system in the soil microbiome and characterize the environmental conditions in which it is effective. By demonstrating the potential of formulated nutritional niches in soil microbiome interventions, we provide significant insights into the field of microbiome engineering. Bioaugmentation is a promising solution to soil contamination, but its practical application is limited due to poor inoculant establishment in the native soil community. This can often be attributed to low nutrient availability and resource competition with native microorganisms. We proposed the use of phosphite as a selective nutrient source to support the growth of a toluene-degrading bacterium, Pseudomonas veronii, in a model soil system. We engineered a strain of this organism that was capable of using phosphite as a phosphorus source and saw that phosphite application enhanced the abundance of the inoculant sixfold within a synthetic soil community. In this study, we present the first investigation of a phosphite selection system in the soil microbiome and characterize the environmental conditions in which it is effective. By demonstrating the potential of formulated nutritional niches in soil microbiome interventions, we provide significant insights into the field of microbiome engineering.

Abstract Human activities cause a global loss of soil microbiome diversity and functionality. One way to reverse this trend is through microbiota transplants, but the processes determining merger outcomes are not well understood. Here, we investigated the roles of habitat filtering and microbiota origin on microbiome development upon mergers, with the hypothesis that native strains are better adapted to their own habitat and will outcompete non-native ones in niche colonization. To test this, we contrasted community development in soil microcosms between two taxa-diverse microbiota originating from either topsoil [SoilCom (SC)] or freshwater lake [LakeCom (LC)], and a defined mixture of 21 soil bacteria (SynCom). When inoculated separately, SC and LC showed similar taxa and colonization patterns contributing to community growth and decline within the soil microcosms. SynCom transplants to either SC or LC under renewed growth conditions permanently altered their community trajectories, and slightly further converged their taxa compositions. Levels of SynCom members in both resident backgrounds decreased from initial 50–80% to below 1% within 2 months. Merged as well as non-merged communities resembled natural soils in comparison to over 81 000 publicly available soil, sediment, and lake microbiomes. Our results show that habitat filtering is dominant over microbiota taxa origin in determining transplant outcomes. Even though the proliferation of SynCom transplants remained limited, their capacity to influence community merger trajectories long term opens new paths for soil microbiome engineering.

Human activities cause global losses of soil microbiome diversity and functionality. Microbiota transplants offer a potential solution, but the factors influencing transplant success remain unclear. We investigated how microbiota origin affects microbiome mergers, hypothesizing that native strains through niche preference are better adapted to their habitat and will outcompete non-native ones. To test this, we contrasted transplants between soil microcosm-cultured topsoil or lakewater communities with a community of 21 soil bacteria (SynCom). In both cases, SynCom transplant increased resident productivity but permanently shifted compositions, although its abundance dropped from an initial 50-80% to <1% within two months. Both merged and non-merged communities resembled natural soil microbiota in comparisons with over 81,000 soil, sediment and lake compositional data. Our results show that habitat filtering and niche competition, more than microbiota origin, determine transplant outcomes. Despite the limited proliferation of SynCom transplants, their capacity to instill lasting community trajectory changes opens new paths for microbiome engineering. TEASER Even transiently present microbiota transplants can alter resident microbiome composition through processes governed by habitat filtering.

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. Bioremediation via microbial inoculation often performs poorly in real-world conditions. Here, the authors show that bacterial inoculants may fail to establish in complex soil microbiomes because they open new niches that facilitate growth of resident microbes.

SUMMARY Communities 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.

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

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. ABSTRACT 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.

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). ABSTRACT 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.

Microbiomes are typically characterised by high species diversity but it is poorly understood how such system-level complexity can be generated and propagated. Here, we used soils as a relevant model to study microbiome development. 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 distinct soil microbiome signatures. Development trajectories and compositional states were different for communities propagated in soils than in liquid suspension. Microbiome 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. TEASER Species-rich soil microbiomes grow and propagate reproducibly despite inherent stochastic complexity, paving the way for soil restoration.

Antimicrobial peptides (AMPs) are molecules with antimicrobial activity and could be a promising alternative to classical antibiotics, whose clinical efficiency is undermined by emergence of resistance. Our group is studying one such antibiotic alternative – the antimicrobial peptide TAT-RasGAP317-326. We recently reported the antimicrobial activity of this peptide against a range of Gram-positive and Gram-negative bacteria. In this article, we show that the presence of divalent cations and low pH levels have an impact on TAT-RasGAP317-326 activity, whereas serum proteins only partially reduce the antibacterial activity of TAT-RasGAP317-326. In addition, we show that iron supplementation reduces TAT-RasGAP317-326 binding to bacteria. Using a transcriptomics approach and screening of bacterial mutant libraries, we map the transcriptional response of bacteria when exposed to TAT-RasGAP317-326 and identify cellular pathways that may play a role in bacterial resistance to TAT-RasGAP317-326. We test combinations of TAT-RasGAP317-326 with other AMPs and detect no evidence for an additive effect between any of the peptide combinations. Finally, we perform a resistance selection screen that reveals differences between bacterial strains with respect to their rate of resistance emergence against the TAT-RasGAP317-326 peptide. Taken together, our findings bring a better understanding of how extracellular factors might impact the antimicrobial activity of TAT-RasGAP317-326 peptide and thus contribute basic biology insight into the mechanisms behind TAT-RasGAP317-326 activity, potentially aiding future strategies to improve the efficiency of this peptide in vivo.