Biology subjects

Valette, O.

Publications and source records attributed to Valette, O..

6 recordsLinked to original sources

bioRxiv2026

A short C-terminal plug motif in a conserved type 4 pilus component controls pilus-tip localisation and filament biogenesis

Type 4a pili (T4aP), the most widespread and functionally versatile subtype of the type 4 filaments (T4F) superfamily, are functionalised at their tip by the adhesin PilC/PilY1. How this unusually large non-pilin protein is stably displayed at the pilus tip, and why is it required for pilus biogenesis, remains unknown. Here, using a multidisciplinary approach in Neisseria meningitidis, we show that the last 12 residues of PilC, representing ~1% of the protein, are both necessary and sufficient for pilus-tip localisation and filament biogenesis. X-ray crystallography reveals that this short "plug" motif binds the PilK subunit (within a complex of four minor pilins that caps the pilus) through {beta}-strand augmentation, a mode of interaction not previously characterised in T4F. Binding assays with synthetic peptides establish the specificity and affinity of this interaction, while the addition of a plug peptide extracellularly to cultures of a{Delta} pilC mutant restores pilus biogenesis. We show, using different methods, that the plug motif markedly stabilises PilK, providing an explanation for the requirement of PilC in pilus biogenesis. Consequently, expression of a PilK protein carrying a fused plug in N. meningitidis bypasses the requirement for PilC in pilus biogenesis. Together, these findings define the molecular basis of PilC/PilY1 function and localisation, reconcile all previous observations, and establish a broadly applicable model for T4aP biogenesis.

microbiology↗

bioRxiv2025

Unravelling the molecular mechanisms of DNA capture by the Com pilus in naturally transformable monoderm bacteria

Transformation is a mechanism of horizontal gene transfer widespread in bacteria. The first step in transformation - capture of exogenous DNA - is mediated by surface-exposed filaments belonging to the type 4 filament (T4F) superfamily. How these protein polymers, composed of major and minor pilin subunits, interact with DNA remains poorly understood. Here, we address this question for the Com pilus, a widespread T4F mediating DNA capture in competent monoderm species. Our functional analysis, performed in Streptococcus sanguinis, was guided by a complete structural model of the Com pilus. We show that the major pilin ComGC does not bind DNA. In contrast, a systematic mutational analysis of electropositive residues exposed at the filament surface in the four minor pilins (ComGD, ComGE, ComGF, ComGG) reveals that the interface between ComGD and ComGF is important for DNA capture. Sequential mutations in these two interacting subunits lead to complete abolition of transformation, without affecting piliation. We further demonstrate the physical interaction between ComGD and ComGF using disulfide crosslinking, upon mutagenesis of two strategically positioned residues into cysteines. A structural model of the Com pilus tip interacting with DNA recapitulates all these findings and highlights a novel mode of DNA-binding, conserved in hundreds of monoderm species. IMPORTANCEBacteria are capable of evolving and diversifying very rapidly by acquiring new genetic material via horizontal gene transfer (HGT). Transformation is a widespread mechanism of HGT in bacteria, which results from the capture of extracellular DNA by surface-exposed pili belonging to the superfamily of type 4 filament (T4F). How T4F - that are composed of major and minor pilins - interact with DNA remains poorly understood, especially in competent monoderm species that use a unique T4F for DNA capture known as Com pilus or T4dP. The significance of this work is in characterizing a novel mode of DNA-binding by showing that the interface between two minor pilins part of a tip-located complex of four pilins - found in many different T4F - have been functionalized in monoderms to capture DNA. This is an evolutionary strategy used by bacteria to promote the exceptional functional versatility of T4F.

microbiology↗

bioRxiv2023

Systematic functional analysis of the Com pilus in Streptococcus sanguinis: a minimalistic type 4 filament dedicated to DNA uptake in monoderm bacteria

Type 4 filaments (T4F) are a superfamily of functionally versatile nanomachines, ubiquitous in prokaryotes, which use similar multi-protein machineries to assemble and operate filamentous polymers of type 4 pilins. The best studied T4F use very complex machineries, which has posed challenges to understanding the mechanisms of both filament assembly and the roles they facilitate. Here, we report the systematic functional analysis of the Com pilus, a widespread T4F mediating DNA uptake during natural transformation in monoderm bacteria. Using Streptococcus sanguinis as a model, we show that Com pili are bona fide type 4 pili (T4P), which represent a new pilus sub-type. We show that with only eight components necessary for their assembly and functioning - all "core" poteins universally conserved across this superfamily - the Com pilus epitomises a minimalistic T4F. We demonstrate that core T4F components are sufficient for filament assembly. Intriguingly, akin to more elaborate T4F, the Com pilus contains four minor pilins forming a complex likely to be situated at the apex of the filaments. Our results have global implications for T4F and make Com pili a model for elucidating the fundamental processes underpinning filament assembly.

microbiology↗

bioRxiv2023

Structure of a heteropolymeric type 4 pilus from a monoderm bacterium

Type 4 pili (T4P) are important virulence factors, which belong to a superfamily of nanomachines ubiquitous in prokaryotes, called type 4 filaments (T4F). T4F are defined as helical polymers of type 4 pilins. Recent advances in cryo-electron microscopy (cryo-EM) led to structures of several T4F. This revealed that the long N-terminal -helix, the trademark of pilins, packs in the centre of the filaments to form a hydrophobic core, which in bacteria is accompanied by the melting (unfolding) of a portion of 1. Since all available bacterial T4F structures are from diderm species, we tested whether this architecture is conserved in phylogenetically distant species by determining the structure of the T4P of the monoderm Streptococcus sanguinis. Our 3.7 A resolution cryo-EM structure of this heteropolymeric T4P, and the resulting full atomic model including all minor pilins, highlight universal features of bacterial T4F and have widespread implications in understanding their biology.

microbiology↗

bioRxiv2023

Sinorhizobium meliloti FcrX coordinates cell cycle and division during free-living growth and symbiosis

Sinorhizobium meliloti is a soil bacterium that establishes a symbiosis within root nodules of legumes (Medicago sativa, for example) where it fixes atmospheric nitrogen into ammonia and obtains in return carbon sources and other nutrients. In this symbiosis, S. meliloti undergoes a drastic cellular change leading to a terminal differentiated form (called bacteroid) characterized by genome endoreduplication, increase of cell size and high membrane permeability. The bacterial cell cycle (mis)regulation is at the heart of this differentiation process. In free-living cells, the master regulator CtrA ensures the progression of cell cycle by activating cell division (controlled by the tubulin-like protein FtsZ) and simultaneously inhibiting supernumerary DNA replication, while on the other hand the downregulation of CtrA and FtsZ is essential for bacteroid differentiation during symbiosis, preventing endosymbiont division and permitting genome endoreduplication. Little is known in S. meliloti about regulators of CtrA and FtsZ, as well as the processes that control bacteroid development. Here, we combine cell biology, biochemistry and bacterial genetics approaches to understand the function(s) of FcrX, a new factor that controls both CtrA and FtsZ, in free-living growth and in symbiosis. Depletion of the essential gene fcrX led to abnormally high levels of FtsZ and CtrA and minicell formation. Using multiple complementary techniques, we showed that FcrX is able to interact physically with FtsZ and CtrA. Moreover, its transcription is controlled by CtrA itself and displays an oscillatory pattern in the cell cycle. We further showed that, despite a weak homology with FliJ-like proteins, only FcrX proteins from closely-related species are able to complement S. meliloti fcrX function. Finally, deregulation of FcrX showed abnormal symbiotic behaviors in plants suggesting a putative role of this factor during bacteroid differentiation. In conclusion, FcrX is the first known cell cycle regulator that acts directly on both, CtrA and FtsZ, thereby controlling cell cycle, division and symbiotic differentiation.

microbiology↗

bioRxiv2019

CcnA, a novel non-coding RNA regulating the bacterial cell cycle

Bacteria are powerful models for understanding how cells divide and accomplish global regulatory programs. In Caulobacter crescentus, a cascade of essential master regulators supervises the correct and sequential activation of DNA replication, cell division and development of different cell types. Among them, the response regulator CtrA plays a crucial role coordinating all those functions. Here, for the first time we describe the role of a novel factor named CcnA, a cell cycle regulated ncRNA located at the origin of replication, presumably activated by CtrA and responsible for the accumulation of CtrA itself. In addition, CcnA may be also involved in the inhibition of translation of the S-phase regulator, GcrA, by interacting with its 5 untranslated region (5-UTR). Performing in vitro experiments and mutagenesis, we propose a mechanism of action of CcnA based on liberation (ctrA) or sequestration (gcrA) of their ribosome-binding site (RBS). Finally, its role may be conserved in other alphaproteobacterial species, such as Sinorhizobium meliloti, representing indeed a potentially conserved process modulating cell cycle in Caulobacterales and Rhizobiales.

microbiology↗