Antibiotic-producing plant-associated bacteria, anti-virulence therapy and microbiome engineering: Integrated approaches in sustainable agriculture.

Plant health is crucial for maintaining the well-being of humans, animals and the environment. Plant pathogens pose significant challenges to agricultural production, global food security and ecosystem biodiversity. This problem is exacerbated by the impact of climate change, which is expected to alter the emergence and evolution of plant pathogens and their interaction with their plant hosts. Traditional approaches to managing phytopathogens involved the use of chemical pesticides, but alternative strategies are needed to address their ongoing decline in performance as well as their negative impact on the environment and public health. Here, we highlight the advancement and effectiveness of biocontrol strategies based on the use of antimicrobial-producing plant-associated bacteria, anti-virulence therapy (e.g. quorum quenching) and microbiome engineering as sustainable biotechnological approaches to promote plant health and foster sustainable agriculture. Notably, Enterobacterales are emerging as important biocontrol agents and as a source of new antimicrobials for potential agricultural use. We analysed here the genomes of over 250 plant-associated enterobacteria to examine their potential to synthesize secondary metabolites. Exploration of the plant microbiome is of major interest in the search for eco-friendly alternatives for reducing the use of chemical pesticides.

Bacterial amino acid chemotaxis: a widespread strategy with multiple physiological and ecological roles.

Chemotaxis is the directed, flagellum-based movement of bacteria in chemoeffector gradients. Bacteria respond chemotactically to a wide range of chemoeffectors, including amino, organic, and fatty acids, sugars, polyamines, quaternary amines, purines, pyrimidines, aromatic hydrocarbons, oxygen, inorganic ions, or polysaccharides. Most frequent are chemotactic responses to amino acids (AAs), which were observed in numerous bacteria regardless of their phylogeny and lifestyle. Mostly chemoattraction responses are observed, although a number of bacteria are repelled from certain AAs. Chemoattraction is associated with the important metabolic value of AAs as growth substrates or building blocks of proteins. However, additional studies revealed that AAs are also sensed as environmental cues. Many chemoreceptors are specific for AAs, and signaling is typically initiated by direct ligand binding to their four-helix bundle or dCache ligand-binding domains. Frequently, bacteria possess multiple AA-responsive chemoreceptors that at times possess complementary AA ligand spectra. The identification of sequence motifs in the binding sites at dCache_1 domains has permitted to define an AA-specific family of dCache_1AA chemoreceptors. In addition, AAs are among the ligands recognized by broad ligand range chemoreceptors, and evidence was obtained for chemoreceptor activation by the binding of AA-loaded solute-binding proteins. The biological significance of AA chemotaxis is very ample including in biofilm formation, root and seed colonization by beneficial bacteria, plant entry of phytopathogens, colonization of the intestine, or different virulence-related features in human/animal pathogens. This review provides insights that may be helpful for the study of AA chemotaxis in other uncharacterized bacteria.

Differential CheR Affinity for Chemoreceptor C-Terminal Pentapeptides Modulates Chemotactic Responses.

Many chemoreceptors contain a C-terminal pentapeptide at the end of a linker. In Escherichia coli, this pentapeptide forms a high-affinity binding site for CheR and phosphorylated CheB, and its removal interferes with chemoreceptor adaptation. Analysis of chemoreceptors revealed significant variation in their pentapeptide sequences, and bacteria often possess multiple chemoreceptors with differing pentapeptides. To assess whether this sequence variation alters CheR affinity and chemotaxis, we used Pectobacterium atrosepticum SCRI1043 as a model. SCRI1043 has 36 chemoreceptors, with 19 of them containing a C-terminal pentapeptide. We show that the affinity of CheR for the different pentapeptides varies up to 11-fold (KD 90 nM to 1 µM). Pentapeptides with the highest and lowest affinities differ only in a single amino acid. Deletion of the cheR gene abolishes chemotaxis. The replacement of the pentapeptide in the PacC chemoreceptor with those of the highest and lowest affinities significantly reduced chemotaxis to its cognate chemoeffector, L-Asp. Altering the PacC pentapeptide also reduced chemotaxis to L-Ser, but not to nitrate, which are responses mediated by the nontethered PacB and PacN chemoreceptors, respectively. Changes in the pentapeptide sequence thus modulate the response of the cognate receptor and that of another chemoreceptor.

Ubiquitous purine sensor modulates diverse signal transduction pathways in bacteria.

Purines and their derivatives control intracellular energy homeostasis and nucleotide synthesis, and act as signaling molecules. Here, we combine structural and sequence information to define a purine-binding motif that is present in sensor domains of thousands of bacterial receptors that modulate motility, gene expression, metabolism, and second-messenger turnover. Microcalorimetric titrations of selected sensor domains validate their ability to specifically bind purine derivatives, and evolutionary analyses indicate that purine sensors share a common ancestor with amino-acid receptors. Furthermore, we provide experimental evidence of physiological relevance of purine sensing in a second-messenger signaling system that modulates c-di-GMP levels.

Auxin-mediated regulation of susceptibility to toxic metabolites, c-di-GMP levels, and phage infection in the rhizobacterium Serratia plymuthica.

The communication between plants and their microbiota is highly dynamic and involves a complex network of signal molecules. Among them, the auxin indole-3-acetic acid (IAA) is a critical phytohormone that not only regulates plant growth and development, but is emerging as an important inter- and intra-kingdom signal that modulates many bacterial processes that are important during interaction with their plant hosts. However, the corresponding signaling cascades remain largely unknown. Here, we advance our understanding of the largely unknown mechanisms by which IAA carries out its regulatory functions in plant-associated bacteria. We showed that IAA caused important changes in the global transcriptome of the rhizobacterium Serratia plymuthica and multidisciplinary approaches revealed that IAA sensing interferes with the signaling mediated by other pivotal plant-derived signals such as amino acids and 4-hydroxybenzoic acid. Exposure to IAA caused large alterations in the transcript levels of genes involved in amino acid metabolism, resulting in significant metabolic alterations. IAA treatment also increased resistance to toxic aromatic compounds through the induction of the AaeXAB pump, which also confers resistance to IAA. Furthermore, IAA promoted motility and severely inhibited biofilm formation; phenotypes that were associated with decreased c-di-GMP levels and capsule production. IAA increased capsule gene expression and enhanced bacterial sensitivity to a capsule-dependent phage. Additionally, IAA induced the expression of several genes involved in antibiotic resistance and led to changes in the susceptibility and responses to antibiotics with different mechanisms of action. Collectively, our study illustrates the complexity of IAA-mediated signaling in plant-associated bacteria. IMPORTANCE: Signal sensing plays an important role in bacterial adaptation to ecological niches and hosts. This communication appears to be particularly important in plant-associated bacteria since they possess a large number of signal transduction systems that respond to a wide diversity of chemical, physical, and biological stimuli. IAA is emerging as a key inter- and intra-kingdom signal molecule that regulates a variety of bacterial processes. However, despite the extensive knowledge of the IAA-mediated regulatory mechanisms in plants, IAA signaling in bacteria remains largely unknown. Here, we provide insight into the diversity of mechanisms by which IAA regulates primary and secondary metabolism, biofilm formation, motility, antibiotic susceptibility, and phage sensitivity in a biocontrol rhizobacterium. This work has important implications for our understanding of bacterial ecology in plant environments and for the biotechnological and clinical applications of IAA, as well as related molecules.

Bacterial sensor evolved by decreasing complexity.

Bacterial receptors feed into multiple signal transduction pathways that regulate a variety of cellular processes including gene expression, second messenger levels and motility. Receptors are typically activated by signal binding to ligand binding domains (LBD). Cache domains are omnipresent LBDs found in bacteria, archaea, and eukaryotes, including humans. They form the predominant family of extracytosolic bacterial LBDs and were identified in all major receptor types. Cache domains are composed of either a single (sCache) or a double (dCache) structural module. The functional relevance of bimodular LBDs remains poorly understood. Here, we identify the PacF chemoreceptor in the phytopathogen Pectobacterium atrosepticum that recognizes formate at the membrane distal module of its dCache domain, triggering chemoattraction. We further demonstrate that a family of formate-specific sCache domains has evolved from a dCache domain, exemplified by PacF, by losing the membrane proximal module. By solving high-resolution structures of two family members in complex with formate, we show that the molecular basis for formate binding at sCache and dCache domains is highly similar, despite their low sequence identity. The apparent loss of the membrane proximal module may be related to the observation that dCache domains bind ligands typically at the membrane distal module, whereas the membrane proximal module is not involved in signal sensing. This work advances our understanding of signal sensing in bacterial receptors and suggests that evolution by reducing complexity may be a common trend shaping their diversity. Significance: Many bacterial receptors contain multi-modular sensing domains indicative of complex sensory processes. The presence of more than one sensing module likely permits the integration of multiple signals, although, the molecular detail and functional relevance for these complex sensors remain poorly understood. Bimodular sensory domains are likely to have arisen from the fusion or duplication of monomodular domains. Evolution by increasing complexity is generally believed to be a dominant force. Here we reveal the opposite - how a monomodular sensing domain has evolved from a bimodular one. Our findings will thus motivate research to establish whether evolution by decreasing complexity is typical of other sensory domains.

Sensing the environment by bacterial plant pathogens: What do their numerous chemoreceptors recognize?

Bacteria have evolved multiple sensing strategies to efficiently adapt to their natural hosts and environments. In the context of plant pathology, chemotaxis allows phytopathogenic bacteria to direct their movement towards hosts through the detection of a landscape of plant-derived molecules, facilitating the initiation of the infective process. The importance of chemotaxis for the lifestyle of phytopathogens is also reflected in the fact that they have, on average, twice as many chemoreceptors as bacteria that do not interact with plants. Paradoxically, the knowledge about the function of plant pathogen chemoreceptors is scarce. Notably, many of these receptors seem to be specific to plant-interacting bacteria, suggesting that they may recognize plant-specific compounds. Here, we highlight the need to advance our knowledge of phytopathogen chemoreceptor function, which may serve as a base for the development of anti-infective therapies for the control of phytopathogens.

Ubiquitous purine sensor modulates diverse signal transduction pathways in bacteria.

Purines and their derivatives are key molecules for controlling intracellular energy homeostasis and nucleotide synthesis. In eukaryotes, including humans, purines also act as signaling molecules that mediate extracellular communication and control key cellular processes, such as proliferation, migration, differentiation, and apoptosis. However, the signaling role of purines in bacteria is largely unknown. Here, by combining structural and sequence information, we define a purine-binding motif, which is present in sensor domains of thousands of bacterial receptors that modulate motility, gene expression, metabolism and second messenger turnover. The screening of compound libraries and microcalorimetric titrations of selected sensor domains validated their ability to specifically bind purine derivatives. The physiological relevance of purine sensing was demonstrated in a second messenger signaling system that modulates c-di-GMP levels.

Systematic mapping of chemoreceptor specificities for Pseudomonas aeruginosa.

IMPORTANCE: Chemotaxis of motile bacteria has multiple physiological functions. It enables bacteria to locate optimal ecological niches, mediates collective behaviors, and can play an important role in infection. These multiple functions largely depend on ligand specificities of chemoreceptors, and the number and identities of chemoreceptors show high diversity between organisms. Similar diversity is observed for the spectra of chemoeffectors, which include not only chemicals of high metabolic value but also bacterial, plant, and animal signaling molecules. However, the systematic identification of chemoeffectors and their mapping to specific chemoreceptors remains a challenge. Here, we combined several in vivo and in vitro approaches to establish a systematic screening strategy for the identification of receptor ligands and we applied it to identify a number of new physiologically relevant chemoeffectors for the important opportunistic human pathogen P. aeruginosa. This strategy can be equally applicable to map specificities of sensory domains from a wide variety of receptor types and bacteria.

Sensing preferences for prokaryotic solute binding protein families.

Solute binding proteins (SBPs) are of central physiological relevance for prokaryotes. These proteins present substrates to transporters, but they also stimulate different signal transduction receptors. SBPs form a superfamily of at least 33 protein Pfam families. To assess possible links between SBP sequence and the ligand recognized, we have inspected manually all SBP three-dimensional structures deposited in the protein data bank and retrieved 748 prokaryotic structures that have been solved in complex with bound ligand. These structures were classified into 26 SBP Pfam families. The analysis of the ligands recognized revealed that most families possess a preference for a compound class. There were three families each that bind preferentially saccharides and amino acids. In addition, we identified families that bind preferentially purines, quaternary amines, iron and iron-chelating compounds, oxoanions, bivalent metal ions or phosphates. Phylogenetic analyses suggest convergent evolutionary events that lead to families that bind the same ligand. The functional link between chemotaxis and compound uptake is reflected in similarities in the ligands recognized by SBPs and chemoreceptors. Associating Pfam families with ligand profiles will be of help to design experimental strategies aimed at the identification of ligands for uncharacterized SBPs.

Accessing nutrients as the primary benefit arising from chemotaxis.

About half of the known bacterial species perform chemotaxis that gains them access to sites that are optimal for growth and survival. The motility apparatus and chemotaxis signaling pathway impose a large energetic and metabolic burden on the cell. There is almost no limit to the type of chemoeffectors that are recognized by bacterial chemoreceptors. For example, they include hormones, neurotransmitters, quorum-sensing molecules, and inorganic ions. However, the vast majority of chemoeffectors appear to be of metabolic value. We review here the experimental evidence indicating that accessing nutrients is the main selective force that led to the evolution of chemotaxis.

The emerging role of auxins as bacterial signal molecules: Potential biotechnological applications.

Microorganisms are exposed in their natural niches to a wide diversity of signal molecules. Specific detection of these signals results in alterations in microbial metabolism and physiology. Auxins like indole-3-acetic acid are key phytohormones that regulate plant growth and development. Nonetheless, auxin biosynthesis is not restricted to plants but is ubiquitous in all kingdoms of life. This wide phylogenetic distribution of auxins production, together with the diversity of regulated cellular processes, have made auxins key intra- and inter-kingdom signal molecules in life modulating, for example microbial physiology, metabolism and virulence. Despite their increasing importance as global signal molecules, the mechanisms by which auxins perform their regulatory functions in microorganisms are largely unknown. In this article, we outline recent research that has advanced our knowledge of the mechanisms of bacterial auxin perception. We also highlight the potential applications of this research in aspects such as antibiotic production, biosensor design, plant microbiome engineering and antivirulence therapies.

Regulation of indole-3-acetic acid biosynthesis and consequences of auxin production deficiency in Serratia plymuthica.

Indole-3-acetic acid (IAA) is emerging as a key intra- and inter-kingdom signal molecule that modulates a wide range of processes of importance during plant-microorganism interaction. However, the mechanisms by which IAA carries out its functions in bacteria as well as the regulatory processes by which bacteria modulate auxin production are largely unknown. Here, we found that IAA synthesis deficiency results in important global transcriptional changes in the broad-range antibiotic-producing rhizobacterium Serratia plymuthica A153. Most pronounced transcriptional changes were observed in various gene clusters for aromatic acid metabolism, including auxin catabolism. To delve into the corresponding molecular mechanisms, different regulatory proteins were biochemically characterized. Among them, a TyrR orthologue was essential for IAA production through the activation of the ipdc gene encoding a key enzyme for IAA biosynthesis. We showed that TyrR specifically recognizes different aromatic amino acids which, in turn, alters the interactions of TyrR with the ipdc promoter. Screening of mutants defective in various transcriptional and post-transcriptional regulators allowed the identification of additional regulators of IAA production, including PigP and quorum sensing-related genes. Advancing our knowledge on the mechanisms that control the IAA biosynthesis in beneficial phytobacteria is of biotechnological interest for improving agricultural productivity and sustainable agricultural development.

Study of NIT domain-containing chemoreceptors from two global phytopathogens and identification of NIT domains in eukaryotes.

Bacterial signal transduction systems are typically activated by the binding of signal molecules to receptor ligand binding domains (LBDs), such as the NIT LBD. We report here the identification of the NIT domain in more than 15,000 receptors that were present in 30 bacterial phyla, but also in 19 eukaryotic phyla, expanding its known phylogenetic distribution. The NIT domain formed part of seven receptor families that either control transcription, mediate chemotaxis or regulate second messenger levels. We have produced the NIT domains from chemoreceptors of the bacterial phytopathogens Pectobacterium atrosepticum (PacN) and Pseudomonas savastanoi (PscN) as individual purified proteins. High-throughput ligand screening using compound libraries revealed a specificity for nitrate and nitrite binding. Isothermal titration calorimetry experiments showed that PacN-LBD bound preferentially nitrate ( K D = 1.9 µM), whereas the affinity of PscN-LBD for nitrite ( K D = 2.1 µM) was 22 times higher than that for nitrate. Analytical ultracentrifugation experiments indicated that PscN-LBD is monomeric in the presence and absence of ligands. The R182A mutant of PscN did not bind nitrate or nitrite. This residue is not conserved in the NIT domain of the Pseudomonas aeruginosa chemoreceptor PA4520, which may be related to its failure to bind nitrate/nitrite. The magnitude of P. atrosepticum chemotaxis towards nitrate was significantly greater than that of nitrite and pacN deletion almost abolished responses to both compounds. This study highlights the important role of nitrate and nitrite as signal molecules in life and advances our knowledge on the NIT domain as universal nitrate/nitrite sensor module.

Biosynthesis of Antifungal Solanimycin May Involve an Iterative Nonribosomal Peptide Synthetase Module.

Dickeya solani, a plant-pathogenic bacterium, produces solanimycin, a potent hybrid polyketide/nonribosomal peptide (PKS/NRPS) anti-fungal compound. The biosynthetic gene cluster responsible for synthesis of this compound has been identified. Because of instability, the complete structure of the compound has not yet been elucidated, but LC-MS2 identified that the cluster produces two main compounds, solanimycin A and B, differing by a single hydroxyl group. The fragmentation pattern revealed that the central part of solanimycin A is a hexapeptide, Gly-Dha-Dha-Dha-Dha-Dha (where Dha is dehydroalanine). This is supported by isotopic labeling studies using labeled serine and glycine. The N-terminal group is a polyketide-derived C16 acyl group containing a conjugated hexaene, a hydroxyl, and an amino group. The additional hydroxyl group in solanimycin B is on the α-carbon of the glycine residue. The incorporation of five sequential Dha residues is unprecedented because there is only one NRPS module in the cluster that is predicted to activate and attach serine (which is subsequently dehydrated to Dha), meaning that this NRPS module must act iteratively. While a few other iterative NRPS modules are known, they all involve iteration of two or three modules. We believe that the repetitive use of a single module makes the solanimycin biosynthetic pathway unique among NRPSs so far reported.

Three unrelated chemoreceptors provide Pectobacterium atrosepticum with a broad-spectrum amino acid sensing capability.

Amino acids are important nutrients and also serve as signals for diverse signal transduction pathways. Bacteria use chemoreceptors to recognize amino acid attractants and to navigate their gradients. In Escherichia coli two likely paralogous chemoreceptors Tsr and Tar detect 9 amino acids, whereas in Pseudomonas aeruginosa the paralogous chemoreceptors PctA, PctB and PctC detect 18 amino acids. Here, we show that the phytobacterium Pectobacterium atrosepticum uses the three non-homologous chemoreceptors PacA, PacB and PacC to detect 19 proteinogenic and several non-proteinogenic amino acids. PacB recognizes 18 proteinogenic amino acids as well as 8 non-proteinogenic amino acids. PacB has a ligand preference for the three branched chain amino acids L-leucine, L-valine and L-isoleucine. PacA detects L-proline next to several quaternary amines. The third chemoreceptor, PacC, is an ortholog of E. coli Tsr and the only one of the 36 P. atrosepticum chemoreceptors that is encoded in the cluster of chemosensory pathway genes. Surprisingly, in contrast to Tsr, which primarily senses serine, PacC recognizes aspartate as the major chemoeffector but not serine. Our results demonstrate that bacteria use various strategies to sense a wide range of amino acids and that it takes more than one chemoreceptor to achieve this goal.

The Cellular Abundance of Chemoreceptors, Chemosensory Signaling Proteins, Sensor Histidine Kinases, and Solute Binding Proteins of Pseudomonas aeruginosa Provides Insight into Sensory Preferences and Signaling Mechanisms.

Chemosensory pathways and two-component systems are important bacterial signal transduction systems. In the human pathogen Pseudomonas aeruginosa, these systems control many virulence traits. Previous studies showed that inorganic phosphate (Pi) deficiency induces virulence. We report here the abundance of chemosensory and two-component signaling proteins of P. aeruginosa grown in Pi deficient and sufficient media. The cellular abundance of chemoreceptors differed greatly, since a 2400-fold difference between the most and least abundant receptors was observed. For many chemoreceptors, their amount varied with the growth condition. The amount of chemoreceptors did not correlate with the magnitude of chemotaxis to their cognate chemoeffectors. Of the four chemosensory pathways, proteins of the Che chemotaxis pathway were most abundant and showed little variation in different growth conditions. The abundance of chemoreceptors and solute binding proteins indicates a sensing preference for amino acids and polyamines. There was an excess of response regulators over sensor histidine kinases in two-component systems. In contrast, ratios of the response regulators CheY and CheB to the histidine kinase CheA of the Che pathway were all below 1, indicative of different signaling mechanisms. This study will serve as a reference for exploring sensing preferences and signaling mechanisms of other bacteria.