skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


  • NSF Public Access
  • Search Results
  • The Protein-Protein Interaction Network Reveals a Novel Role of the Signal Transduction Protein PII in the Control of c-di-GMP Homeostasis in Azospirillum brasilense
Title: The Protein-Protein Interaction Network Reveals a Novel Role of the Signal Transduction Protein PII in the Control of c-di-GMP Homeostasis in Azospirillum brasilense
ABSTRACT The PII family comprises a group of widely distributed signal transduction proteins ubiquitous in prokaryotes and in the chloroplasts of plants. PII proteins sense the levels of key metabolites ATP, ADP, and 2-oxoglutarate, which affect the PII protein structure and thereby the ability of PII to interact with a range of target proteins. Here, we performed multiple ligand fishing assays with the PII protein orthologue GlnZ from the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense to identify 37 proteins that are likely to be part of the PII protein-protein interaction network. Among the PII targets identified were enzymes related to nitrogen and fatty acid metabolism, signaling, coenzyme synthesis, RNA catabolism, and transcription. Direct binary PII-target complex was confirmed for 15 protein complexes using pulldown assays with recombinant proteins. Untargeted metabolome analysis showed that PII is required for proper homeostasis of important metabolites. Two enzymes involved in c-di-GMP metabolism were among the identified PII targets. A PII-deficient strain showed reduced c-di-GMP levels and altered aerotaxis and flocculation behavior. These data support that PII acts as a major metabolic hub controlling important enzymes and the homeostasis of key metabolites such as c-di-GMP in response to the prevailing nutritional status. IMPORTANCE The PII proteins sense and integrate important metabolic signals which reflect the cellular nutrition and energy status. Such extraordinary ability was capitalized by nature in such a way that the various PII proteins regulate different facets of metabolism by controlling the activity of a range of target proteins by protein-protein interactions. Here, we determined the PII protein interaction network in the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense . The interactome data along with metabolome analysis suggest that PII functions as a master metabolic regulator hub. We provide evidence that PII proteins act to regulate c-di-GMP levels in vivo and cell motility and adherence behaviors.  more » « less
Award ID(s):
1715185
PAR ID:
10226222
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ;
Editor(s):
Porto, Carla
Date Published:
Journal Name:
mSystems
Volume:
5
Issue:
6
ISSN:
2379-5077
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; (, 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 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. 
    more » « less
  2. ; ; ; (, BMC Bioinformatics)
    null (Ed.)
    Abstract Background Second messengers, c-di-GMP and (p)ppGpp, are vital regulatory molecules in bacteria, influencing cellular processes such as biofilm formation, transcription, virulence, quorum sensing, and proliferation. While c-di-GMP and (p)ppGpp are both synthesized from GTP molecules, they play antagonistic roles in regulating the cell cycle. In C. crescentus , c-di-GMP works as a major regulator of pole morphogenesis and cell development. It inhibits cell motility and promotes S-phase entry by inhibiting the activity of the master regulator, CtrA. Intracellular (p)ppGpp accumulates under starvation, which helps bacteria to survive under stressful conditions through regulating nucleotide levels and halting proliferation. (p)ppGpp responds to nitrogen levels through RelA-SpoT homolog enzymes, detecting glutamine concentration using a nitrogen phosphotransferase system (PTS Ntr ). This work relates the guanine nucleotide-based second messenger regulatory network with the bacterial PTS Ntr system and investigates how bacteria respond to nutrient availability. Results We propose a mathematical model for the dynamics of c-di-GMP and (p)ppGpp in C. crescentus and analyze how the guanine nucleotide-based second messenger system responds to certain environmental changes communicated through the PTS Ntr system. Our mathematical model consists of seven ODEs describing the dynamics of nucleotides and PTS Ntr enzymes. Our simulations are consistent with experimental observations and suggest, among other predictions, that SpoT can effectively decrease c-di-GMP levels in response to nitrogen starvation just as well as it increases (p)ppGpp levels. Thus, the activity of SpoT (or its homologues in other bacterial species) can likely influence the cell cycle by influencing both c-di-GMP and (p)ppGpp. Conclusions In this work, we integrate current knowledge and experimental observations from the literature to formulate a novel mathematical model. We analyze the model and demonstrate how the PTS Ntr system influences (p)ppGpp, c-di-GMP, GMP and GTP concentrations. While this model does not consider all aspects of PTS Ntr signaling, such as cross-talk with the carbon PTS system, here we present our first effort to develop a model of nutrient signaling in C. crescentus . 
    more » « less
  3. ; ; ; ; ; ; ; ; ; ; 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. We 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. 
    more » « less
  4. ; ; ; ; ; (, Frontiers in Sustainable Food Systems)
    Plant growth-promoting bacteria (PGPB) are valuable for supporting sustainable food production and may alleviate the negative impacts of chemical fertilizers on human health and the environment. While single-strain inoculations have proven unreliable due to poor survival and colonization in the rhizosphere, application of PGPB in multispecies consortia has the potential to improve these outcomes. Here, we describe a new approach for screening and identifying bacterial consortia that improve the growth of corn relative to plants inoculated with a single strain. The method uses the microwell recovery array (MRA), a microfabricated high-throughput screening device, to rapidly explore the maize ( Zea mays L .) rhizobiome for higher-order combinations of bacteria that promote the growth and colonization of the nitrogen-fixing PGPB, Azospirillum brasilense . The device simultaneously generates thousands of random, unique combinations of bacteria that include A. brasilense and members of the maize rhizobiome, then tracks A. brasilense growth in each combination during co-culture. Bacteria that show the highest levels of A. brasilense growth promotion are then recovered from the device using a patterned light extraction technique and are identified. With this approach, the screen uncovered growth-promoting consortia consisting primarily of bacteria from the Acinetobacter - Enterobacter - Serratia genera, which were then co-inoculated with A. brasilense on axenic maize seedlings that were monitored inside a plant growth chamber. Compared to maize plants inoculated with A. brasilense alone, plants that were co-inoculated with these consortia showed accelerated growth after 15 days. Follow-up root colonization assays revealed that A. brasilense colonized at higher levels on roots from the co-inoculated seedlings. These findings demonstrate a new method for rapid bioprospecting of root and soil communities for complementary PGPB and for developing multispecies consortia with potential use as next-generation biofertilizers. 
    more » « less
  5. ; ; ; ; ; ; ; ; ; ; et al (, Proceedings of the National Academy of Sciences)
    Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogenAgrobacterium tumefaciensproduces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase, is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are produced via a nonessential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering c-di-GMP breakdown and dampening its synthesis. Pterins are excreted, and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in apruAmutant. PruR and DcpA are encoded in an operon with wide conservation among diverse Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a pterin-responsive regulatory mechanism that controls biofilm formation and related c-di-GMP-dependent phenotypes inA. tumefaciensand potentially acts more widely in multiple proteobacterial lineages. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email