Periodic Reporting for period 1 - D3NAs (Coping with Inhospitable Sweetness: Insights from a (Diversity)^3 Study of Nectar Acinetobacters) Reporting period: 2017-08-01 to 2019-07-31 Summary of the context and overall objectives of the project Microorganisms find in plants heterogeneous and highly dynamic habitats which can differ widely at macro- and microscales in physico-chemical conditions and nutrient availability. Most previous studies on plant microbiology have focused on describing the astonishing diversity of microorganisms occurring in the rhizosphere and the phyllosphere, whereas other plant parts like flowers as microbial habitats have remained underexplored until recently. In this regard, an emerging focus of research is the study of floral nectar as a natural habitat for different highly adapted microorganisms that are able to withstand elevated sugar contents and the presence of plant secondary metabolites with defensive functions, including a study of the ecological role of these nectar inhabitants. The species of the genus Acinetobacter (also known as “acinetobacters”) rank among the most frequent bacterial nectar-dwellers, but the diversity and ecological role(s) of these microorganisms still remain poorly characterized.The overall objective of this Marie Skłodowska-Curie Action (MSCA) was to provide novel insights on why the acinetobacters can thrive so well in floral nectar and animal pollinators. To this end, we followed a multidisciplinary approach that dealt with different levels of study of the diversity of the nectar acinetobacters (“D3 approach”), namely phylogenetic, phenotypic and genomic diversity. Notably, our research approach combined several disciplines, including microbial ecology, population phylogenetics, bacterial physiology, phenomics and genomics, so as to provide the experienced researcher (ER) with numerous opportunities to extend his knowledge in such disciplines, and to establish strong international and multidisciplinary collaborations with several scientists from both the host institution and other organizations. Additionally, through the implementation of this action, the ER aimed to boost his potential for reaching a position of professional maturity by getting additional training in management, communication, dissemination and exploitation of results, and networking. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Work packages and main resultsThe activities performed during the implementation of this MSCA were organized into three experimental work packages, one per study level of the diversity of the nectar acinetobacters:1) Phylogenetic diversity: development of a multilocus sequence analysis scheme for phylogenetic analysis of the nectar acinetobacters.A collection of 82 strains obtained from floral nectar and insects in different countries (Spain, Belgium, USA and Japan) was successfully typed by a three-loci scheme comprised by the 16S rRNA gene and two different regions (zone 1 and zone 2) of the rpoB gene, which encodes the beta subunit of RNA polymerase. Detailed phylogenetic analysis of the nucleotide sequences revealed the presence of some clades that may represent new species within the genus Acinetobacter. 2) Phenotypic level: high-throughput screening of phenotypic characteristics of the nectar acinetobacters (Fig. 1).This work package involved the developed of a 96-well plate assays based on Biolog’s phenotype microarray technology to test for assimilation of carbon and nitrogen compounds (30 and 24 assays, respectively), and tolerance to different stressors commonly found in floral nectar (28 assays). Analysis of the phenotypic data obtained for the collection of 82 nectar- and insect-associated acinetobacters by such technology identified seven different profiles for carbon assimilation, 32 different profiles for nitrogen assimilation and ten different profiles for tolerance to inhibitors. Surprisingly, even when they are frequently encountered in floral nectars of high sugar concentration, tested strains could only resist moderate osmotic pressures. Moreover, clustering of strains according to their nitrogen assimilation patterns (but not to their carbon assimilation and tolerance profiles) was linked to species identity. Such clade-driven distribution suggests niche partitioning for assimilation nitrogen resources that may allow coexistence in floral nectar and other habitats.3) Genomic diversity: whole genome sequencing and analysis of select strains.This work package was carried out in close collaboration with other research groups that were contacted during the implementation of this MSCA. A set of 11 Acinetobacter strains belonging to different rpoB clades and phenotype clusters were sequenced, and the results confirmed the existence of new species of nectar- and insect-associated acinetobacters among the studied strains.Non-scientific management activitiesApart from the leading role of the ER in the scientific aspects of the project (experimental work, data analysis and manuscript drafting), he was actively involved in the management aspects of the MSCA, including financial aspects, grant writing and fundraising, networking, and teaching and mentoring of bachelor and master students.Exploitation and dissemination of resultsThe results obtained in this MSCA have been disseminated through the participation of the ER in different conferences, the production of some publications for peer-reviewed journals (one published paper and three manuscripts in preparation), and other outreach activities. Moreover, all relevant output coming from this MSCA (publications, pictures, useful links, etc.) has been disseminated through the website and Twitter accounts of the host lab and ER. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The genetic and phenotypic results obtained during the implementation of this MSCA may be used in future scientific projects dealing with diversity studies of the genus Acinetobacter or, in general, plant-associated microorganisms. The potential application of the results of this project in the industrial setting is difficult to assess at this point, as the use of the nectar acinetobacters in industrial or biomedical applications has not yet been explored in detail. It remains also to be investigated whether the nectar acinetobacters can be used to increase pollination success. Pollinator decline is a global problem, and both academics and industry are urgently seeking for innovative tools to increase pollination efficiency. The application of pollinator-attracting nectar microbes may be one such solution.Regarding the impact of this MSCA to the host lab, it certainly reinforced its leading position in nectar microbiology research, and opened new avenues for future collaboration in the field. Furthermore, the ER contributed to develop new protocols and analysis methods that are now incorporated in the routine of the host lab. At the same time, the technical and management skills acquired by the ER from the host lab during the implementation of this MSCA have greatly improved his career perspectives. Fig. 1: Overview of the methods used in Work Package 2 for phenotyping of the nectar acinetobacters.
