Chemical Proportionality within Molecular Networks.

Molecular networking of non-targeted tandem mass spectrometry data connects structurally related molecules based on similar fragmentation spectra. Here, we report the Chemical Proportionality (ChemProp) contextualization of molecular networks. ChemProp scores the changes of abundance between two connected nodes over sequential data series (e.g., temporal or spatial relationships), which can be displayed as a direction within the network to prioritize potential biological and chemical transformations or proportional changes of (biosynthetically) related compounds. We tested the ChemProp workflow on a ground truth data set of a defined mixture and highlighted the utility of the tool to prioritize specific molecules within biological samples, including bacterial transformations of bile acids, human drug metabolism, and bacterial natural products biosynthesis. The ChemProp workflow is freely available through the Global Natural Products Social Molecular Networking (GNPS) environment.

More than words: the chemistry behind the interactions in the plant holobiont.

Plants and microbes have evolved sophisticated ways to communicate and coexist. The simplest interactions that occur in plant-associated habitats, i.e., those involved in disease detection, depend on the production of microbial pathogenic and virulence factors and the host's evolved immunological response. In contrast, microbes can also be beneficial for their host plants in a number of ways, including fighting pathogens and promoting plant growth. In order to clarify the mechanisms directly involved in these various plant-microbe interactions, we must still deepen our understanding of how these interkingdom communication systems, which are constantly modulated by resident microbial activity, are established and, most importantly, how their effects can span physically separated plant compartments. Efforts in this direction have revealed a complex and interconnected network of molecules and associated metabolic pathways that modulate plant-microbe and microbe-microbe communication pathways to regulate diverse ecological responses. Once sufficiently understood, these pathways will be biotechnologically exploitable, for example, in the use of beneficial microbes in sustainable agriculture. The aim of this review is to present the latest findings on the dazzlingly diverse arsenal of molecules that efficiently mediate specific microbe-microbe and microbe-plant communication pathways during plant development and on different plant organs.

Global transcriptional response of solvent-sensitive and solvent-tolerant Pseudomonas putida strains exposed to toluene.

Pseudomonas putida strains are generally recognized as solvent tolerant, exhibiting varied sensitivity to organic solvents. Pan-genome analysis has revealed that 30% of genes belong to the core-genome of Pseudomonas. Accessory and unique genes confer high degree of adaptability and capabilities for the degradation and synthesis of a wide range of chemicals. For the use of these microbes in bioremediation and biocatalysis, it is critical to understand the mechanisms underlying these phenotypic differences. In this study, RNA-seq analysis compared the short- and long-term responses of the toluene-sensitive KT2440 strain and the highly tolerant DOT-T1E strain. The sensitive strain activates a larger number of genes in a higher magnitude than DOT-T1E. This is expected because KT2440 bears one toluene tolerant pump, while DOT-T1E encodes three of these pumps. Both strains activate membrane modifications to reduce toluene membrane permeability. The KT2440 strain activates the TCA cycle to generate energy, while avoiding energy-intensive processes such as flagellar biosynthesis. This suggests that KT2440 responds to toluene by focusing on survival mechanisms. The DOT-T1E strain activates toluene degradation pathways, using toluene as source of energy. Among the unique genes encoded by DOT-T1E is a 70 kb island composed of genes of unknown function induced in response to toluene.

Pseudomonas putida as a platform for the synthesis of aromatic compounds.

Aromatic compounds such as l-phenylalanine, 2-phenylethanol and trans-cinnamate are aromatic compounds of industrial interest. Current trends support replacement of chemical synthesis of these compounds by 'green' alternatives produced in microbial cell factories. The solvent-tolerant Pseudomonas putida DOT-T1E strain was genetically modified to produce up to 1 g l-1 of l-phenylalanine. In order to engineer this strain, we carried out the following stepwise process: (1) we selected random mutants that are resistant to toxic phenylalanine analogues; (2) we then deleted up to five genes belonging to phenylalanine metabolism pathways, which greatly diminished the internal metabolism of phenylalanine; and (3) in these mutants, we overexpressed the pheAfbr gene, which encodes a recombinant variant of PheA that is insensitive to feedback inhibition by phenylalanine. Furthermore, by introducing new genes, we were able to further extend the diversity of compounds produced. Introduction of histidinol phosphate transferase (PP_0967), phenylpyruvate decarboxylase (kdc) and an alcohol dehydrogenase (adh) enabled the strain to produce up to 180 mg l-1 2-phenylethanol. When phenylalanine ammonia lyase (pal) was introduced, the resulting strain produced up to 200 mg l-1 of trans-cinnamate. These results demonstrate that P. putida can serve as a promising microbial cell factory for the production of l-phenylalanine and related compounds.

GtrS and GltR form a two-component system: the central role of 2-ketogluconate in the expression of exotoxin A and glucose catabolic enzymes in Pseudomonas aeruginosa.

In the human pathogen Pseudomonas aeruginosa, the GltR regulator is required for glucose transport, whereas GtrS is a sensor kinase that plays a key role in mediating bacteria-host interaction and pathogen dissemination in the host. We show that GtrS and GltR form a two-component system that regulates the expression from the promoters Pedd/gap-1, PoprB and Pglk, which control the expression of genes involved in glucose metabolism and transport. In addition, the GtrS/GltR pair regulates the expression of toxA that encodes exotoxin A, the primary virulence factor. Microcalorimetry-based ligand screening of the recombinant GtrS ligand-binding domain revealed specific binding of 2-ketogluconate (2-KG) (KD=5 µM) and 6-phosphogluconate (KD=98 µM). These effectors accelerate GtrS autophosphorylation, with concomitant transphosphorylation of GltR leading to a three-fold increase in transcription. Surprisingly, in vivo a similar increase in expression from the above promoters was observed for the mutant deficient in GltR regardless of the presence of effectors. The GltR operator site was found to contain the consensus sequence 5'-tgGTTTTTc-3'. We propose that 2-KG is a key metabolite in the stringent transcriptional control of genes involved in virulence and glucose metabolism. We show that GltR is a transcriptional repressor that is released from DNA upon phosphorylation.

Differential transcriptional response to antibiotics by Pseudomonas putida†DOT-T1E.

Multi-drug resistant bacteria are a major threat to humanity, especially because the current battery of known antibiotics is not sufficient to combat infections produced by these microbes. Therefore, the study of how current antibiotics act and how bacteria defend themselves against antibiotics is of critical importance. Pseudomonas putida DOT-T1E exhibits an impressive array of RND efflux pumps, which confer this microorganism high resistance to organic solvents and antibiotics that would kill most other microorganisms. We have chosen DOT-T1E as a model microbe to study the microbial responses to a wide battery of antibiotics (chloramphenicol, rifampicin, tetracycline, ciprofloxacin, ampicillin, kanamycin, spectinomycin and gentamicin). Ribonucleic acid sequencing (RNA)-seq analyses revealed that each antibiotic provokes a unique transcriptional response profile in DOT-T1E. While many of the genes identified were related to known antibiotic targets, others were unrelated or encoded hypothetical proteins. These results indicate that our knowledge of antibiotic resistance mechanisms is still partial. We also identified 138 new small RNAs (sRNAs) in DOT-T1E, dramatically adding to the 16 that have been previously described. Importantly, our results reveal that a correlation exists between the expression of messenger RNA and sRNA, indicating that some of these sRNAs are likely involved in fine tuning the expression of antibiotic resistance genes. Taken together, these findings open new frontiers in the fight against multi-drug resistant bacteria and point to the potential use of sRNAs as novel antimicrobial targets.

Identification of new residues involved in intramolecular signal transmission in a prokaryotic transcriptional repressor.

TtgV is a member of the IclR family of transcriptional regulators. This regulator controls its own expression and that of the ttgGHI operon, which encodes an RND efflux pump. TtgV has two domains: a GAF-like domain harboring the effector-binding pocket and a helix-turn-helix (HTH) DNA-binding domain, which are linked by a long extended helix. When TtgV is bound to DNA, a kink at residue 86 in the extended helix gives rise to 2 helices. TtgV contacts DNA mainly through a canonical recognition helix, but its three-dimensional structure bound to DNA revealed that two residues, R19 and S35, outside the HTH motif, directly contact DNA. Effector binding to TtgV releases it from DNA; when this occurs, the kink at Q86 is lost and residues R19 and S35 are displaced due to the reorganization of the turn involving residues G44 and P46. Mutants of TtgV were generated at positions 19, 35, 44, 46, and 86 by site-directed mutagenesis to further analyze their role. Mutant proteins were purified to homogeneity, and differential scanning calorimetry (DSC) studies revealed that all mutants, except the Q86N mutant, unfold in a single event, suggesting conservation of the three-dimensional organization. All mutant variants bound effectors with an affinity similar to that of the parental protein. R19A, S35A, G44A, Q86N, and Q86E mutants did not bind DNA. The Q86A mutant was able to bind to DNA but was only partially released from its target operator in response to effectors. These results are discussed in the context of intramolecular signal transmission from the effector binding pocket to the DNA binding domain.

Interspecies signalling: Pseudomonas putida efflux pump TtgGHI is activated by indole to increase antibiotic resistance.

In Gram-negative bacteria, multidrug efflux pumps are responsible for the extrusion of chemicals that are deleterious for growth. Some of these efflux pumps are induced by endogenously produced effectors, while abiotic or biotic signals induce the expression of other efflux pumps. In Pseudomonas putida, the TtgABC efflux pump is the main antibiotic extrusion system that respond to exogenous antibiotics through the modulation of the expression of this operon mediated by TtgR. The plasmid-encoded TtgGHI efflux pump in P. putida plays a minor role in antibiotic resistance in the parental strain; however, its role is critical in isogenic backgrounds deficient in TtgABC. Expression of ttgGHI is repressed by the TtgV regulator that recognizes indole as an effector, although P. putida does not produce indole itself. Because indole is not produced by Pseudomonas, the indole-dependent antibiotic resistance seems to be part of an antibiotic resistance programme at the community level. Pseudomonas putida recognizes indole added to the medium or produced by Escherichia coli in mixed microbial communities. Transcriptomic analyses revealed that the indole-specific response involves activation of 43 genes and repression of 23 genes. Indole enhances not only the expression of the TtgGHI pump but also a set of genes involved in iron homeostasis, as well as genes for amino acid catabolism. In a ttgABC-deficient P. putida, background ampicillin and other bactericidal compounds lead to cell death. Co-culture of E. coli and P. putida ΔttgABC allowed growth of the P. putida mutant in the presence of ampicillin because of induction of the indole-dependent efflux pump.

Unravelling the impact of environmental factors in shaping plant microbiomes.

This article emphasizes the significant role of environmental factors in shaping the plant microbiome, highlighting how bacterial and fungal communities influence plant responses to water stress, and how environmental factors shape fungal communities in crops. Furthermore, recent studies describe how different genotypes and levels of water stress affect the composition of bacterial communities associated with quinoa plants, as well as the relationship between environmental factors and the structure of fungal communities in apple fruit. These findings underscore the importance of understanding plant microbiome dynamics in developing effective crop protection strategies and improving agricultural sustainability with the objective of advance towards a microbiome-based strategy which allows us to improve crop tolerance to abiotic stresses.

Sporulation Activated via σW Protects Bacillus from a Tse1 Peptidoglycan Hydrolase Type VI Secretion System Effector.

Within bacterial communities, community members engage in interactions employing diverse offensive and defensive tools to reach coexistence. Extracellular-matrix production and sporulation are defensive mechanisms used by Bacillus subtilis cells when they interact with Pseudomonas chlororaphis strains expressing a type VI secretion system (T6SS). Here, we define Tse1 as the main toxin mobilized by the Pseudomonas chlororaphis T6SS that triggers sporulation in Bacillus subtilis. We characterize Tse1 as a peptidoglycan hydrolase that indirectly alters the dynamics and functionality of the Bacillus cell membrane. We also delineate the response of Bacillus cells to Tse1, which through the coordinated actions of the extracellular sigma factor σW and the cytoplasmic histidine kinases KinA and KinB, culminates in activation of the sporulation cascade. We propose that this cellular developmental response permits bacilli to defend against the toxicity of T6SS-mobilized Tse1 effector. IMPORTANCE The study of bacterial interactions is helping to define species-specific strategies used to modulate the competition dynamics underlying the development of community compositions. In this study, we deciphered the role of Pseudomonas T6SS when competing with Bacillus and the mechanism by which a T6SS-toxin modifies Bacillus physiology. We found that Pseudomonas triggers Bacillus sporulation by injecting through T6SS a toxin that we called Tse1. We found that Tse1 is a hydrolase that degrades Bacillus peptidoglycan and indirectly damages Bacillus membrane functionality. In addition, we demonstrated the mechanism by which Bacillus cells increase the sporulation rate upon recognition of the presence of Tse1. Interestingly, asporogenic Bacillus cells are more sensitive to T6SS activity, which led us to propose sporulation as a last resort of bacilli to overcome this family of toxins.

Mechanisms of resistance to chloramphenicol in Pseudomonas putida KT2440.

Pseudomonas putida KT2440 is a chloramphenicol-resistant bacterium that is able to grow in the presence of this antibiotic at a concentration of up to 25 µg/ml. Transcriptomic analyses revealed that the expression profile of 102 genes changed in response to this concentration of chloramphenicol in the culture medium. The genes that showed altered expression include those involved in general metabolism, cellular stress response, gene regulation, efflux pump transporters, and protein biosynthesis. Analysis of a genome-wide collection of mutants showed that survival of a knockout mutant in the TtgABC resistance-nodulation-division (RND) efflux pump and mutants in the biosynthesis of pyrroloquinoline (PQQ) were compromised in the presence of chloramphenicol. The analysis also revealed that an ABC extrusion system (PP2669/PP2668/PP2667) and the AgmR regulator (PP2665) were needed for full resistance toward chloramphenicol. Transcriptional arrays revealed that AgmR controls the expression of the pqq genes and the operon encoding the ABC extrusion pump from the promoter upstream of open reading frame (ORF) PP2669.

A Noninvasive Method for Time-Lapse Imaging of Microbial Interactions and Colony Dynamics.

Complex interactions between microbial populations can greatly affect the overall properties of a microbial community, sometimes leading to cooperation and mutually beneficial coexistence, or competition and the death or displacement of organisms or subpopulations. Interactions between different biofilm populations are highly relevant in diverse scientific areas, from antimicrobial resistance to microbial ecology. The utilization of modern microscopic techniques has provided a new and interesting insight into how bacteria interact at the cellular level to form and maintain microbial biofilms. However, our ability to follow complex intraspecies and interspecies interactions in vivo at the microscopic level has remained somewhat limited. Here, we detailed BacLive, a novel noninvasive method for tracking bacterial growth and biofilm dynamics using high-resolution fluorescence microscopy and an associated ImageJ processing macro (https://github.com/BacLive) for easier data handling and image analysis. Finally, we provided examples of how BacLive can be used in the analysis of complex bacterial communities. IMPORTANCE Communication and interactions between single cells are continuously defining the structure and composition of microbial communities temporally and spatially. Methods routinely used to study these communities at the cellular level rely on sample manipulation which makes microscopic time-lapse experiments impossible. BacLive was conceived as a method for the noninvasive study of the formation and development of bacterial communities, such as biofilms, and the formation dynamics of specialized subpopulations in time-lapse experiments at a colony level. In addition, we developed a tool to simplify the processing and analysis of the data generated by this method.

Native metabolomics identifies the rivulariapeptolide family of protease inhibitors.

The identity and biological activity of most metabolites still remain unknown. A bottleneck in the exploration of metabolite structures and pharmaceutical activities is the compound purification needed for bioactivity assignments and downstream structure elucidation. To enable bioactivity-focused compound identification from complex mixtures, we develop a scalable native metabolomics approach that integrates non-targeted liquid chromatography tandem mass spectrometry and detection of protein binding via native mass spectrometry. A native metabolomics screen for protease inhibitors from an environmental cyanobacteria community reveals 30 chymotrypsin-binding cyclodepsipeptides. Guided by the native metabolomics results, we select and purify five of these compounds for full structure elucidation via tandem mass spectrometry, chemical derivatization, and nuclear magnetic resonance spectroscopy as well as evaluation of their biological activities. These results identify rivulariapeptolides as a family of serine protease inhibitors with nanomolar potency, highlighting native metabolomics as a promising approach for drug discovery, chemical ecology, and chemical biology studies.

Bacterial extracellular matrix as a natural source of biotechnologically multivalent materials.

The extracellular matrix (ECM) is an intricate megastructure made by bacterial cells to form architecturally complex biostructures called biofilms. Protection of cells, modulation of cell-to-cell signalling, cell differentiation and environmental sensing are functions of the ECM that reflect its diverse chemical composition. Proteins, polysaccharides and eDNA have specific functionalities while cooperatively interacting to sustain the architecture and biological relevance of the ECM. The accumulated evidence on the chemical heterogeneity and specific functionalities of ECM components has attracted attention because of their potential biotechnological applications, from agriculture to the water and food industries. This review compiles information on the most relevant bacterial ECM components, the biophysical and chemical features responsible for their biological roles, and their potential to be further translated into biotechnological applications.

Chemical interplay and complementary adaptative strategies toggle bacterial antagonism and co-existence.

Bacterial communities are in a continuous adaptive and evolutionary race for survival. In this work we expand our knowledge on the chemical interplay and specific mutations that modulate the transition from antagonism to co-existence between two plant-beneficial bacteria, Pseudomonas chlororaphis PCL1606 and Bacillus amyloliquefaciens FZB42. We reveal that the bacteriostatic activity of bacillaene produced by Bacillus relies on an interaction with the protein elongation factor FusA of P. chlororaphis and how mutations in this protein lead to tolerance to bacillaene and other protein translation inhibitors. Additionally, we describe how the unspecific tolerance of B. amyloliquefaciens to antimicrobials associated with mutations in the glycerol kinase GlpK is provoked by a decrease of Bacillus cell membrane permeability, among other pleiotropic responses. We conclude that nutrient specialization and mutations in basic biological functions are bacterial adaptive dynamics that lead to the coexistence of two primary competitive bacterial species rather than their mutual eradication.

Chemical fertilization: a short-term solution for plant productivity?

The effect of long-term chemical fertilization on plant-microorganisms and microbe-microbe interactions.

Dual functionality of the amyloid protein TasA in Bacillus physiology and fitness on the phylloplane.

Bacteria can form biofilms that consist of multicellular communities embedded in an extracellular matrix (ECM). In Bacillus subtilis, the main protein component of the ECM is the functional amyloid TasA. Here, we study further the roles played by TasA in B. subtilis physiology and biofilm formation on plant leaves and in vitro. We show that ΔtasA cells exhibit a range of cytological symptoms indicative of excessive cellular stress leading to increased cell death. TasA associates to the detergent-resistant fraction of the cell membrane, and the distribution of the flotillin-like protein FloT is altered in ΔtasA cells. We propose that, in addition to a structural function during ECM assembly and interactions with plants, TasA contributes to the stabilization of membrane dynamics as cells enter stationary phase.