Conjugative Coupling Proteins and the Role of Their Domains in Conjugation, Secondary Structure and in vivo Subcellular Location

Type IV Coupling Proteins (T4CPs) are essential elements in many type IV secretion systems (T4SSs). The members of this family display sequence, length, and domain architecture heterogeneity, being the conserved Nucleotide-Binding Domain the motif that defines them. In addition, most T4CPs contain a Transmembrane Domain (TMD) in the amino end and an All-Alpha Domain facing the cytoplasm. Additionally, a few T4CPs present a variable domain at the carboxyl end. The structural paradigm of this family is TrwB(R388), the T4CP of conjugative plasmid R388. This protein has been widely studied, in particular the role of the TMD on the different characteristics of TrwB(R388). To gain knowledge about T4CPs and their TMD, in this work a chimeric protein containing the TMD of TraJ(pKM101)and the cytosolic domain of TrwB(R388)has been constructed. Additionally, one of the few T4CPs of mobilizable plasmids, MobB(CloDF13)of mobilizable plasmid CloDF13, together with its TMD-less mutant MobB Delta TMD have been studied. Mating studies showed that the chimeric protein is functionalin vivoand that it exerted negative dominance against the native proteins TrwB(R388)and TraJ(pKM101). Also, it was observed that the TMD of MobB(CloDF13)is essential for the mobilization of CloDF13 plasmid. Analysis of the secondary structure components showed that the presence of a heterologous TMD alters the structure of the cytosolic domain in the chimeric protein. On the contrary, the absence of the TMD in MobB(CloDF13)does not affect the secondary structure of its cytosolic domain. Subcellular localization studies showed that T4CPs have a unipolar or bipolar location, which is enhanced by the presence of the remaining proteins of the conjugative system. Unlike what has been described for TrwB(R388), the TMD is not an essential element for the polar location of MobB(CloDF13). The main conclusion is that the characteristics described for the paradigmatic TrwB(R388)T4CP should not be ascribed to the whole T4CP family. Specifically, it has been proven that the mobilizable plasmid-related MobB(CloDF13)presents different characteristics regarding the role of its TMD. This work will contribute to better understand the T4CP family, a key element in bacterial conjugation, the main mechanism responsible for antibiotic resistance spread.This work was in part supported by grants from the University of the Basque Country (GIU18/229 and COLAB19/08) and the Industry Department of the Basque Government (ELKARTEK 2020 KK-2020/00007). IA-R was a pre-doctoral student supported by the Basque Government

Study of the role transmembrane domain of Type IV Coupling Proteins

345 p.La conjugación bacteriana es el mecanismo más importante en la diseminación de genes de resistencia a antibióticos entre bacterias. Este proceso se realiza mediante unos complejos macromoleculares denominados Sistemas de Secreción de Tipo IV. Las proteínas acopladoras forman parte de estos sistemas, siendo una pieza clave en el proceso conjugativo.El objetivo de esta tesis doctoral ha sido caracterizar el funcionamiento de diferentes proteínas acopladoras, centrándose en el papel de su dominio transmembrana. Para ello se han seleccionado proteínas acopladoras de diferentes sistemas y características. Además se han creado proteínas mutantes (tanto quimeras como de delección) con las que estudiar la función del dominio transmembrana.En concreto se ha estudiado la actividad y la localización subcelular de las diferentes proteínas in vivo. Además se han desarrollado protocolos de purificación obteniendo muestras con las que realizar ensayos in vitro. De esta manera se ha caracterizado la estabilidad de las proteínas, su unión a moléculas de ADN, sus patrones de oligomerización, su estructura secundaria y su actividad ATP hidrolasa. Finalmente, se ha realizado la puesta a punto de técnicas de reconstitución en membranas modelo de las proteínasacopladoras, con el fin de realizar ensayos single-molecule en el futuro.En esta tesis se ha demostrado que las características descritas para la proteína acopladora TrwBR388, el paradigma de esta familia, no son extrapolables a todas las proteínas de la familia, remarcando así la necesidad de más estudios en este campo