The interaction of the F-like plasmid-encoded TraN isoforms with their cognate outer membrane receptors

Abstract

Horizontal gene transfer via conjugation plays a major role in bacterial evolution. In F-like plasmids, efficient DNA transfer is mediated by close association between donor and recipient bacteria. This process, known as mating pair stabilization (MPS), is mediated by interactions between the plasmid-encoded outer membrane (OM) protein TraN in the donor and chromosomally-encoded OM proteins in the recipient. We have recently reported the existence of seven TraN sequence types, which are grouped into four structural types, we named TraNα, TraNβ, TraNγ, TraNδ. Moreover, we have shown specific pairing between TraNα and OmpW, TraNβ and OmpK36 of Klebsiella pneumoniae, TraNγ and OmpA and TraNδ and OmpF. In this study we found that although structurally similar, TraNα encoded by the pSLT plasmid (TraNα2) binds OmpW in both Escherichia coli and Citrobacter rodentium while TraNα encoded by the R100-1 plasmid (TraNα1) only binds OmpW in E. coli. AlphaFold2 predictions suggested that this specificity is mediated by a single amino acid difference in loop 3 of OmpW, which we confirmed experimentally. Moreover, we show that single amino acids insertions into loop 3 of OmpK36 affect TraNβ-mediated conjugation efficiency of the K. pneumoniae resistance plasmid pKpQIL. Lastly, we report that TraNβ can also mediate MPS by binding OmpK35, making it the first TraN variant that can bind more than one OM protein in the recipient. Together, these data show that subtle differences in the OM receptors can impact TraN-mediated conjugation efficiency.

A Whole-Cell Screening Platform to Discover Cell Adhesion Molecules that Enable Programmable Bacterial Cell-Cell Adhesion

“…The bacteria nanobody display library (100 ng) was then transformed into the E. coli MG1655 ΔompW strain via electroporation. We selected the Δ ompW strain to minimize the interactions between OmpW and TraN, as suggested by Low et al 21, 41 . The cells were harvested and resuspended in LB with 10% glycerol for storage at -80 °C.…”