Biology subjects

Viollier, P. H.

Publications and source records attributed to Viollier, P. H..

15 recordsLinked to original sources

bioRxiv2026

Plasmid-encoded host reprogramming promotes plasmid dissemination

Conjugative plasmids are major drivers of antibiotic resistance dissemination, yet how newly transferred plasmids establish in recipient cells remains poorly understood. Here we investigate YfjB, a previously uncharacterized conserved leading-region protein, which is zygotically induced immediately after plasmid entry and acts specifically during the earliest post-transfer stages. Multi-omics analyses reveal that YfjB reprograms host transcription, triggering extensive metabolic rewiring that compensates the transient fitness cost of plasmid acquisition. Structural analyses show that YfjB is a ParB-like protein containing a CTP-binding domain and a helix-turn-helix DNA-binding motif, linked to a previously uncharacterized dimerization module that forms a V-shaped clamp-like architecture compatible with DNA loading. Consistently, live-cell imaging reveals nucleoid-associated foci in transconjugants, and ChIP-seq identifies multiple chromosomal binding sites. We therefore rename the protein HerB (Host Expression Reprogrammer, ParB-like). More broadly, our findings reveal how mobile genetic elements facilitate their dissemination by transiently subverting host physiology.

microbiology↗

bioRxiv2025

Enterococcus faecalis alters antibiotic susceptibility in Pseudomonas aeruginosa mixed species biofilms.

Bacterial infections often occur in polymicrobial biofilms where nutrient limitation and interspecies interactions can profoundly shape microbial physiology. Enterococcus faecalis can antagonize Pseudomonas aeruginosa growth under conditions of iron limitation, a known host defense mechanism. We report here that this growth antagonism uncovers surviving P. aeruginosa cells capable of surviving antibiotic challenge, including ampicillin, cefepime, and ciprofloxacin, when grown in iron-restricted biofilms with E. faecalis. Transcriptomic profiling of P. aeruginosa revealed a distinctive response characterized by broad downregulation of biosynthetic, metabolic, and virulence pathways, alongside selective induction of membrane remodeling proteins, transport systems, and biofilm-associated genes. Induction of arnT in P. aeruginosa, required for lipid A modification, correlated with enhanced antibiotic survival to ampicillin, cefepime, and ciprofloxacin. Additionally, the diguanylate cyclase SiaD and efflux transporter MfsC in P. aeruginosa were implicated in decreased antibiotic susceptibility to the same antibiotics above. This transcriptional response was unique to the dual stress of iron deprivation and microbial competition with E. faecalis, illustrating how interspecies interactions can simultaneously inhibit and protect P. aeruginosa, shedding light on potential persistence mechanisms in iron-limited polymicrobial environments. IMPORTANCEThis study addresses antibiotic susceptibility in Pseudomonas aeruginosa, a major opportunistic ESKAPE pathogen, within polymicrobial biofilms and under host-relevant iron-restricted conditions. Polymicrobial biofilm-associated infections are notoriously difficult to treat due to complex interspecies interactions and increased antibiotic resistance. We demonstrate that Enterococcus faecalis not only antagonizes P. aeruginosa growth under iron limitation but also induces a unique transcriptional profile enhancing P. aeruginosa survival during antibiotic challenge. This shift involves broad transcriptional reprogramming in P. aeruginosa, characterized by global metabolic downregulation and activation of envelope remodeling pathways, including the arn operon. These findings reveal how interspecies interactions under iron stress can both suppress and protect bacterial pathogens and underscore the importance of considering community context in treatment strategies for persistent infections.

microbiology↗

bioRxiv2025

Electron Cryo-Tomography Reveals the Caulobacter vibrioidesTight Adherence Pilus Architecture

The type IV filament superfamily is a widespread group of molecular machines involved in natural transformation, motility, adhesion, nutrient uptake, and secretion of a wide spectrum of protein substrates. The gram-negative bacterium Caulobacter vibrioides expresses the tight adherence (Tad) pilus, a type IV machine involved in surface colonization. Here we investigated the proteins involved in Tad pilus production by {Phi}CbK resistance screening and the order of machine assembly and its polar remnant by fluorescence tagging. Using electron cryo-tomography and subtomogram averaging of wild-type and mutant strains, we resolved the Tad pilus machine architecture and built an integrative model of its structure. The resulting model suggests the individual roles of multiple Tad proteins. Together the data also reveals the Tad pilus machines assembly order.

cell biology↗

bioRxiv2023

Regulation of the transcription factor CdnL promotes adaptation to nutrient stress in Caulobacter

In response to nutrient deprivation, bacteria activate a conserved stress response pathway called the stringent response (SR). During SR activation in Caulobacter crescentus, SpoT synthesizes the secondary messengers (p)ppGpp, which affect transcription by binding RNA polymerase to downregulate anabolic genes. (p)ppGpp also impacts expression of anabolic genes by controlling the levels and activities of their transcriptional regulators. In Caulobacter, a major regulator of anabolic genes is the transcription factor CdnL. If and how CdnL is controlled during the SR and why that might be functionally important is unclear. Here, we show that CdnL is downregulated post-translationally during starvation in a manner dependent on SpoT and the ClpXP protease. Inappropriate stabilization of CdnL during starvation causes misregulation of ribosomal and metabolic genes. Functionally, we demonstrate that the combined action of SR transcriptional regulators and CdnL clearance allows for rapid adaptation to nutrient repletion. Moreover, cells that are unable to clear CdnL during starvation are outcompeted by wild-type cells when subjected to nutrient fluctuations. We hypothesize that clearance of CdnL during the SR, in conjunction with direct binding of (p)ppGpp and DksA to RNAP, is critical for altering the transcriptome in order to permit cell survival during nutrient stress. SIGNIFICANCEThe stringent response (SR) is a ubiquitous bacterial stress response that promotes adaptation to nutrient deprivation. While it is known that SR activation affects RNA polymerase activity to reprogram the transcriptome, the impact of the SR on other transcriptional regulators is not well understood. Here, we show that a conserved transcription factor, CdnL, is cleared upon activation of the SR, and that its clearance is important for cells to efficiently adapt to nutrient fluctuations. Our results suggest that CdnL regulation enables adaptation by transcriptionally downregulating ribosome biosynthesis and flux through metabolic pathways, thereby promoting survival during nutrient stress. As CdnL homologs are broadly found, we hypothesize that CdnL regulation is a conserved mechanism of bacterial adaptation to stress.

microbiology↗

bioRxiv2023

Interconversion of tripartite and bipartite systems for site-specific O-sialylation of flagellin in Gram-negative and Gram-positive bacteria

Many bacteria decorate flagellin with sialic acid-like sugars such as pseudaminic acid (Pse) by O-glycosylation on serine or threonine residues. Evidence for sufficiency of sialylation by a conserved flagellin glycosyltransferase (fGT) system is lacking, presumably because of (a) missing component(s). Here, we reconstituted two Maf-type fGTs from the Gram-negative bacterium Shewanella oneidensis MR-1 in a heterologous host producing a Pse donor sugar. While Maf-1 is sufficient for flagellin glycosylation, Maf-2 reconstitution requires a newly identified, cis-encoded and conserved specificity factor GlfM, predicted to form a four-helix bundle. While GlfM binds Maf-2 to form a ternary complex with flagellin, the C-terminal tetratricopeptide repeat (TPR) domain of Maf-1 confers flagellin acceptor and O-glycosylation specificity at preferred serine residues. GlfM from Gram-negative and Gram-positive bacteria are functional, providing evidence for convergent evolution of specialized flagellin modification systems with acceptor serine selectivity, while also shaping the interconversion of bacterial tripartite and bipartite O-glycosylation systems.

microbiology↗

bioRxiv2023

Co-chaperone-mediated post-translational control of efflux pump induction underlies adaptive β-lactam resistance in Caulobacter crescentus.

The acquisition of multi-drug resistance (MDR) determinants jeopardizes treatment of bacterial infections with antibiotics. The tripartite efflux pump AcrAB-NodT confers adaptive MDR in the non-pathogenic -proteobacterium Caulobacter crescentus via transcriptional induction by first-generation quinolone antibiotics. We discovered that overexpression of AcrAB-NodT by mutation or exogenous inducers confers resistance to cephalosporin and penicillin ({beta}-lactam) antibiotics. Combining two-step mutagenesis-sequencing (Mut-Seq) and cephalosporin-resistant point mutants, we dissected how TipR uses a common operator of the divergent tipR and acrAB-nodT promoter for adaptive and/or potentiated AcrAB-NodT-directed efflux. Chemical screening identified compounds that interfere with DNA-binding by TipR or induce its dependent proteolytic turnover. We found that long-term induction of AcrAB-NodT disfigures the envelope and that homeostatic control by TipR includes co-induction of the DnaJ-like co-chaperone DjlA, to boost pump assembly and/or capacity in anticipation of envelope stress. Thus, the adaptive MDR regulatory circuitry reconciles drug efflux with co-chaperone function for trans-envelope assemblies and maintenance.

microbiology↗

bioRxiv2022

Genetic glyco-profiling and rewiring of insulated flagellin glycosylation pathways

Glycosylation of surface structures diversifies cells chemically and physically. Sialic acids commonly serve as glycosyl donors, particularly pseudaminic (Pse) or legionaminic acid (Leg) that prominently decorate eubacterial and archaeal surface layers or appendages. We investigated a new class of FlmG protein glycosyltransferases that modify flagellin, the structural subunit of the flagellar filament. Functional insulation of orthologous Pse and Leg biosynthesis pathways accounted for the flagellin glycosylation specificity and motility conferred by the cognate FlmG in the -proteobacteria Caulobacter crescentus and Brevundimonas subvibrioides, respectively. Exploiting these functions, we conducted genetic glyco-profiling to classify Pse or Leg biosynthesis pathways and we used heterologous reconstitution experiments to unearth a signature determinant of Leg biosynthesis in eubacteria and archaea. These findings and our chimeric FlmG analyses reveal two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that accepts either Leg or Pse and that uses specialized flagellin-binding domain to identify the substrate.

microbiology↗

bioRxiv2021

The CTPase activity of ParB acts as a timing mechanism to control the dynamics and function of prokaryotic DNA partition complexes

DNA partitioning CTPases of the ParB family mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromeric region of target DNA molecules and then spread laterally to form large nucleoprotein complexes that serve as docking points for the DNA segregation machinery. Here, we identify conformational changes that underlie the CTP- and parS-dependent closure of ParB clamps. Moreover, we solve crystal structures of ParB in the pre- and post-hydrolysis state and provide insights into the catalytic mechanism underlying nucleotide hydrolysis. The characterization of CTPase-deficient ParB variants reveals that CTP hydrolysis serves as a timing mechanism to control the sliding time of ParB. Hyperstable clamps are trapped on the DNA, leading to excessing spreading and severe chromosome segregation defects in vivo. These findings clarify the role of the ParB CTPase cycle in partition complex dynamics and function and thus complete our understanding of this prototypic CTP-dependent molecular switch.

microbiology↗

bioRxiv2020

Cryptosporulation in Kurthia spp. forces a rethinking of asporogenesis in Firmicutes

Sporulation is a complex morphophysiological process resulting in a cellular structure that is more resistant than the vegetative form. In Firmicutes, this structure is produced within the mother cell, and is called an endospore. Endospore formation is thought to have evolved in the common ancestor of Firmicutes. However, sporulation has apparently been lost in some extant lineages that are defined as asporogenic. We isolated strain 11kri321, a representative of the genus Kurthia, from an oligotrophic geothermal reservoir. While Kurthia spp. is considered to comprise only asporogenic species, strain 11kri321 produced spores. Genomic reconstruction of the sporulation pathway shows elements typical of sporulation in Bacilli, including the signaling for sporulation onset. However, key genes were missing, including those involved in engulfment and dipicolinic acid synthesis. Based on the results for strain 11kri321, sporulation was investigated in other Kurthia strains. Genes involved in signaling, cell division and spore coat formation were detected in three available Kurthia genomes. Moreover, endosporulation was clearly visualized in at least two of the four strains tested. These results show that Kurthia is an endospore-forming Firmicute lineage. However, the genetic background of sporulation in this genus deviates from the known sporulation pathway in Firmicutes and even within Bacilli, suggesting that a revision of the minimal set of genes used for genomic detection of sporulation is required. Based on our findings we propose the term cryptosporulant to refer to putative asporogenic Firmicutes for which a detailed genomic and physiological characterization of sporulating is missing. ImportanceEndospore-forming Firmicutes include many environmental and medical relevant bacterial clades. In these microorganisms, the ability to produce endospores is essential for survival in the environment and even for pathogenesis. The minimum core of genes required to produce a viable and resistant spore, the distinction between endospore-forming and asporogenic groups, as well as the evolution of sporulation have been a subject of investigation and debate for decades. Here, we demonstrate endosporulation in the genus Kurthia, considered as asporogenic. Morphological, physiological and genomic analyses were undertaken to demonstrate that sporulation is not lost within this lineage. Based on our results we propose a re-examination of the minimal genetic requirements of sporulation and the use of the term cryptosporulant to describe lineages of Firmicutes that have not previously been observed to sporulate, but for which a detailed analysis is still missing.

microbiology↗

bioRxiv2019

Bacterial cell cycle control by citrate synthase independent of enzymatic activity

Coordination of cell cycle progression with central metabolism is fundamental to all cell types and likely underlies differentiation into dispersal cells in bacteria. How central metabolism is monitored to regulate cell cycle functions is poorly understood. A forward genetic selection for cell cycle regulators in the polarized alpha-proteobacterium Caulobacter crescentus unearthed the uncharacterized CitA citrate synthase, a TCA (tricarboxylic acid) cycle enzyme, as unprecedented checkpoint regulator of the G1[->]S transition. We show that loss of the CitA protein provokes a (p)ppGpp alarmone-dependent G1-phase arrest without apparent metabolic or energy insufficiency. While S-phase entry is still conferred when CitA is rendered catalytically inactive, the paralogous CitB citrate synthase has no overt role other than sustaining TCA cycle activity when CitA is absent. With eukaryotic citrate synthase paralogs known to fulfill regulatory functions, our work extends the moonlighting paradigm to citrate synthase coordinating central (TCA) metabolism with development and perhaps antibiotic tolerance in bacteria.

cell biology↗

bioRxiv2019

Loss of Bacterial Cell Pole Stabilization in Caulobacter crescentus Sensitizes to Outer Membrane Stress and Peptidoglycan-Directed Antibiotics

Rod-shaped bacteria frequently localise proteins to one or both cell poles in order to regulate processes such as chromosome replication or polar organelle development. However, the role of such polar factors in responses to extracellular stimuli has been generally unexplored. We employed chemical-genetic screening to probe the interaction between one such factor from Caulobacter crescentus, TipN, and extracellular stress and found that TipN is required for normal tolerance of cell envelope-directed antibiotics, including vancomycin that does not normally inhibit growth of Gram-negative bacteria. Forward genetic screening for suppressors of vancomycin sensitivity in the absence of TipN revealed the TonB-dependent receptor ChvT as the mediator of vancomycin tolerance. Loss of ChvT improved resistance to vancomycin and cefixime in the otherwise sensitive {Delta}tipN strain. The activity of the two-component system regulating ChvT (ChvIG) was increased in {Delta}tipN cells relative to wild type under some, but not all, cell wall stress conditions that this strain was sensitised to, in particular cefixime and detergent exposure. Together, these results indicate that the ChvIG two-component system has been co-opted as a sensor of cell wall stress and that TipN can influence cell envelope stability and ChvIG-mediated signaling in addition to its roles in intracellular development.\n\nAuthor summaryMaintenance of an intact cell envelope is essential for free-living bacteria to survive harsh conditions they may encounter in their environment. In the case of rod-shaped bacteria, the poles of the cell are potential weak points in the cell envelope due to the high curvature of the layers and the need to break and re-form parts of the cell envelope at the division plane in order to form new poles as the cells replicate and divide. We have found that TipN, a factor required for correct division and cell pole development in the rod-shaped bacterium, Caulobacter crescentus, is also needed for maintaining normal levels of resistance to cell wall-targeting antibiotics such as vancomycin and cefixime, which interfere with peptidoglycan synthesis. We also identified an outer membrane receptor, ChvT, that was responsible for allowing vancomycin access to the cells and found that the two-component system that negatively regulates ChvT production was activated by various kinds of cell wall stress. Presence or absence of TipN influenced how active this system was in the presence of cefixime or of the membrane-disrupting detergent sodium deoxycholate. Since TipN is normally located at the poles of the cell and at the division plane just before cells complete division, our results suggest that it is involved in stabilisation of these weak points of the cell envelope as well as its other roles inside the cell.

microbiology↗

bioRxiv2019

Molecular architecture of the DNA-binding sites of the P-loop ATPases MipZ and ParA from Caulobacter crescentus

Two related P-loop ATPases, ParA and MipZ, mediate the spatiotemporal regulation of chromosome segregation and cell division in Caulobacter crescentus. Both of these proteins share the ability to form dynamic concentration gradients that control the positioning of regulatory targets within the cell. Their proper localization relies on their nucleotide-dependent cycling between a monomeric and a dimeric state, driven by interaction with the chromosome partitioning protein ParB, and on the ability of the dimeric species to associate non-specifically with the nucleoid. In this study, we use a combination of genetic screening, biochemical analysis and hydrogen/deuterium exchange mass spectrometry to identify the residues mediating the interaction of MipZ with DNA. Our results show that the DNA-binding activity of MipZ relies on a series of positively charged and hydrophobic residues lining both sides of the dimer interface. MipZ thus appears to associate with DNA in a sequence-independent manner through electrostatic interactions with the DNA phosphate backbone. In support of this hypothesis, chromatin immunoprecipitation analyses did not reveal any specific target sites in vivo. When extending our analysis to ParA, we found that the architectures of the MipZ and ParA DNA-binding sites are markedly different, although their relative positions on the dimer surface and their mode of DNA binding are conserved. Importantly, bioinformatic analysis suggests that the same principles apply to other members of the P-loop ATPase family. ParA-like ATPases thus share common mechanistic features, although their modes of action have diverged considerably during the course of evolution.\n\nSIGNIFICANCEParA-like P-loop ATPases are involved in a variety of cellular processes in bacteria, including chromosome and plasmid segregation, chemoreceptor and carboxysome positioning, and division site placement. Many members of this large protein family depend on the ability to bind non-specific DNA for proper function. Although previous studies have yielded insights in the DNA-binding properties of some ParA-like ATPases, a comprehensive view of the underlying mechanisms is still lacking. Here, we combine state-of-the-art cell biological, biochemical and biophysical approaches to localize the DNA-binding regions of the ParA-like ATPases MipZ and ParA from Caulobacter crescentus. We show that the two proteins use the same interface and mode of action to associate with DNA, suggesting that the mechanistic basis of DNA binding may be conserved in the ParA-like ATPase family.

microbiology↗

bioRxiv2019

Bacterial cell cycle and growth phase switch by the essential transcriptional regulator CtrA

Many bacteria acquire dissemination and virulence traits in G1-phase. CtrA, an essential and conserved cell cycle transcriptional regulator identified in the dimorphic alpha-proteobacterium Caulobacter crescentus, first activates promoters in late S-phase and then mysteriously switches to different target promoters in G1-phase. We uncovered a highly conserved determinant in the DNA-binding domain (DBD) of CtrA uncoupling this promoter switch. We also show that it reprograms CtrA occupancy in stationary cells inducing a (p)ppGpp alarmone signal perceived by the RNA polymerase beta subunit. A simple side chain modification in a critical residue within the core DBD imposes opposing developmental phenotypes and transcriptional activities of CtrA. A naturally occurring polymorphism in the rickettsial DBD resembles a mutation that drives CtrA towards activation of the dispersal (G1-phase) program in Caulobacter. Hence, we propose that this determinant dictates promoter reprogramming during the growth transition of obligate intracellular rickettsia differentiating from replicative cells into dispersal cells.

microbiology↗

bioRxiv2019

A bifunctional ATPase drives tad pilus extension and retraction

Molecular motors convert chemical energy directly into mechanical work1 and are found in all domains of life2. These motors are critical to intracellular transport3, motility4,5, macromolecular protein assembly3,6, and many essential processes7. A wide-spread class of related bacterial motors drive the dynamic activity of extracellular fibers, such as type IV pili (T4P), that are extended and retracted using so-called secretion motor ATPases. Among these, the tight adherence (tad) pili are critical for surface sensing, surface attachment, and biofilm formation8-10. How tad pili undergo dynamic cycles of extension and retraction8 despite lacking a dedicated retraction motor ATPase has remained a mystery. Here we find that a bifunctional pilus motor ATPase, CpaF, drives both activities through ATP hydrolysis. Specifically, we show that mutations within the ATP hydrolysis active site of Caulobacter crescentus CpaF result in a correlated reduction in the rates of extension and retraction. Moreover, a decrease in the rate of ATP hydrolysis directly scales with a decrease in the force of retraction and reduced dynamics in these CpaF mutants. This mechanism of motor protein bifunctionality extends to another genus of tad-bearing bacteria. In contrast, the T4aP subclass of pili possess dedicated extension and retraction motor ATPase paralogs. We show that these processes are uncoupled using a slow ATP hydrolysis mutation in the extension ATPase of competence T4aP of Vibrio cholerae that decreases the rate of extension but has no effect on the rate of retraction. Thus, a single motor ATPase is able to drive the bidirectional processes of pilus fiber extension and retraction.

microbiology↗

bioRxiv2016

Cell Cycle Constraints and Environmental Control of Local DNA Hypomethylation in Alpha-Proteobacteria

Heritable DNA methylation imprints are ubiquitous and underlie genetic variability from bacteria to humans. In microbial genomes, DNA methylation has been implicated in gene transcription, DNA replication and repair, nucleoid segregation, transposition and virulence of pathogenic strains. Despite the importance of local (hypo)methylation at specific loci, how and when these patterns are established during the cell cycle remains poorly characterized. Taking advantage of the small genomes and the synchronizability of -proteobacteria, we discovered that conserved determinants of the cell cycle transcriptional circuitry establish specific hypomethylation patterns in the cell cycle model system Caulobacter crescentus. We used genome-wide methyl-N6-adenine (m6A-) analyses by restriction-enzyme-cleavage sequencing (REC-Seq) and single-molecule real-time (SMRT) sequencing to show that MucR, a transcriptional regulator that represses virulence and cell cycle genes in S-phase but no longer in G1-phase, occludes 5-GANTC-3 sequence motifs that are methylated by he DNA adenine methyltransferase CcrM. Constitutive expression of CcrM or heterologous methylases in at least two different -proteobacteria homogenizes m6A patterns even when MucR is present and affects promoter activity. Environmental stress (phosphate limitation) can override and reconfigure local hypomethylation patterns imposed by the cell cycle circuitry that dictate when and where local hypomethylation is instated.\n\nAuthor SummaryDNA methylation is the post-replicative addition of a methyl group to a base by a methyltransferase that recognise a specific sequence, and represents an epigenetic regulatory mechanism in both eukaryotes and prokaryotes. In microbial genomes, DNA methylation has been implicated in gene transcription, DNA replication and repair, nucleoid segregation, transposition and virulence of pathogenic strains. CcrM is a conserved, cell cycle regulated adenine methyltransferase that methylates GANTC sites in -proteobacteria. N6-methyl-adenine (m6A) patterns generated by CcrM can change the affinity of a given DNA-binding protein for its target sequence, and therefore affect gene expression. Here, we combine restriction enzyme cleavage-deep sequencing (REC-Seq) with SMRT sequencing to identify hypomethylated 5-GANTC-3 (GANTCs) in -proteobacterial genomes instated by conserved cell cycle factors. By comparing SMRT and REC-Seq data with chromatin immunoprecipitation-deep sequencing data (ChIP-Seq) we show that a conserved transcriptional regulator, MucR, induces local hypomethylation patterns by occluding GANTCs to the CcrM methylase and we provide evidence that this competition occurs during S-phase, but not in G1-phase cells. Furthermore, we find that environmental signals (such as phosphate depletion) are superimposed to the cell cycle control mechanism and can override the specific hypomethylation pattern imposed by the cell cycle transcriptional circuitry.

microbiology↗