Characterization of the nucleoprotein complex interconnecting the divisome and the terminal replication region of the Xanthomonas citri chromosome

• Grant number: 24/10539-9
• Support Opportunities: Scholarships in Brazil - Doctorate
• Effective date (Start): October 01, 2024
• Effective date (End): June 30, 2028
• Field of knowledge: Agronomical Sciences - Agronomy - Plant Health
• Principal Investigator: Henrique Ferreira
• Grantee: Natália Alleoni
• Host Institution: Instituto de Biociências (IB). Universidade Estadual Paulista (UNESP). Campus de Rio Claro. Rio Claro , SP, Brazil
• Associated research grant: 21/10577-0 - Biology of Bacteria and Bacteriophages Research Center, AP.CEPID

Abstract

The bacterial cell cycle can be understood as the interval between two eventssubsequent cell division. During this period, bacteria must double theirgenetic material and segregate chromosomes simultaneously with theDNA replication. Division, in turn, begins during thereplication/segregation and must be completed after complete partition of chromosomesnewly duplicated files, avoiding their guillotining. In this way, a highorganization of chromosomes within cells influences their mobility andcorrect portion of these to the daughter cells. There are several forces that can operatesegregation of bacterial replicons and, among them, we mention DNA compaction bycondensins (e.g. SMC), the action of partition proteins (e.g. ParABS) and forcesentropic, where polymers (DNA) undergo separation into cylindrical compartments withdefined characteristics to maximize entropy. Furthermore, the terminal region ofChromosome replication appears to be involved in both the segregation process,regarding the functioning of the division. To date, two systems ofDNA organization of bacterial chromosome replication terms: MatP-ZapBZapA from Escherichia coli (E. coli) and ZapT-ZauP-ZapA from Caulobacter crescentus (C.growing). MatP and ZapT are proteins that bind to DNA in the terminal region ofreplication of chromosomes, occurring in their compaction. Subsequently, this complexnucleoprotein is anchored to the division via interaction with the ZapB or ZauP proteins, whichin turn bind to ZapA (FtsZ accessory protein, capable of binding and stabilizing theZ-ring). During the proponent's master's degree (FAPESP 2022/01768-9), we used coimmunoprecipitation to identify GFP-ZapB interactors in Xanthomonas citri (X.citri) and we investigated the existence of a likely end-end DNA organization systemof replication in this bacterium. Among the proteins identified we mention AMD14_RS13615,a homologue of ZapT from C. crescentus, and AMD14_RS08395, a protein with functionunknown. In the current proposal, studying the function of genes thatwe code for these proteins using deletion/complementation techniques and orgene depletion. We will perform subcellular localization assays for fusions of theseGFP proteins and variants to investigate their roles in segregation dynamics andcell division. Finally, purify the AMD14_RS1361 andAMD14_RS08395 for biochemical and interaction studies with their probable DNAstarget, in Chip-seq assays