Periodic Reporting for period 1 - LuxSoM (LuxR solos as major proteobacterial players of cell-cell signaling in the plant microbiome)
Periodo di rendicontazione: 2024-11-01 al 2025-10-31
Sintesi del contesto e degli obiettivi generali del progetto
About 60 to 70% of soils in the EU are subject to severe degradation processes: erosion, pollution, loss of biodiversity, salinisation and sealing; this is the result of unsustainable land use and overexploitation of agrochemicals. Microbial-based solutions, in agriculture, are now becoming important alternatives as an environmentally friendly strategy to agrochemical additives. However, microbially-based inoculants are often unstable or lose efficacy over time due to poor colonization mainly because of the limited knowledge of the mechanisms underlying the formation and maintenance of plant-associated microbial communities. To develop more effective microbial-based strategies we need to increase the knowledge on: (i) microbial communities behaviour in natural environments and (ii) bacterial cell-cell interaction networks that influence community composition, dynamics, and stability. Numerous mechanisms of cell-cell interactions have been studied in laboratory set-ups (mainly pure cultures), but their roles in natural environments (e.g root-microbiome) remain elusive, preventing concrete applications. The practical exploitation of these mechanisms will very likely lead to novel solutions for a sustainable agriculture, in order to fight the upcoming challenge of climate change. Main members of the root microbiome are Proteobacteria, as they account for 50% of the bacterial population, and LuxSoM aims to generate critical insights on their assembly and cell-cell communication mechanisms via a well-defined and targeted approach. LuxR solos, which evolved from cell-cell signaling quorum sensing systems (QS), are very widespread and almost exclusively found in Proteobacteria. They are a wide family of transcriptional regulators that respond to endogenous or exogenous (also of plant origin) signals. We intend to study the LuxR solos by genomics, genetics, molecular biology, analytical and molecular chemistry, biochemistry, microbiome analysis and state-of -the-art mass spectrometry based technologies. The main aim of this project is to explore the importance of bacterial LuxR solos in the plant-microbiome network, understanding their influence on plant host physiology and microbial community dynamics. This research seeks to develop a community of interacting plant-beneficial strains that will serve as a probiotic solution for plants to enhance plant health and sustainable agricultural productivity. Moreover, the discovery of novel bacterial signal molecules will also open a realm of possibilities for the utilization of these compounds as prebiotics in order to influence plant microbiomes and thus devise new ways to enhance plant health in a sustainable way. The study of LuxR solos-based systems will bridge the gap of our understanding of how bacteria engage in contact-independent cell-cell communication, allowing also the design of new probiotic and prebiotic solutions for agriculture. This project will unravel the first major cell-cell signal players for plant microbiome establishment.
Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti
The activities are performed under the following 4 axis:
1)R&IO1- Establishment of an inclusive list of LuxR solos candidates through in silico genome mining : Through an exhaustive bioinformatic analysis of genomes from plant-beneficial bacteria, the goal is to pinpoint at least 5 subclasses of LuxR solo candidates that represent diverse cell-cell signaling systems responding to different signals and cues.
2)R&IO2- Generation of luxR solo gene knock-out mutants and LuxR solo-biosensor representatives: Through genomic mutant construction along with transcriptional activity studies (promoter studies, qPCR etc.) of each luxR solo and nearby located small-molecule biosynthetic gene clusters and generation of LuxR-solo biosensor vectors, the goal is to create a collection of molecular tools to validate our hypothesis.
3)R&IO3 - High-throughput identification of novel LuxR solo signal molecules in plant-associated bacteria by mass spectrometry(MS) and LuxR solo-based biosensors : Through mass spectrometry(MS)spectra comparison between WT and relative LuxR and associated BGC mutants, the goal is to discover new BCGs which synthesize novel small molecular weight molecules. These newly discovered molecules might also play a role in signaling between different species. In addition, the application of these newly identified molecules might trigger an enhanced resistance state, also termed as induced systemic resistance (ISR), in the plant-host, against a broad range of pathogens, being in this way involved in interkingdom signaling. Test whether the LuxR solo representative are also able to detect exogenous ligands produced by the SynCom rather than individual bacterial strains, by using LuxR solo-based biosensors.
4) Integration of metabolic networks data with microbiome data to design efficient probiotic and prebiotic solutions : The goal is to investigate the function of the newly identified LuxR solo classes involved in signaling amongst members of a SynCom (generated R&IO3) by using metabolic network approaches. This will be achieved by: (i) comparing metabolite signatures of simplified communities formed by luxR-deficient and luxR-wild-type members matching tandem mass spectrometry data from the two conditions tested; (ii) utilizing MS imaging techniques for in situ visualization of the spatial distribution of chemical molecules in 2D and 3D; (iii) conducting 16S rRNA community studies to validate the SynCom profile on plant roots, confirming the presence and activity of the targeted bacterial members. Finally, once identified specific bacterial members interacting via a type of LuxR solo-based system, I will generate a consortium of plant-beneficial strains to be used as plant probiotic. A similar approach will be used to target the effect on the microbiome of the newly identified LuxR solo-responding signals, to design prebiotic solutions.
Last update: February 2025
• R&IO1- Establishment of an inclusive list of LuxR solos candidates through in silico genome mining (M4, target: at least 10 subclasses of LuxR solo candidates, means of verification: release of bioinformatic scripts and results. In silico genome mining and classification of LuxR solos → fully completed. A dataset of 26,577 LuxR solos from 16,683 genomes was analyzed; ~62 % show adjacent operons/BGCs, yielding 584 operon-type clusters. M1. Bioinformatic scripts and table results are deposited in GitHub repository under the link: D1.2(si apre in una nuova finestra)
• R&IO2 - Generation of luxR solo gene knock-out mutants and LuxR solo-biosensors (M6, target: 10 knock-out mutants representing 10 different subclasses of LuxR solos involved in intraspecies and interspecies signaling as explained below; molecular vectors needed for the study, means of verification: bacterial collection of wild-type strains + knock-out mutants; generation of a series of LuxR-solo biosensor vectors): Knock-out and biosensor construction → partially completed. 13 mutants have been generated and molecularly validated, multiple promoter–reporter constructs have been generated and will be soon used to confirm the regulatory pathways of the candidates LuxR solos under studied (M2). A collection of molecular knock-out mutants for the selected bacterial strains has been generated and saved at -80C in ICGEB. (D2)
• R&IO3 - High-throughput identification of novel LuxR solo signal molecules in plant-associated bacteria by mass spectrometry(MS) and LuxR solo-based biosensors (M15, target: identification of the chemical signals binding each of the 10 selected LuxR solo; means of verification: 2 publications; generation of a SynCom model; release of (MS/MS) spectrometry data: Metabolomic profiling and signal discovery → advanced. Comprehensive LC-MS/MS untargeted pipeline established and validated across five model strains. Identification of putative signal metabolites (i.e. phenazine derivatives, isocoumarin-derivatives and N-acetyltyramine derivatives) to be fully validated via retention time matching and NMR (M3.1). Untargeted metabolomics raw data have been released in GNPS (Task ID: 39a791c811f743b2be07efe42d9fb9f7) and MassIVE (MassIVE MSV000098307) (D3.2). One manuscript has been submitted to Cell Reports and the other one is under finalization.
• R&IO4 - Integration of metabolic networks data with microbiome data to design efficient probiotic and prebiotic solutions (M24, target: comprehensive understanding of the function of LuxR solo circuits in a microbiome context, mean of verification: 1 publication; development of at least one probiotic consortium and prebiotic compound): Integration of metabolomic data with microbiome networks → initiated. The strains to be used in the plant-inspired SynCom have been chosen, collected and partially characterized physiochemically.
Last update: October 2025
1)R&IO1- Establishment of an inclusive list of LuxR solos candidates through in silico genome mining : Through an exhaustive bioinformatic analysis of genomes from plant-beneficial bacteria, the goal is to pinpoint at least 5 subclasses of LuxR solo candidates that represent diverse cell-cell signaling systems responding to different signals and cues.
2)R&IO2- Generation of luxR solo gene knock-out mutants and LuxR solo-biosensor representatives: Through genomic mutant construction along with transcriptional activity studies (promoter studies, qPCR etc.) of each luxR solo and nearby located small-molecule biosynthetic gene clusters and generation of LuxR-solo biosensor vectors, the goal is to create a collection of molecular tools to validate our hypothesis.
3)R&IO3 - High-throughput identification of novel LuxR solo signal molecules in plant-associated bacteria by mass spectrometry(MS) and LuxR solo-based biosensors : Through mass spectrometry(MS)spectra comparison between WT and relative LuxR and associated BGC mutants, the goal is to discover new BCGs which synthesize novel small molecular weight molecules. These newly discovered molecules might also play a role in signaling between different species. In addition, the application of these newly identified molecules might trigger an enhanced resistance state, also termed as induced systemic resistance (ISR), in the plant-host, against a broad range of pathogens, being in this way involved in interkingdom signaling. Test whether the LuxR solo representative are also able to detect exogenous ligands produced by the SynCom rather than individual bacterial strains, by using LuxR solo-based biosensors.
4) Integration of metabolic networks data with microbiome data to design efficient probiotic and prebiotic solutions : The goal is to investigate the function of the newly identified LuxR solo classes involved in signaling amongst members of a SynCom (generated R&IO3) by using metabolic network approaches. This will be achieved by: (i) comparing metabolite signatures of simplified communities formed by luxR-deficient and luxR-wild-type members matching tandem mass spectrometry data from the two conditions tested; (ii) utilizing MS imaging techniques for in situ visualization of the spatial distribution of chemical molecules in 2D and 3D; (iii) conducting 16S rRNA community studies to validate the SynCom profile on plant roots, confirming the presence and activity of the targeted bacterial members. Finally, once identified specific bacterial members interacting via a type of LuxR solo-based system, I will generate a consortium of plant-beneficial strains to be used as plant probiotic. A similar approach will be used to target the effect on the microbiome of the newly identified LuxR solo-responding signals, to design prebiotic solutions.
Last update: February 2025
• R&IO1- Establishment of an inclusive list of LuxR solos candidates through in silico genome mining (M4, target: at least 10 subclasses of LuxR solo candidates, means of verification: release of bioinformatic scripts and results. In silico genome mining and classification of LuxR solos → fully completed. A dataset of 26,577 LuxR solos from 16,683 genomes was analyzed; ~62 % show adjacent operons/BGCs, yielding 584 operon-type clusters. M1. Bioinformatic scripts and table results are deposited in GitHub repository under the link: D1.2(si apre in una nuova finestra)
• R&IO2 - Generation of luxR solo gene knock-out mutants and LuxR solo-biosensors (M6, target: 10 knock-out mutants representing 10 different subclasses of LuxR solos involved in intraspecies and interspecies signaling as explained below; molecular vectors needed for the study, means of verification: bacterial collection of wild-type strains + knock-out mutants; generation of a series of LuxR-solo biosensor vectors): Knock-out and biosensor construction → partially completed. 13 mutants have been generated and molecularly validated, multiple promoter–reporter constructs have been generated and will be soon used to confirm the regulatory pathways of the candidates LuxR solos under studied (M2). A collection of molecular knock-out mutants for the selected bacterial strains has been generated and saved at -80C in ICGEB. (D2)
• R&IO3 - High-throughput identification of novel LuxR solo signal molecules in plant-associated bacteria by mass spectrometry(MS) and LuxR solo-based biosensors (M15, target: identification of the chemical signals binding each of the 10 selected LuxR solo; means of verification: 2 publications; generation of a SynCom model; release of (MS/MS) spectrometry data: Metabolomic profiling and signal discovery → advanced. Comprehensive LC-MS/MS untargeted pipeline established and validated across five model strains. Identification of putative signal metabolites (i.e. phenazine derivatives, isocoumarin-derivatives and N-acetyltyramine derivatives) to be fully validated via retention time matching and NMR (M3.1). Untargeted metabolomics raw data have been released in GNPS (Task ID: 39a791c811f743b2be07efe42d9fb9f7) and MassIVE (MassIVE MSV000098307) (D3.2). One manuscript has been submitted to Cell Reports and the other one is under finalization.
• R&IO4 - Integration of metabolic networks data with microbiome data to design efficient probiotic and prebiotic solutions (M24, target: comprehensive understanding of the function of LuxR solo circuits in a microbiome context, mean of verification: 1 publication; development of at least one probiotic consortium and prebiotic compound): Integration of metabolomic data with microbiome networks → initiated. The strains to be used in the plant-inspired SynCom have been chosen, collected and partially characterized physiochemically.
Last update: October 2025
Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)
Expected results include:
1) The plant microbiome is primarily composed of bacteria belonging to four phyla (Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes) with Proteobacteria being greatly predominant in the root microbiome and certain proteobacterial genera being members of a universal core plant microbiome. However, little is known on the mechanisms underlying the formation and maintenance of these well-structured microbial communities. Based on my recent publication, all of these genera contain multiple LuxR solos genes, indicating diverse binding properties and biological roles. Investigating the prevalence and significance of LuxR solos in these genera will most likely reveal the first major regulatory family involved in dynamic cell-cell signaling in the plant microbiome.
2) Until now, only a limited number of molecular and genetic studies have investigated the mechanism of action of few classes of LuxR solos, notably the subfamilies of PAB (Plant Associated Bacteria) which respond to a plant signal and SdiA which responds to exogenous AHLs. However, there are many more classes which will play other major roles in cell-cell signaling and all need to be studied and mapped in a microbiome-context in order to understand their role in shaping and forming the plant-root microbiome, as proposed in this project. R&IO2 aims to bridge this knowledge gap by establishing a robust genetic and molecular foundation for the project. These resources will not only be valuable for the current study but will also serve as valuable tools for future research.
3) Recently, I have discovered a LuxR solo located adjacent to a large BGC operon which regulates the solo gene transcriptionally creating a positive feedback loop that lead to intra-species signaling (unpublished data). Moreover, I preliminarily observed that a considerable number of luxR solos subclasses are situated adjacent to uncharacterized BGC clusters, suggesting a link between them and the adjacent regulators. Building on this finding, R&IO3 will focus on a set of LuxR solos near uncharacterized BGCs, to discover novel endogenously produced signals representing new cell-cell signaling circuits. This area of research is relatively unexplored, with only two known instances of LuxR solos responding to endogenous molecules [16,17]. The uncharacterized BGC clusters which synthesize new small molecular-weight molecules hold significant potential for applications as for example using them as prebiotics in order to affect/control plant microbiomes as well as testing them for enhancing plant-host immunity by ISR. So far, several plant beneficial microbes derived molecules are known to stimulate plant-defense responses and help plants to obtain broad-spectrum disease resistance. Moreover, limited information is available on the involvement of LuxR solos in inter-species bacterial communication and the types of molecules they can bind and respond to. This innovative approach provides insight into interbacterial cell-cell signaling via a major predominant novel family of regulators and in a pertinent set-up resembling what occurs in planta. Furthermore, microbial compounds produced in a microbiome environment can be used to manipulate the plant/soil resident microbiome devising environmentally friendly approaches to sustainable agriculture
4)A current major challenge is to understand the cell-cell signaling interactions among bacteria to devise ways to harness the microbiome. Hardly any studies/data is available on the role of cell-cell communication processes between bacteria in a plant microbiome context. Cell-cell signals can also be synthesized only in a microbiome context as bacterial species produce them by responding to microbiome and/or other environmental cues; this very important aspect is currently not been addressed as most studies are being performed in simple/pure culture laboratory set-ups. By using advanced mass spectrometry methods and data processing, I will analyze the metabolomic profiles of synthetic communities (SynComs) and study the effects of LuxR solo-based signaling on the microbiome. This approach will provide a currently unique deeper understanding of microbial communities' functions and implications. Until now, very few multi-strain consortia have been employed as probiotics to enhance plant yield and health. Most of these showed limited activity or efficacy, likely also due to the lack of understanding on the inter-bacterial interactions among the different members of the consortia. I will develop a consortium comprised of plant-beneficial strains that are compatible and can undergo LuxR solo-based cell-cell signaling for improved interspecies as well plant-bacteria interactions.
1) The plant microbiome is primarily composed of bacteria belonging to four phyla (Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes) with Proteobacteria being greatly predominant in the root microbiome and certain proteobacterial genera being members of a universal core plant microbiome. However, little is known on the mechanisms underlying the formation and maintenance of these well-structured microbial communities. Based on my recent publication, all of these genera contain multiple LuxR solos genes, indicating diverse binding properties and biological roles. Investigating the prevalence and significance of LuxR solos in these genera will most likely reveal the first major regulatory family involved in dynamic cell-cell signaling in the plant microbiome.
2) Until now, only a limited number of molecular and genetic studies have investigated the mechanism of action of few classes of LuxR solos, notably the subfamilies of PAB (Plant Associated Bacteria) which respond to a plant signal and SdiA which responds to exogenous AHLs. However, there are many more classes which will play other major roles in cell-cell signaling and all need to be studied and mapped in a microbiome-context in order to understand their role in shaping and forming the plant-root microbiome, as proposed in this project. R&IO2 aims to bridge this knowledge gap by establishing a robust genetic and molecular foundation for the project. These resources will not only be valuable for the current study but will also serve as valuable tools for future research.
3) Recently, I have discovered a LuxR solo located adjacent to a large BGC operon which regulates the solo gene transcriptionally creating a positive feedback loop that lead to intra-species signaling (unpublished data). Moreover, I preliminarily observed that a considerable number of luxR solos subclasses are situated adjacent to uncharacterized BGC clusters, suggesting a link between them and the adjacent regulators. Building on this finding, R&IO3 will focus on a set of LuxR solos near uncharacterized BGCs, to discover novel endogenously produced signals representing new cell-cell signaling circuits. This area of research is relatively unexplored, with only two known instances of LuxR solos responding to endogenous molecules [16,17]. The uncharacterized BGC clusters which synthesize new small molecular-weight molecules hold significant potential for applications as for example using them as prebiotics in order to affect/control plant microbiomes as well as testing them for enhancing plant-host immunity by ISR. So far, several plant beneficial microbes derived molecules are known to stimulate plant-defense responses and help plants to obtain broad-spectrum disease resistance. Moreover, limited information is available on the involvement of LuxR solos in inter-species bacterial communication and the types of molecules they can bind and respond to. This innovative approach provides insight into interbacterial cell-cell signaling via a major predominant novel family of regulators and in a pertinent set-up resembling what occurs in planta. Furthermore, microbial compounds produced in a microbiome environment can be used to manipulate the plant/soil resident microbiome devising environmentally friendly approaches to sustainable agriculture
4)A current major challenge is to understand the cell-cell signaling interactions among bacteria to devise ways to harness the microbiome. Hardly any studies/data is available on the role of cell-cell communication processes between bacteria in a plant microbiome context. Cell-cell signals can also be synthesized only in a microbiome context as bacterial species produce them by responding to microbiome and/or other environmental cues; this very important aspect is currently not been addressed as most studies are being performed in simple/pure culture laboratory set-ups. By using advanced mass spectrometry methods and data processing, I will analyze the metabolomic profiles of synthetic communities (SynComs) and study the effects of LuxR solo-based signaling on the microbiome. This approach will provide a currently unique deeper understanding of microbial communities' functions and implications. Until now, very few multi-strain consortia have been employed as probiotics to enhance plant yield and health. Most of these showed limited activity or efficacy, likely also due to the lack of understanding on the inter-bacterial interactions among the different members of the consortia. I will develop a consortium comprised of plant-beneficial strains that are compatible and can undergo LuxR solo-based cell-cell signaling for improved interspecies as well plant-bacteria interactions.