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A PilZ-Containing Chemotaxis Receptor Mediates Oxygen and Wheat Root Sensing in Azospirillum brasilense.

ABSTRACT: Chemotactic bacteria sense environmental changes via dedicated receptors that bind to extra- or intracellular cues and relay this signal to ultimately alter direction of movement toward beneficial cues and away from harmful environments. In complex environments, such as the rhizosphere, bacteria must be able to sense and integrate diverse cues. Azospirillum brasilense is a microaerophilic motile bacterium that promotes growth of cereals and grains. Root surface colonization is a prerequisite for the beneficial effects on plant growth but how motile A. brasilense navigates the rhizosphere is poorly studied. Previously only 2 out of 51 A. brasilense chemotaxis receptors have been characterized, AerC and Tlp1, and only Tlp1 was found to be essential for wheat root colonization. Here we describe another chemotaxis receptor, named Aer, that is homologous to the Escherichia coli Aer receptor, likely possesses an FAD cofactor and is involved in aerotaxis (taxis in an air gradient). We also found that the A. brasilense Aer contributes to sensing chemical gradients originating from wheat roots. In addition to A. brasilense Aer having a putative N-terminal FAD-binding PAS domain, it possesses a C-terminal PilZ domain that contains all the conserved residues for binding c-di-GMP. Mutants lacking the PilZ domain of Aer are altered in aerotaxis and are completely null in wheat root colonization and they also fail to sense gradients originating from wheat roots. The PilZ domain of Aer is also vital in integrating Aer signaling with signaling from other chemotaxis receptors to sense gradients from wheat root surfaces and colonizing wheat root surfaces.

SUBMITTER: O'Neal L

PROVIDER: S-EPMC6406031 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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A PilZ-Containing Chemotaxis Receptor Mediates Oxygen and Wheat Root Sensing in <i>Azospirillum brasilense</i>.

O'Neal Lindsey L Akhter Shehroze S Alexandre Gladys G

Frontiers in microbiology 20190301

Chemotactic bacteria sense environmental changes via dedicated receptors that bind to extra- or intracellular cues and relay this signal to ultimately alter direction of movement toward beneficial cues and away from harmful environments. In complex environments, such as the rhizosphere, bacteria must be able to sense and integrate diverse cues. <i>Azospirillum brasilense</i> is a microaerophilic motile bacterium that promotes growth of cereals and grains. Root surface colonization is a prerequis ...[more]

PMID: 30881352

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Project description:Plant roots shape the rhizosphere community by secreting compounds that recruit diverse bacteria. Colonization of various plant roots by the motile alphaproteobacterium Azospirillum brasilense causes increased plant growth, root volume, and crop yield. Bacterial chemotaxis in this and other motile soil bacteria is critical for competitive colonization of the root surfaces. The role of chemotaxis in root surface colonization has previously been established by endpoint analyses of bacterial colonization levels detected a few hours to days after inoculation. More recently, microfluidic devices have been used to study plant-microbe interactions, but these devices are size limited. Here, we use a novel slide-in chamber that allows real-time monitoring of plant-microbe interactions using agriculturally relevant seedlings to characterize how bacterial chemotaxis mediates plant root surface colonization during the association of A. brasilense with Triticum aestivum (wheat) and Medicago sativa (alfalfa) seedlings. We track A. brasilense accumulation in the rhizosphere and on the root surfaces of wheat and alfalfa. A. brasilense motile cells display distinct chemotaxis behaviors in different regions of the roots, including attractant and repellent responses that ultimately drive surface colonization patterns. We also combine these observations with real-time analyses of behaviors of wild-type and mutant strains to link chemotaxis responses to distinct chemicals identified in root exudates to specific chemoreceptors that together explain the chemotactic response of motile cells in different regions of the roots. Furthermore, the bacterial second messenger c-di-GMP modulates these chemotaxis responses. Together, these findings illustrate dynamic bacterial chemotaxis responses to rhizosphere gradients that guide root surface colonization.IMPORTANCE Plant root exudates play critical roles in shaping rhizosphere microbial communities, and the ability of motile bacteria to respond to these gradients mediates competitive colonization of root surfaces. Root exudates are complex chemical mixtures that are spatially and temporally dynamic. Identifying the exact chemical(s) that mediates the recruitment of soil bacteria to specific regions of the roots is thus challenging. Here, we connect patterns of bacterial chemotaxis responses and sensing by chemoreceptors to chemicals found in root exudate gradients and identify key chemical signals that shape root surface colonization in different plants and regions of the roots.

Project description:Bacterial chemotaxis affords motile bacteria the ability to navigate the environment to locate niches for growth and survival. At the molecular level, chemotaxis depends on chemoreceptor signaling arrays that interact with cytoplasmic proteins to control the direction of movement. In Azospirillum brasilense, chemotaxis is mediated by two distinct chemotaxis pathways: Che1 and Che4. Both Che1 and Che4 are critical in the A. brasilense free-living and plant-associated lifestyles. Here, we use whole-cell proteomics and metabolomics to characterize the role of chemotaxis in A. brasilense physiology. We found that mutants lacking CheA1 or CheA4 or both are affected in nonchemotaxis functions, including major changes in transcription, signaling transport, and cell metabolism. We identify specific effects of CheA1 and CheA4 on nitrogen metabolism, including nitrate assimilation and nitrogen fixation, that may depend, at least, on the transcriptional control of rpoN, which encodes RpoN, a global regulator of metabolism, including nitrogen. Consistent with proteomics, the abundance of several nitrogenous compounds (purines, pyrimidines, and amino acids) changed in the metabolomes of the chemotaxis mutants relative to the parental strain. Further, we uncover novel, and yet uncharacterized, layers of transcriptional and posttranscriptional control of nitrogen metabolism regulators. Together, our data reveal roles for CheA1 and CheA4 in linking chemotaxis and nitrogen metabolism, likely through control of global regulatory networks.IMPORTANCE Bacterial chemotaxis is widespread in bacteria, increasing competitiveness in diverse environments and mediating associations with eukaryotic hosts ranging from commensal to beneficial and pathogenic. In most bacteria, chemotaxis signaling is tightly linked to energy metabolism, with this coupling occurring through the sensory input of several energy-sensing chemoreceptors. Here, we show that in A. brasilense the chemotaxis proteins have key roles in modulating nitrogen metabolism, including nitrate assimilation and nitrogen fixation, through novel and yet unknown regulations. These results are significant given that A. brasilense is a model bacterium for plant growth promotion and free-living nitrogen fixation and is used as a bio-inoculant for cereal crops. Chemotaxis signaling in A. brasilense thus links locomotor behaviors to nitrogen metabolism, allowing cells to continuously and reciprocally adjust metabolism and chemotaxis signaling as they navigate gradients.

Project description:A successful microbial inoculant can increase root colonization and establish a positive interaction with native microorganisms to promote growth and productivity of cereal crops. Zinc (Zn) is an intensively reported deficient nutrient for maize and wheat production in Brazilian Cerrado. It can be sustainably managed by inoculation with plant growth-promoting bacteria and their symbiotic association with other microorganisms such as arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE). The objective of this study was to evaluate the effect of Azospirillum brasilense inoculation and residual Zn rates on root colonization and grain yield of maize and wheat in succession under the tropical conditions of Brazil. These experiments were conducted in a randomized block design with four replications and arranged in a 5 × 2 factorial scheme. The treatments consisted of five Zn rates (0, 2, 4, 6 and 8 kg ha−1) applied from zinc sulfate in maize and residual on wheat and without and with seed inoculation of A. brasilense. The results indicated that root colonization by AMF and DSE in maize–wheat cropping system were significantly increased with interaction of Zn rates and inoculation treatments. Inoculation with A. brasilense at residual Zn rates of 4 kg ha−1 increased root colonization by AMF under maize cultivation. Similarly, inoculation with A. brasilense at residual Zn rates of 2 and 4 kg ha−1 reduced root colonization by DSE under wheat in succession. The leaf chlorophyll index and leaf Zn concentration were increased with inoculation of the A. brasilense and residual Zn rates. The inoculation did not influence AMF spore production and CO2-C in both crops. The grain yield and yield components of maize–wheat were increased with the inoculation of A. brasilense under residual Zn rates of 3 to 4 kg ha−1 in tropical savannah conditions. Inoculation with A. brasilense under residual Zn rates up to 4 kg ha−1 promoted root colonization by AMF and DSE in the maize cropping season. While the inoculation with A. brasilense under 2 and 4 kg ha−1 residual Zn rates reduced root colonization by AMF and DSE in the wheat cropping season. Therefore, inoculation with A. brasilense in combination with Zn fertilization could consider a sustainable approach to increase the yield and performance of the maize–wheat cropping system in the tropical savannah conditions of Brazil.

Project description:Chemotaxis is the movement of cells in response to gradients of diverse chemical cues. Motile bacteria utilize a conserved chemotaxis signal transduction system to bias their motility and navigate through a gradient. A central regulator of chemotaxis is the histidine kinase CheA. This cytoplasmic protein interacts with membrane-bound receptors, which assemble into large polar arrays, to propagate the signal. In the alphaproteobacterium Azospirillum brasilense, Che1 controls transient increases in swimming speed during chemotaxis, but it also biases the cell length at division. However, the exact underlying molecular mechanisms for Che1-dependent control of multiple cellular behaviors are not known. Here, we identify specific domains of the CheA1 histidine kinase implicated in modulating each of these functions. We show that CheA1 is produced in two isoforms: a membrane-anchored isoform produced as a fusion with a conserved seven-transmembrane domain of unknown function (TMX) at the N terminus and a soluble isoform similar to prototypical CheA. Site-directed and deletion mutagenesis combined with behavioral assays confirm the role of CheA1 in chemotaxis and implicate the TMX domain in mediating changes in cell length. Fluorescence microscopy further reveals that the membrane-anchored isoform is distributed around the cell surface while the soluble isoform localizes at the cell poles. Together, the data provide a mechanism for the role of Che1 in controlling multiple unrelated cellular behaviors via acquisition of a new domain in CheA1 and production of distinct functional isoforms.IMPORTANCE Chemotaxis provides a significant competitive advantage to bacteria in the environment, and this function has been transferred laterally multiple times, with evidence of functional divergence in different genomic contexts. The molecular principles that underlie functional diversification of chemotaxis in various genomic contexts are unknown. Here, we provide a molecular mechanism by which a single CheA protein controls two unrelated functions: chemotaxis and cell length. Acquisition of this multifunctionality is seemingly a recent evolutionary event. The findings illustrate a mechanism by which chemotaxis function may be co-opted to regulate additional cellular functions.

Project description:Azospirillum is a plant growth promoting rhizobacteria (PGPR) with ability to produce several phytohormones such as auxins, mainly indole-3-acetic acid (IAA). The positive interaction of Azospirillum with plants has been simplified and explained through the bacterial capacity to produce IAA. Typical changes on root architecture by promoting the number of lateral roots and hair formation, and reducing the primary root length were established in inoculated plants. These changes increase the root surface improving the water and nutrients acquisition, and thus the growth of the whole plant. The mechanisms by which Azospirillum induces such changes fails to be explained only by the bacterial capacity to produce IAA. In this work, we have evaluated the root architecture and gene expression changes occurred in Arabidopsis thaliana inoculated with A. brasilense Az39 and the IAA-deficient mutant (Az39 ipdC-), or treated with exogenous IAA solution to confirm both, the IAA-dependent and IAA-independent Azospirillum´s pathways to promote the root growth. Our results demonstrate the ability of Az39 to modify the primary root development through IAA biosynthesis, while other IAA-independent mechanisms were related to an increase in the lateral roots development and the root hairs number. Jasmonates, ethylene and salicylic acid were increased in the IAA-deficient bacterial treatments, as the ipdC mutant significantly up-regulated transcription of genes enriched of these phytohormones signaling after 7 days. Further, the physical presence of the inactive bacteria (Az39φ) seems to mediate the development of root hairs, a mechanism common to other non-PGPR as E. coli DH5α. Our results suggest that Az39 inoculation induces morphological changes in root architecture through both IAA-dependent and independent mechanism. The IAA biosynthesis by Az39 reduces the primary root length; while the cells contact with the roots increases the root hairs production. Both the synthesis of active IAA and the presence of metabolically active Az39 cells increase the growth and development of lateral roots.

Dataset Information

A PilZ-Containing Chemotaxis Receptor Mediates Oxygen and Wheat Root Sensing in Azospirillum brasilense.

Publications

A PilZ-Containing Chemotaxis Receptor Mediates Oxygen and Wheat Root Sensing in <i>Azospirillum brasilense</i>.

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