Title: Multiple CheY Proteins Control Surface-Associated Lifestyles of Azospirillum brasilense
Bacterial chemotaxis is the directed movement of motile bacteria in gradients of chemoeffectors. This behavior is mediated by dedicated signal transduction pathways that couple environment sensing with changes in the direction of rotation of flagellar motors to ultimately affect the motility pattern. Azospirillum brasilense uses two distinct chemotaxis pathways, named Che1 and Che4, and four different response regulators (CheY1, CheY4, CheY6, and CheY7) to control the swimming pattern during chemotaxis. Each of the CheY homologs was shown to differentially affect the rotational bias of the polar flagellum and chemotaxis. The role, if any, of these CheY homologs in swarming, which depends on a distinct lateral flagella system or in attachment is not known. Here, we characterize CheY homologs’ roles in swimming, swarming, and attachment to abiotic and biotic (wheat roots) surfaces and biofilm formation. We show that while strains lacking CheY1 and CheY6 are still able to navigate air gradients, strains lacking CheY4 and CheY7 are chemotaxis null. Expansion of swarming colonies in the presence of gradients requires chemotaxis. The induction of swarming depends on CheY4 and CheY7, but the cells’ organization as dense clusters in productive swarms appear to depend on functional CheYs but not chemotaxis per se . more »
Similarly, functional CheY homologs but not chemotaxis, contribute to attachment to both abiotic and root surfaces as well as to biofilm formation, although these effects are likely dependent on additional cell surface properties such as adhesiveness. Collectively, our data highlight distinct roles for multiple CheY homologs and for chemotaxis on swarming and attachment to surfaces. « less
ABSTRACT The alphaproteobacterium Sinorhizobium meliloti secretes two acidic exopolysaccharides (EPSs), succinoglycan (EPSI) and galactoglucan (EPSII), which differentially enable it to adapt to a changing environment. Succinoglycan is essential for invasion of plant hosts and, thus, for the formation of nitrogen-fixing root nodules. Galactoglucan is critical for population-based behaviors such as swarming and biofilm formation and can facilitate invasion in the absence of succinoglycan on some host plants. The biosynthesis of galactoglucan is not as completely understood as that of succinoglycan. We devised a pipeline to identify putative pyruvyltransferase and acetyltransferase genes, construct genomic deletions in strains engineered to produce eithermore »succinoglycan or galactoglucan, and analyze EPS from mutant bacterial strains. EPS samples were examined by 13 C cross-polarization magic-angle spinning (CPMAS) solid-state nuclear magnetic resonance (NMR). CPMAS NMR is uniquely suited to defining chemical composition in complex samples and enables the detection and quantification of distinct EPS functional groups. Galactoglucan was isolated from mutant strains with deletions in five candidate acyl/acetyltransferase genes ( exoZ , exoH , SMb20810 , SMb21188 , and SMa1016 ) and a putative pyruvyltransferase ( wgaE or SMb21322 ). Most samples were similar in composition to wild-type EPSII by CPMAS NMR analysis. However, galactoglucan produced from a strain lacking wgaE exhibited a significant reduction in pyruvylation. Pyruvylation was restored through the ectopic expression of plasmid-borne wgaE . Our work has thus identified WgaE as a galactoglucan pyruvyltransferase. This exemplifies how the systematic combination of genetic analyses and solid-state NMR detection is a rapid means to identify genes responsible for modification of rhizobial exopolysaccharides. IMPORTANCE Nitrogen-fixing bacteria are crucial for geochemical cycles and global nitrogen nutrition. Symbioses between legumes and rhizobial bacteria establish root nodules, where bacteria convert dinitrogen to ammonia for plant utilization. Secreted exopolysaccharides (EPSs) produced by Sinorhizobium meliloti (succinoglycan and galactoglucan) play important roles in soil and plant environments. The biosynthesis of galactoglucan is not as well characterized as that of succinoglycan. We employed solid-state nuclear magnetic resonance (NMR) to examine intact EPS from wild-type and mutant S. meliloti strains. NMR analysis of EPS isolated from a wgaE gene mutant revealed a novel pyruvyltransferase that modifies galactoglucan. Few EPS pyruvyltransferases have been characterized. Our work provides insight into the biosynthesis of an important S. meliloti EPS and expands the knowledge of enzymes that modify polysaccharides.« less
Ganusova, Elena E.; Vo, Lam T.; Abraham, Paul E.; O’Neal Yoder, Lindsey; Hettich, Robert L.; Alexandre, Gladys(
, mSystems)
Petersen, Jillian Michelle
(Ed.)
ABSTRACT 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 nonchemotaxismore »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.« less
Kitts, Giordan; Giglio, Krista M.; Zamorano-Sánchez, David; Park, Jin Hwan; Townsley, Loni; Cooley, Richard B.; Wucher, Benjamin R.; Klose, Karl E.; Nadell, Carey D.; Yildiz, Fitnat H.; et al(
, mBio)
ABSTRACT The dinucleotide second messenger c-di-GMP has emerged as a central regulator of reversible cell attachment during bacterial biofilm formation. A prominent cell adhesion mechanism first identified in pseudomonads combines two c-di-GMP-mediated processes: transcription of a large adhesin and its cell surface display via posttranslational proteolytic control. Here, we characterize an orthologous c-di-GMP effector system and show that it is operational in Vibrio cholerae , where it regulates two distinct classes of adhesins. Through structural analyses, we reveal a conserved autoinhibition mechanism of the c-di-GMP receptor that controls adhesin proteolysis and present a structure of a c-di-GMP-bound receptor module. Wemore »further establish functionality of the periplasmic protease controlled by the receptor against the two adhesins. Finally, transcription and functional assays identify physiological roles of both c-di-GMP-regulated adhesins in surface attachment and biofilm formation. Together, our studies highlight the conservation of a highly efficient signaling effector circuit for the control of cell surface adhesin expression and its versatility by revealing strain-specific variations. IMPORTANCE Vibrio cholerae , the causative agent of the diarrheal disease cholera, benefits from a sessile biofilm lifestyle that enhances survival outside the host but also contributes to host colonization and infectivity. The bacterial second messenger c-di-GMP has been identified as a central regulator of biofilm formation, including in V. cholerae ; however, our understanding of the pathways that contribute to this process is incomplete. Here, we define a conserved signaling system that controls the stability of large adhesion proteins at the cell surface of V. cholerae , which are important for cell attachment and biofilm formation. Insight into the regulatory circuit underlying biofilm formation may inform targeted strategies to interfere with a process that renders this bacterium remarkably adaptable to changing environments.« less
Kamei, Masayuki; Ameri, Abigail J.; Ferraro, Aileen R.; Bar-Peled, Yael; Zhao, Fangzhou; Ethridge, Christina L.; Lail, Kathleen; Amirebrahimi, Mojgan; Lipzen, Anna; Ng, Vivian; et al(
, Proceedings of the National Academy of Sciences)
Polycomb Group (PcG) proteins are part of an epigenetic cell memory system that plays essential roles in multicellular development, stem cell biology, X chromosome inactivation, and cancer. In animals, plants, and many fungi, Polycomb Repressive Complex 2 (PRC2) catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) to assemble transcriptionally repressed facultative heterochromatin. PRC2 is structurally and functionally conserved in the model fungusNeurospora crassa, and recent work in this organism has generated insights into PRC2 control and function. To identify components of the facultative heterochromatin pathway, we performed a targeted screen ofNeurosporadeletion strains lacking individual ATP-dependent chromatin remodeling enzymes. We foundmore »theNeurosporahomolog of IMITATION SWITCH (ISW) is critical for normal transcriptional repression, nucleosome organization, and establishment of typical histone methylation patterns in facultative heterochromatin domains. We also found that stable interaction between PRC2 and chromatin depends on ISW. A functional ISW ATPase domain is required for gene repression and normal H3K27 methylation. ISW homologs interact with accessory proteins to form multiple complexes with distinct functions. Using proteomics and molecular approaches, we identified three distinctNeurosporaISW-containing complexes. A triple mutant lacking three ISW accessory factors and disrupting multiple ISW complexes led to widespread up-regulation of PRC2 target genes and altered H3K27 methylation patterns, similar to an ISW-deficient strain. Taken together, our data show that ISW is a key component of the facultative heterochromatin pathway inNeurospora, and that distinct ISW complexes perform an apparently overlapping role to regulate chromatin structure and gene repression at PRC2 target domains.
ABSTRACT Plant roots shape the rhizosphere community by secreting compounds that recruit diverse bacteria. Colonization of various plant roots by the motile alphaproteobacterium Azospirillum brasilens e 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 usemore »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. brasilens e 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.« less
Ganusova, Elena E., Vo, Lam T., Mukherjee, Tanmoy, and Alexandre, Gladys. Multiple CheY Proteins Control Surface-Associated Lifestyles of Azospirillum brasilense. Retrieved from https://par.nsf.gov/biblio/10226225. Frontiers in Microbiology 12. Web. doi:10.3389/fmicb.2021.664826.
Ganusova, Elena E., Vo, Lam T., Mukherjee, Tanmoy, & Alexandre, Gladys. Multiple CheY Proteins Control Surface-Associated Lifestyles of Azospirillum brasilense. Frontiers in Microbiology, 12 (). Retrieved from https://par.nsf.gov/biblio/10226225. https://doi.org/10.3389/fmicb.2021.664826
Ganusova, Elena E., Vo, Lam T., Mukherjee, Tanmoy, and Alexandre, Gladys.
"Multiple CheY Proteins Control Surface-Associated Lifestyles of Azospirillum brasilense". Frontiers in Microbiology 12 (). Country unknown/Code not available. https://doi.org/10.3389/fmicb.2021.664826.https://par.nsf.gov/biblio/10226225.
@article{osti_10226225,
place = {Country unknown/Code not available},
title = {Multiple CheY Proteins Control Surface-Associated Lifestyles of Azospirillum brasilense},
url = {https://par.nsf.gov/biblio/10226225},
DOI = {10.3389/fmicb.2021.664826},
abstractNote = {Bacterial chemotaxis is the directed movement of motile bacteria in gradients of chemoeffectors. This behavior is mediated by dedicated signal transduction pathways that couple environment sensing with changes in the direction of rotation of flagellar motors to ultimately affect the motility pattern. Azospirillum brasilense uses two distinct chemotaxis pathways, named Che1 and Che4, and four different response regulators (CheY1, CheY4, CheY6, and CheY7) to control the swimming pattern during chemotaxis. Each of the CheY homologs was shown to differentially affect the rotational bias of the polar flagellum and chemotaxis. The role, if any, of these CheY homologs in swarming, which depends on a distinct lateral flagella system or in attachment is not known. Here, we characterize CheY homologs’ roles in swimming, swarming, and attachment to abiotic and biotic (wheat roots) surfaces and biofilm formation. We show that while strains lacking CheY1 and CheY6 are still able to navigate air gradients, strains lacking CheY4 and CheY7 are chemotaxis null. Expansion of swarming colonies in the presence of gradients requires chemotaxis. The induction of swarming depends on CheY4 and CheY7, but the cells’ organization as dense clusters in productive swarms appear to depend on functional CheYs but not chemotaxis per se . Similarly, functional CheY homologs but not chemotaxis, contribute to attachment to both abiotic and root surfaces as well as to biofilm formation, although these effects are likely dependent on additional cell surface properties such as adhesiveness. Collectively, our data highlight distinct roles for multiple CheY homologs and for chemotaxis on swarming and attachment to surfaces.},
journal = {Frontiers in Microbiology},
volume = {12},
author = {Ganusova, Elena E. and Vo, Lam T. and Mukherjee, Tanmoy and Alexandre, Gladys},
}
