The molecular basis of FimT-mediated DNA uptake during bacterial natural transformation

Abstract

Naturally competent bacteria encode sophisticated protein machinery for the uptake and translocation of exogenous DNA into the cell. If this DNA is integrated into the bacterial genome, the bacterium is said to be naturally transformed. Most competent bacterial species utilise type IV pili for the initial DNA uptake step. These proteinaceous cell-surface structures are composed of thousands of pilus subunits (pilins), designated as major or minor according to their relative abundance in the pilus. Here, we show that the minor pilin FimT plays an important role in the natural transformation of Legionella pneumophila. We use NMR spectroscopy, in vitro DNA binding assays and in vivo transformation assays to understand the molecular basis of FimT’s role in this process. FimT binds to DNA via an electropositive patch, rich in arginines, several of which are well-conserved and located in a conformationally flexible C-terminal tail. FimT orthologues from other Gammaproteobacteria share the ability to bind to DNA. Our results suggest that FimT plays an important role in DNA uptake in a wide range of competent species.

Complete functional analysis of type IV pilus components of a reemergent plant pathogen reveals neofunctionalization of paralog genes

“…Additionally, R160 was always preceded by a glycine residue, constituting a highly conserved GRxR motif (Fig. S15b), as seen elsewhere 46 . Although amino acid identities ranged from 95.60% to 100% within X. fastidiosa , and from 15.5% to 80% compared to other species, only 41 out of 1,416 FimT3 sequences encoded by the Xanthomonadaceae do not carry the GRxR motif (Table S5).…”

“…Moreover, alignment of FimT3 with a FimT ortholog that is essential for natural competence of Acinetobacter baylyi 45 (28% identity and 46% similarity over 91% of query sequence) showed alignment of R162 of FimT3 to an arginine of FimT from A. baylyi . Likewise, FimT3 shares 27% identity (48% similarity) over 80% of query sequence with a recently described DNA-binding FimT ortholog of Legionella pneumophila , in which the aligned R160 and R162 have been demonstrated as important to bind DNA 46 (Fig. S14c).…”

“…Likewise, FimT3 shares 27% identity (48% similarity) over 80% of query sequence with a recently described DNA-binding FimT ortholog of Legionella pneumophila, in which the aligned R160 and R162 have been demonstrated as important to bind DNA 46 (Fig. S14c).…”

“…However, the binding appears to be non-specific, which is in contrast to ComP 43 . FimT from the human pathogen L. pneumophila 46 , which is a homologue of the FimT3 studied here, was recently identified as a pilin with DNA-binding ability.…”

“…encoded by other members of the Xanthomonadaceae family. Previously, DNA-binding homologues of FimT3 have been found in many representative species of γ-Proteobacteria, including the Xanthomonadaceae family to which X. fastidiosa belongs 46 . We performed searches using the Conditional Reciprocal Best BLAST (CRB-BLAST) 47 , as well as phylogenetic analyses, to further explore the distribution of the fimT paralogs within this family.…”