Phosphatidylinositol 5-phosphatases (5PTases) that hydrolyze the 5' position of the inositol ring are key components of membrane trafficking system. Recently, we reported that mutation in At5PTase7 gene reduced production of reactive oxygen species (ROS) and decreased expression of stress-responsive genes, resulting in increased salt sensitivity. Here, we describe an even more salt-sensitive 5ptase mutant, At5ptase9, which also hydrolyzes the 5' phosphate groups specifically from membrane-bound phosphatidylinositides. Interestingly, the mutants were more tolerant to osmotic stress. We analyzed the main cellular processes that may be affected by the mutation, such as production of ROS, influx of calcium, and induction of salt-response genes. The At5ptase9 mutants showed reduced ROS production and Ca(2+) influx, as well as decreased fluid-phase endocytosis. Inhibition of endocytosis by phenylarsine oxide or Tyrphostin A23 in wild-type plants blocked these responses. Induction of salt-responsive genes in wild-type plants was also suppressed by the endocytosis inhibitors. Thus, inhibition of endocytosis in wild-type plants mimicked the salt stress responses, observed in the At5ptase9 mutants. In summary, our results show a key non-redundant role of At5PTase7 and 9 isozymes, and underscore the localization of membrane-bound PtdIns in regulating plant salt tolerance by coordinating the endocytosis, ROS production, Ca(2+) influx, and induction of stress-responsive genes.

Microbial ecosystems tightly associated with a eukaryotic host are widespread in nature. The genetic and metabolic networks of the eukaryotic hosts and the associated microbes have coevolved to form a symbiotic relationship. Both the Gram-positive Bacillus subtilis and the Gram-negative Serratia plymuthica can form biofilms on plant roots and thus can serve as a model system for the study of interspecies interactions in a host-associated ecosystem. We found that B. subtilis biofilms expand collectively and asymmetrically toward S. plymuthica, while expressing a nonribosomal antibiotic bacillaene and an extracellular protease. As a result, B. subtilis biofilms outcompeted S. plymuthica for successful colonization of the host. Strikingly, the plant host was able to enhance the efficiency of this killing by inducing bacillaene synthesis. In turn, B. subtilis biofilms increased the resistance of the plant host to pathogens. These results provide an example of how plant-bacterium symbiosis promotes the immune response of the plant host and the fitness of the associated bacteria. IMPORTANCE Our study sheds mechanistic light on how multicellular biofilm units compete to successfully colonize a eukaryote host, using B. subtilis microbial communities as our lens. The microbiota and its interactions with its host play various roles in the development and prevention of diseases. Using competing beneficial biofilms that are essential microbiota members on the plant host, we found that B. subtilis biofilms activate collective migration to capture their prey, followed by nonribosomal antibiotic synthesis. Plant hosts increase the efficiency of antibiotic production by B. subtilis biofilms, as they activate the synthesis of polyketides; therefore, our study provides evidence of a mechanism by which the host can indirectly select for beneficial microbiota members.on July 10, 2020 by guest http://aem.asm.org/ Downloaded from RESULTSB. subtilis biofilm actively expands toward a competing S. plymuthica colony. When grown on a solid biofilm-inducing medium, B. sutbilis biofilms form symmetrical, circular colonies. To determine the effect of a competing S. plymuthica colony on the development of a B. subtilis biofilm, the two species were inoculated next to each other on a solid biofilm medium. After 2 days, the B. subtilis colony reached S. plymuthica, forming a thick wrinkle around its edge and penetrating toward its center. By the third day, B. subtilis biofilm completely engulfed the S. plymuthica colony, covering it with a thin, unstructured film and enclosing it within the circular wrinkle (Fig. 1A). The location and shape of the B. subtilis center of the biofilm did not change during the interaction with S. plymuthica. However, the biofilm colony advanced asymmetrically toward the S. plymuthica colony, breaking from its usual circular shape ( Fig. 1A).We next examined the mechanisms that could mediate this asymmetric expansion toward a competitor. Many bacterial cells are capable of directional swimming using their flagella (2...

Beneficial and probiotic bacteria play an important role in conferring immunity of their hosts to a wide range of bacterial, viral, and fungal diseases. Bacillus subtilis is a Gram-positive bacterium that protects the plant from various pathogens due to its capacity to produce an extensive repertoire of antibiotics. At the same time, the plant microbiome is a highly competitive niche, with multiple microbial species competing for space and resources, a competition that can be determined by the antagonistic potential of each microbiome member. Therefore, regulating antibiotic production in the rhizosphere is of great importance for the elimination of pathogens and establishing beneficial host-associated communities. In this work, we used B. subtilis as a model to investigate the role of plant colonization in antibiotic production. Flow cytometry and imaging flow cytometry (IFC) analysis supported the notion that Arabidopsis thaliana specifically induced the transcription of the biosynthetic clusters for the non-ribosomal peptides surfactin, bacilysin, plipastatin, and the polyketide bacillaene. IFC was more robust in quantifying the inducing effects of A. thaliana, considering the overall heterogeneity of the population. Our results highlight IFC as a useful tool to study the effect of association with a plant host on bacterial gene expression. Furthermore, the common regulation of multiple biosynthetic clusters for antibiotic production by the plant can be translated to improve the performance and competitiveness of beneficial members of the plant microbiome.

Lactobacillaceae are Gram-positive rods, facultative anaerobes, and belong to the lactic acid bacteria (LAB) that frequently serve as probiotics. We systematically compared five LAB strains for the effects of different carbohydrates on their free-living and biofilm lifestyles. We found that fermentable sugars triggered a heterogeneous response in LAB strains, frequently manifested specifically in altered carrying capacity during planktonic growth and colony development. The fermentation capacities of the strains were compatible and could not account for heterogeneity in their differential carrying capacity in liquid and on a solid medium. Among tested LAB strains, L. paracasei, and L. rhamanosus GG survived self-imposed acid stress while L. acidophilus was extremely sensitive to its own glucose utilization acidic products. The addition of a buffering system during growth on a solid medium significantly improved the survival of most tested probiotic strains during fermentation. We suggest that the optimal performance of the beneficial microbiota members belonging to lactobacilli is heterogeneous and varies as a function of the growth model and the dependency on a buffering system.

Lactobacillaceae are Gram-positive rods, facultative anaerobes, and belong to the lactic acid bacteria (LAB) that frequently serve as probiotics. We systematically compared five LAB strains for the effects of different carbohydrates on their free-living and biofilm lifestyles. We found that fermentable sugars triggered an altered carrying capacity with strain specificity during planktonic growth. In addition, heterogeneous response to fermentable sugar was manifested in microbial aggregation (measured by imaging flow cytometry), colony development, and attachment to mucin. The acid production capacities of the strains were compatible and could not account for heterogeneity in their differential carrying capacity in liquid and on a solid medium. Among tested LAB strains, L. paracasei, and L. rhamnosus GG survived self-imposed acid stress while L. acidophilus was extremely sensitive to its own glucose utilization acidic products. The addition of a buffering system during growth on a solid medium significantly improved the survival of most tested probiotic strains during fermentation, but the formation of biofilms and aggregation capacity were responsive to the carbohydrate provided rather than to the acidity. We suggest that the optimal performance of the beneficial microbiota members belonging to Lactobacillaceae varies as a function of the growth model and the dependency on a buffering system.