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1. Examining effects of rhizobacteria in relieving abiotic crop stresses using carbon‐11 radiotracing

   https://doi.org/10.1111/ppl.13675  

   Powell, Avery ; Wilder, Stacy L. ; Housh, Alexandra B. ; Scott, Stephanie ; Benoit, Mary ; Powell, Garren ; Waller, Spenser ; Guthrie, James M. ; Schueller, Michael J. ; Ferrieri, Richard A. ( March 2022 , Physiologia Plantarum)

   In agriculture, plant growth promoting bacteria (PGPB) are increasingly used for reducing environmental stress‐related crop losses through mutualistic actions of these microorganisms, activating physiological and biochemical responses, building tolerances within their hosts. Here we report the use of radioactive carbon‐11 (t_½20.4 min) to examine the metabolic and physiological responses ofZea maystoAzospirillum brasilense(HM053) inoculation while plants were subjected to salinity and low nitrogen stresses. Host metabolism of “new” carbon resources (as¹¹C) and physiology including [¹¹C]‐photosynthate translocation were measured in response to imposed growth conditions. Salinity stress caused shortened, dense root growth with a 6‐fold increase in foliar [¹¹C]‐raffinose, a potent osmolyte. ICP‐MS analyses revealed increased foliar Na⁺levels at the expense of K⁺. HM053 inoculation relieved these effects, reinstating normal root growth, lowering [¹¹C]‐raffinose levels while increasing [¹¹C]‐sucrose and its translocation to the roots. Na⁺levels remained elevated with inoculation, but K⁺levels were boosted slightly. Low nitrogen stress yielded longer roots possessing high levels of anthocyanins. Metabolic analysis revealed significant shifts in “new” carbon partitioning into the amino acid pool under low nitrogen stress, with significant increases in foliar [¹¹C]‐glutamate, [¹¹C]‐aspartate, and [¹¹C]‐asparagine, a noted osmoprotectant.¹¹CO₂fixation and [¹¹C]‐photosynthate translocation also decreased, limiting carbon supply to roots. However, starch levels in roots were reduced under nitrogen limitation, suggesting that carbon repartitioning could be a compensatory action to support root growth. Finally, inoculation with HM053 re‐instated normal root growth, reduced anthocyanin, boosted root starch, and returned¹¹C‐allocation levels back to those of unstressed plants.

    

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2. Screening the maize rhizobiome for consortia that improve Azospirillum brasilense root colonization and plant growth outcomes

   https://doi.org/10.3389/fsufs.2023.1106528  

   Barua, Niloy ; Clouse, Kayla M. ; Ruiz Diaz, Dorivar A. ; Wagner, Maggie R. ; Platt, Thomas G. ; Hansen, Ryan R. ( April 2023 , 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. 

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3. The Azospirillum brasilense Core Chemotaxis Proteins CheA1 and CheA4 Link Chemotaxis Signaling with Nitrogen Metabolism

   https://doi.org/10.1128/mSystems.01354-20  

   Ganusova, Elena E. ; Vo, Lam T. ; Abraham, Paul E. ; O’Neal Yoder, Lindsey ; Hettich, Robert L. ; Alexandre, Gladys ( February 2021 , 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. 

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4. Specific Root Exudate Compounds Sensed by Dedicated Chemoreceptors Shape Azospirillum brasilense Chemotaxis in the Rhizosphere

   https://doi.org/10.1128/AEM.01026-20  

   O’Neal, Lindsey ; Vo, Lam ; Alexandre, Gladys ; Parales, Rebecca E. ( July 2020 , Applied and Environmental Microbiology)

   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 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. 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. 

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5. A Putative Acetylation System in Vibrio cholerae Modulates Virulence in Arthropod Hosts

   https://doi.org/10.1128/AEM.01113-18  

   Liimatta, Kalle ; Flaherty, Emily ; Ro, Gabby ; Nguyen, Duy K. ; Prado, Cecilia ; Purdy, Alexandra E. ; Liu, Shuang-Jiang ( November 2018 , Applied and Environmental Microbiology)

   ABSTRACT Acetylation is a broadly conserved mechanism of covalently modifying the proteome to precisely control protein activity. In bacteria, central metabolic enzymes and regulatory proteins, including those involved in virulence, can be targeted for acetylation. In this study, we directly link a putative acetylation system to metabolite-dependent virulence in the pathogen Vibrio cholerae . We demonstrate that the cobB and yfiQ genes, which encode homologs of a deacetylase and an acetyltransferase, respectively, modulate V. cholerae metabolism of acetate, a bacterially derived short-chain fatty acid with important physiological roles in a diversity of host organisms. In Drosophila melanogaster , a model arthropod host for V. cholerae infection, the pathogen consumes acetate within the gastrointestinal tract, which contributes to fly mortality. We show that deletion of cobB impairs growth on acetate minimal medium, delays the consumption of acetate from rich medium, and reduces virulence of V. cholerae toward Drosophila . These impacts can be reversed by complementing cobB or by introducing a deletion of yfiQ into the Δ cobB background. We further show that cobB controls the accumulation of triglycerides in the Drosophila midgut, which suggests that cobB directly modulates metabolite levels in vivo . In Escherichia coli K-12, yfiQ is upregulated by cAMP-cAMP receptor protein (CRP), and we identified a similar pattern of regulation in V. cholerae , arguing that the system is activated in response to similar environmental cues. In summary, we demonstrate that proteins likely involved in acetylation can modulate the outcome of infection by regulating metabolite exchange between pathogens and their colonized hosts. IMPORTANCE The bacterium Vibrio cholerae causes severe disease in humans, and strains can persist in the environment in association with a wide diversity of host species. By investigating the molecular mechanisms that underlie these interactions, we can better understand constraints affecting the ecology and evolution of this global pathogen. The Drosophila model of Vibrio cholerae infection has revealed that bacterial regulation of acetate and other small metabolites from within the fly gastrointestinal tract is crucial for its virulence. Here, we demonstrate that genes that may modify the proteome of V. cholerae affect virulence toward Drosophila , most likely by modulating central metabolic pathways that control the consumption of acetate as well as other small molecules. These findings further highlight the many layers of regulation that tune bacterial metabolism to alter the trajectory of interactions between bacteria and their hosts. 

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