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The root-associated microbiota plays an important role in the response to environmental stress. However, the underlying mechanisms controlling the interaction between salt-stressed plants and microbiota are poorly understood. Here, by focusing on a salt-tolerant plant wild soybean ( Glycine soja ), we demonstrate that highly conserved microbes dominated by Pseudomonas are enriched in the root and rhizosphere microbiota of salt-stressed plant. Two corresponding Pseudomonas isolates are confirmed to enhance the salt tolerance of wild soybean. Shotgun metagenomic and metatranscriptomic sequencing reveal that motility-associated genes, mainly chemotaxis and flagellar assembly, are significantly enriched and expressed in salt-treated samples. We further find that roots of salt stressed plants secreted purines, especially xanthine, which induce motility of the Pseudomonas isolates. Moreover, exogenous application for xanthine to non-stressed plants results in Pseudomonas enrichment, reproducing the microbiota shift in salt-stressed root. Finally, Pseudomonas mutant analysis shows that the motility related gene cheW is required for chemotaxis toward xanthine and for enhancing plant salt tolerance. Our study proposes that wild soybean recruits beneficial Pseudomonas species by exudating key metabolites (i.e., purine) against salt stress. Root-associated microbiota confers benefits to plant in responding to environmental stress. Here, the authors show that wild soybean secretes purines under salt stress, reshapes the microbiota and recruits Pseudomonas.

In this work, a recombinant chemotaxis CheW protein from Thermotoga petrophila RKU-1 (TpeCheW) and its mutant homologue (TpeCheW-mut) have been obtained. Despite the low homology with the CheW protein of the Escherichia coli bacteria, these proteins do not cause metabolic overload and are well expressed by E. coli laboratory strains. A wide range of features and parameters important for isolation of the TpeCheW-mut protein, such as stability over a wide range of temperatures and pH values, high level of expression, solubility, and the possibility of using simple low-stage purification schemes, including heat pretreatment, has been recognized. Possible directions of using this protein in the practice of scientific and applied research have been formulated and justified.

Chemoreceptor arrays are supra molecular transmembrane machines of unknown structure that allow bacteria to sense their surroundings and respond by chemotaxis. We have combined X-ray crystallography of purified proteins with electron cryotomography of native arrays inside cells to reveal the arrangement of the component transmembrane receptors, histidine kinases (CheA) and CheW coupling proteins. Trimers of receptor dimers lie at the vertices of a hexagonal lattice in a \"two-facing-two\" configuration surrounding a ring of alternating CheA regulatory domains (P5) and CheW couplers. Whereas the CheA kinase domains (P4) project downward below the ring, the CheA dimerization domains (P3) link neighboring rings to form an extended, stable array. This highly interconnected protein architecture underlies the remarkable sensitivity and cooperative nature of transmembrane signaling in bacterial chemotaxis.

The gastric pathogen Helicobacter pylori requires a noncanonical cytosolic chemoreceptor transducer-like protein D (TlpD) for efficient colonization of the mammalian stomach. Here, we reconstituted a complete chemotransduction signaling complex in vitro with TlpD and the chemotaxis (Che) proteins CheW and CheA, enabling quantitative assays for potential chemotaxis ligands. We found that TlpD is selectively sensitive at micromolar concentrations to bleach (hypochlorous acid, HOCl), a potent antimicrobial produced by neutrophil myeloperoxidase during inflammation. HOCl acts as a chemoattractant by reversibly oxidizing a conserved cysteine within a 3His/1Cys Zn-binding motif in TlpD that inactivates the chemotransduction signaling complex. We found that H. pylori is resistant to killing by millimolar concentrations of HOCl and responds to HOCl in the micromolar range by increasing its smooth-swimming behavior, leading to chemoattraction to HOCl sources. We show related protein domains from Salmonella enterica and Escherichia coli possess similar reactivity toward HOCl. We propose that this family of proteins enables host-associated bacteria to sense sites of tissue inflammation, a strategy that H. pylori uses to aid in colonizing and persisting in inflamed gastric tissue.

ABSTRACT Flagellar motility plays a central role in the bacterial foodborne pathogen Campylobacter jejuni, as flagellar motility is required for reaching the intestinal epithelium and subsequent colonisation or disease. Flagellar proteins also contribute strongly to biofilm formation during transmission. Chemotaxis is the process directing flagellar motility in response to attractant and repellent stimuli, but its role in biofilm formation of C. jejuni is not well understood. Here we show that inactivation of the core chemotaxis genes cheVAWY in C. jejuni strain NCTC 11168 affects both chemotactic motility and biofilm formation. Inactivation of any of the core chemotaxis genes (cheA, cheY, cheV or cheW) impaired chemotactic motility but did not affect flagellar assembly or growth. The ∆cheY mutant swam in clockwise loops, while complementation restored normal motility. Inactivation of the core chemotaxis genes interfered with the ability to form a discrete biofilm at the air-media interface, and the ∆cheY mutant displayed reduced dispersal/shedding of bacteria into the planktonic fraction. This suggests that while the chemotaxis system is not required for biofilm formation per se, it is necessary for organized biofilm formation. Hence interference with the Campylobacter chemotaxis system at any level disrupts optimal chemotactic motility and transmission modes such as biofilm formation. Chemotaxis system is required for directed motility towards or away from specific environmental stimuli, but if inactivated, also affects the capacity to build and leave a structured biofilm.

CheV is one of the least understood chemosensory signaling proteins. Our demonstration that CheV interacts only with certain chemoreceptors offers fundamental new insights. These findings, combined with the observation that CheV is present in bacteria with numerous chemoreceptors, suggest that CheV plays a role in coordinating chemotactic outputs in complex chemosensory systems. Understanding the mechanisms by which chemotactic responses are defined in bacteria with a high number of chemoreceptors is a major research priority in the field of chemotaxis. While previous studies, including this one, show that the ability to be phosphorylated is crucial for CheV function, the molecular consequences of CheV phosphorylation have remained unclear. Our discovery that phosphorylation is essential for CheV binding to certain chemoreceptors fills in this critical gap in understanding the molecular mechanism of CheV. This study is likely to inspire further research into CheV function in other bacteria using similar approaches.

Motile bacteria employ conserved chemotaxis networks to detect chemical gradients in their surroundings and effectively regulate their locomotion, enabling the location of essential nutrients and other important biological niches. The sensory apparatus of the chemotaxis pathway is an array of core-signaling units (CSUs) composed of transmembrane chemoreceptors, the histidine kinase CheA and an adaptor protein, CheW. Although chemotaxis pathways represent the best understood signaling systems, a detailed mechanistic understanding of signal transduction has been hindered by the lack of a complete structural picture of the CSU and extended array. In this study, we present the structure of the complete CSU from phage φX174 E protein lysed Escherichia coli cells, determined using cryo-electron tomography and sub-tomogram averaging to 12-Å resolution. Using AlphaFold2, we further predict the atomic structures of the CSU’s constituent proteins as well as key protein-protein interfaces, enabling the assembly an all-atom CSU model, which we conformationally refine using our cryo-electron tomography map. Molecular dynamics simulations of the resulting model provide new insight into the periplasmic organization of the complex, including novel interactions between neighboring receptor ligand-binding domains. Our results further elucidate previously unresolved interactions between individual CheA domains, including an anti-parallel P1 dimer and non-productive binding mode between P1 and P4, enhancing our understanding of the structural mechanisms underlying CheA signaling and regulation. Bacterial chemotaxis is a ubiquitous behavior that enables cell movement toward or away from specific chemicals. It serves as an important model for understanding cell sensory signal transduction and motility. Characterization of the molecular mechanisms underlying chemotaxis is of fundamental interest and requires a high-resolution structural picture of the sensing machinery, the chemosensory array. In this study, we combine cryo-electron tomography and molecular simulation to present the complete structure of the core signaling unit, the basic building block of chemosensory arrays, from Escherichia coli . Our results provide new insight into previously poorly-resolved regions of the complex and offer a structural basis for designing new experiments to test mechanistic hypotheses.

The metabolomic and genomic characterization of an endophytic Bacillus safensis Ni7 was carried out in this study. This strain has previously been isolated from the xerophytic plant Nerium indicum L. and reported to enhance the drought tolerance in Capsicum annuum L. seedlings. The effects of drought stress on the morphology, biofilm production, and metabolite production of B. safensis Ni7 are analyzed in the current study. From the results obtained, the organism was found to have multiple strategies such as aggregation and clumping, robust biofilm production, and increased production of surfactin homologues under the drought induced condition when compared to non-stressed condition. Further the whole genome sequencing (WGS) based analysis has demonstrated B. safensis Ni7 to have a genome size of 3,671,999 bp, N50 value of 3,527,239, and a mean G+C content of 41.58%. Interestingly the organism was observed to have the presence of various stress-responsive genes (13, 20U, 16U,160, 39, 17M, 18, 26, and ctc) and genes responsible for surfactin production (srfAA, srfAB, srfAC, and srfAD), biofilm production (epsD, epsE, epsF, epsG, epsH, epsI, epsK, epsL, epsM, epsN, and pel), chemotaxis (cheB_1, cheB_2, cheB_3, cheW_1, cheW_2 cheR, cheD, cheC, cheA, cheY, cheV, and cheB_4), flagella synthesis (flgG_1, flgG_2, flgG_3, flgC, and flgB) as supportive to the drought tolerance. Besides these, the genes responsible for plant growth promotion (PGP), including the genes for nitrogen (nasA, nasB, nasC, nasD, and nasE) and sulfur assimilation (cysL_1&L_2, cysI) and genes for phosphate solubilization (phoA, phoP_1& phoP_2, and phoR) could also be predicted. Along with the same, the genes for catalase, superoxide dismutase, protein homeostasis, cellular fitness, osmoprotectants production, and protein folding could also be predicted from its WGS data. Further pan-genome analysis with plant associated B. safensis strains available in the public databases revealed B. safensis Ni7 to have the presence of a total of 5391 gene clusters. Among these, 3207 genes were identified as core genes, 954 as shell genes and 1230 as cloud genes. This variation in gene content could be taken as an indication of evolution of strains of Bacillus safensis as per specific conditions and hence in the case of B. safensis Ni7 its role in habitat adaptation of plant is well expected. This diversity in endophytic bacterial genes may attribute its role to support the plant system to cope up with stress conditions. Overall, the study provides genomic evidence on Bacillus safensis Ni7 as a stress alleviating microbial partner in plants.

The assembly of chemotaxis receptors and signaling proteins into polar arrays is universal in motile chemotactic bacteria. Comparative genome analyses indicate that most motile bacteria possess multiple chemotaxis signaling systems, and experimental evidence suggests that signaling from distinct chemotaxis systems is integrated. Here, we identify one such mechanism. We show that paralogs from two chemotaxis systems assemble together into chemoreceptor arrays, forming baseplates comprised of proteins from both chemotaxis systems. These mixed arrays provide a straightforward mechanism for signal integration and coordinated response output from distinct chemotaxis systems. Given that most chemotactic bacteria encode multiple chemotaxis systems and the propensity for these systems to be laterally transferred, this mechanism may be common to ensure chemotaxis signal integration occurs. Most chemotactic motile bacteria possess multiple chemotaxis signaling systems, the functions of which are not well characterized. Chemotaxis signaling is initiated by chemoreceptors that assemble as large arrays, together with chemotaxis coupling proteins (CheW) and histidine kinase proteins (CheA), which form a baseplate with the cytoplasmic tips of receptors. These cell pole-localized arrays mediate sensing, signaling, and signal amplification during chemotaxis responses. Membrane-bound chemoreceptors with different cytoplasmic domain lengths segregate into distinct arrays. Here, we show that a bacterium, Azospirillum brasilense , which utilizes two chemotaxis signaling systems controlling distinct motility parameters, coordinates its chemotactic responses through the production of two separate membrane-bound chemoreceptor arrays by mixing paralogs within chemotaxis baseplates. The polar localization of chemoreceptors of different length classes is maintained in strains that had baseplate signaling proteins from either chemotaxis system but was lost when both systems were deleted. Chemotaxis proteins (CheA and CheW) from each of the chemotaxis signaling systems (Che1 and Che4) could physically interact with one another, and chemoreceptors from both classes present in A. brasilense could interact with Che1 and Che4 proteins. The assembly of paralogs from distinct chemotaxis pathways into baseplates provides a straightforward mechanism for coordinating signaling from distinct pathways, which we predict is not unique to this system given the propensity of chemotaxis systems for horizontal gene transfer. IMPORTANCE The assembly of chemotaxis receptors and signaling proteins into polar arrays is universal in motile chemotactic bacteria. Comparative genome analyses indicate that most motile bacteria possess multiple chemotaxis signaling systems, and experimental evidence suggests that signaling from distinct chemotaxis systems is integrated. Here, we identify one such mechanism. We show that paralogs from two chemotaxis systems assemble together into chemoreceptor arrays, forming baseplates comprised of proteins from both chemotaxis systems. These mixed arrays provide a straightforward mechanism for signal integration and coordinated response output from distinct chemotaxis systems. Given that most chemotactic bacteria encode multiple chemotaxis systems and the propensity for these systems to be laterally transferred, this mechanism may be common to ensure chemotaxis signal integration occurs.

Background Plant biostimulants are an emerging class of agricultural inputs known to enhance plant growth and improve their tolerance to abiotic stress. While biostimulants are widely used, their mechanisms of action remain poorly understood. This study investigates the effects of an Ascophyllum nodosum -based biostimulant (ANE) on the rhizosphere bacterial communities of corn ( Zea mays ). Results Root exudates from ANE root-treated plants promoted the swarming motility of Pseudomonas protegens (CHA0), a plant growth-promoting rhizobacterium. Gene expression analysis showed that root exudates from 0.01% ANE-treated plants up-regulated P. protegens CHA0 genes associated with chemotaxis ( cheW , cheV ), pyoverdine ( pvdS ), pyrrolnitrin ( prnD ), and hydrogen cyanide ( hcnA ) biosynthesis compared to controls. ANE also significantly altered rhizosphere microbiome composition, increasing the abundance of genera such as Chryseolinea , Pseudoxanthomonas , Novosphingobium , Quadrisphaera , Turneriella , and Kitasatospora . Liquid chromatography-high resolution mass spectrometry (LC–HRMS) and partial least squares-discriminant analysis (PLS-DA) revealed distinct chemical profiles in the root extracts of ANE-treated plants. Specifically, ANE increased the concentrations of benzoxazinoids, including 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and 6-methoxybenzoxazolinone (MBOA) in maize roots by approximately 1.4-fold and 1.76-fold, respectively. Conclusion Overall, these findings suggest that ANE modifies the rhizosphere microbiome by influencing the chemical composition of both root tissue and root exudates. Graphical abstract