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Introduction: Bacterial two-partner secretion (TPS) systems are involved in processes such as biofilm formation; adhesion and binding to host cells, and contact-dependent growth inhibition (CDI). TPS are described in many pathogenic bacteria, e.g. Enterobacteria, Pseudomonas, Burkholderia and Xanthomonas species. Previously characterized CDI locus in Burkholderia, encode a TpsB transporter and large exported TpsA proteins, and short downstream genes. Objectives: For this research project I aim to characterize the CDI loci in Ralstonia solanacearum species complex (RSSC), an aggressive soil-borne plant pathogen that causes disease in diverse plant species. Especially I focus on a novel lineage of R. solanacearum. The novel lineage, called 4NPB, was first detected in the island of Martinique in early 2000s. It is characterized by a higher pathogenicity and an expanded host range. Materials & methods: 500 RSSC strains were sampled in Martinique and French Guiana during 4NPB emergence. Sequencing, genome assembly, phylogenetic and pangenomic analysis were done to detect genomic changes linked with the emergence of the new lineage. The dataset was supplemented with 400 genomes from NCBI database. TPS proteins from Burkholderia were aligned against all the genomes of the dataset, in order to detect TPS homologs. Structures of the putative TPS proteins were predicted using AlphaFold2 and HMMER. The loci were clustered and assigned to groups by sequence and structure similarity. I will next measure how strains that share or vary in putative CDI loci perform in direct competition in vitro and in planta using a ddPCR assay. Results: I have found an unusually large number of potential TpsA and TpsB homologs in RSSC strains. The putative TPS loci exhibit both conservation and patterns of recent recombination events typical for TPS systems. The loci can be divided into four groups which have diverse locus structures. Analysis of the 4NPB population revealed recombination hotspots found between 4NPB and endemic strains. Recombination and gene gain/loss events were found in various toxin-antitoxin systems, including putative TPS loci. Conclusion: This work will contribute to our understanding of how TPS systems mediate pathogenicity and interbacterial interactions in Ralstonia solanacearum and how TPS systems can potentially be involved into emergence of novel bacterial lineages.
