Biology subjects

Fournes, F.

Publications and source records attributed to Fournes, F..

6 recordsLinked to original sources

bioRxiv2026

Genome replication and cell division control by the methylation-sensitive GcrA regulator in Agrobacterium tumefaciens

Bacteria with complex genomes including secondary chromosomes and/or megaplasmids face unique challenges when controlling their cell cycle: they not only need to coordinate genome replication with other cell cycle events such as cell growth and cell division, but also to synchronize the replication of different replicons. In Alphaproteobacteria, replication and maintenance of large extrachromosomal replicons are usually dependent on RepABC modules, as seen for the plant pathogen Agrobacterium tumefaciens. In this study, we demonstrate that the conserved GcrA protein is an essential methylation-dependent transcriptional regulator playing at least two critical roles during the A. tumefaciens cell cycle. First, GcrA is required for the on-time and synchronized firing of the three RepABC-dependent origins of replication of A. tumefaciens that are needed for chromosomal and megaplasmid replication. Second, GcrA triggers the expression of several essential cell division genes for the timely assembly of a functional divisome. These findings highlight how this conserved global regulator can control and then synchronize a variety of essential cell cycle events in Alphaproteobacteria with complex genomes, notably coordinating the maintenance of several RepABC-dependent replicons with cell division. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=47 SRC="FIGDIR/small/728657v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@c7f0f7org.highwire.dtl.DTLVardef@9cf8f1org.highwire.dtl.DTLVardef@19baa05org.highwire.dtl.DTLVardef@b73437_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

A LexA-like repressor and global H-NS-like regulators enable the fine-tuning of R-tailocin expression in environmental Pseudomonas

Bacteria rely on an arsenal of weapons to challenge their opponents in highly competitive environments. To specifically counter closely related bacteria, specialized weapons with a narrow activity spectrum are deployed, particularly contractile phage tail-like particles or R-tailocins. Their production leads to the lysis of the producing cells, indicating that their expression must be carefully orchestrated so that only a small percentage of cells produce R-tailocins for the benefit of the entire population. In this study, we set out to better understand how the production of these phage tail-like weapons is regulated in environmental pseudomonads using the competitive plant root colonizer and environmental model Pseudomonas protegens CHA0. Using an RNA sequencing (RNA-seq) approach, we found that genes involved in DNA repair, particularly the SOS response program, are upregulated following exposure of the pseudomonad to the DNA-damaging agents mitomycin C and hydrogen peroxide, while genes involved in cell division and primary metabolism are downregulated. The R-tailocin and prophage gene clusters were also upregulated in response to these DNA damaging agents. By combining reverse genetics, transcriptional reporters and chromatin immunoprecipitation sequencing (ChIP-seq), we show that the R-tailocin locus-specific LexA-like regulator PrtR1 represses R-tailocin gene expression by binding directly to the promoter region of the cluster, while the histone-like nucleoid structuring (H-NS) proteins MvaT and MvaV act as master regulators that indirectly regulate R-tailocin cluster expression. Our results suggest that at least these three regulators operate in concert to ensure tight control of R-tailocin expression and cell lytic release in environmental Pseudomonas strains. Author SummaryTo face their opponents in highly competitive environments, bacteria rely on an arsenal of weapons. To specifically target closely related bacterial strains, specialized weapons with a narrow activity spectrum are produced, notably contractile phage tail-like particles such as R-tailocins. Since production of these particles leads to the lysis and death of the producing cell, their expression must be tightly regulated. Here, we set out to better understand how the expression of these phage tail-like weapons is regulated in environmental bacteria using the competitive plant root colonizer and environmental model Pseudomonas protegens CHA0. We found that following DNA-damaging stress, which activates DNA repair systems and downregulates primary metabolism, the expression of gene clusters encoding viral-like particles such as the R-tailocins is upregulated. We identified cluster-specific and global regulators that are either directly or indirectly involved in the control of R-tailocin cluster expression. Our results contribute to a better understanding of the regulatory mechanisms of viral-like particle production in environmental Pseudomonas bacteria.

microbiology↗

bioRxiv2024

A mechanistic step in the genesis of secondary chromosomes from plasmids

Bacterial genomes contain a plethora of secondary replicons of divergent size. Circular replicons must carry a system for resolving dimeric forms, resulting from recombination between sister copies. These systems use site-specific recombinases. Among these, the XerCD recombinase resolves dimers of chromosomes and certain plasmids using different controls. We have analyzed the dimer resolution functions in enterobacterial secondary replicons and show that, in addition to the main chromosomes, XerCD is preferentially used by small plasmids and by the largest secondary replicons, megaplasmids and secondary chromosomes. Indeed, all replicons longer than 250 kb host an active XerCD recombination site. These sites, in contrast to those of small plasmids, use the same control as chromosomes, coupled to cell division by the FtsK protein. We conclude that a chromosome-like mode of dimer resolution is mandatory for the faithful inheritance of large plasmids and chromids, its acquisition being a prerequisite for the genesis of secondary chromosomes from plasmids.

microbiology↗

bioRxiv2023

DNA methylation by CcrM contributes to genome maintenance in the Agrobacterium tumefaciens plant pathogen

The cell cycle-regulated DNA methyltransferase CcrM is conserved in most Alphaproteobacteria, but its role in bacteria with complex or multicentric genomes remains unexplored. Here, we compare the methylome, the transcriptome and the phenotypes of wild-type and CcrM-depleted Agrobacterium tumefaciens cells with a dicentric genome with two essential replication origins. We find that DNA methylation has a pleiotropic impact on motility, biofilm formation and viability. Remarkably, CcrM promotes the expression of the repABCCh2 operon, encoding proteins required for replication initiation/partitioning at ori2, and inhibits gcrA, encoding a conserved global cell cycle regulator. Imaging ori1 and ori2 in live cells, we show that replication from ori2 is often delayed in cells with a hypo-methylated genome, while ori2 over-initiates in cells with a hyper-methylated genome. We thus propose that methylation by CcrM stimulates RepABC-dependent chromosomal origins, uncovering a novel and original connection between CcrM-dependent DNA methylation and genome maintenance in an Alphaproteobacterial pathogen.

microbiology↗

bioRxiv2022

Genetic drivers of chromosomal integron stability

Integrons are adaptive bacterial devices that rearrange promoter less gene cassettes into variable ordered arrays under stress conditions, to sample combinatorial phenotypic diversity. Chromosomal integrons often carry hundreds of silent gene cassettes, with integrase-mediated recombination leading to rampant DNA excision and integration, posing a potential threat to genome integrity. How this activity is regulated and controlled, particularly through selective pressures, to maintain such large cassette arrays is unknown. Here we show a key role of promoter-containing toxin-antitoxin (TA) cassettes as abortive systems that kill the cell when the overall cassette excision rate is too high. These results highlight the importance of TA cassettes regulating the cassette recombination dynamics and provide insight into the evolution and success of integrons in bacterial genomes. TeaserThe accumulation of cassette functions in integrons is ensured by toxin-antitoxin systems which kill the cell when the cassette excision rate is too high.

genetics↗

bioRxiv2020

Cassette recruitment in the chromosomal Integron of Vibrio cholerae

Integrons are genetic systems conferring to bacteria a rapid adaptation capability. The integron integrase is able to capture, stockpile and shuffle novel functions embedded in cassettes. This involves the recognition of both substrates, the attI site, and the cassette associated attC sites. Integrons can be sedentary and chromosomally located (SCI) or, carried by conjugative plasmids (Mobile Integron, MI), hence favoring their dissemination among bacteria. Here, for the first time, we investigate the cassette recruitment in the Vibrio cholerae SCI during conjugation and natural transformation. We demonstrated that horizontally transferred cassette can be recruited inside the chromosomal integron. The endogenous integrase expression is sufficiently triggered, after SOS response induction mediated by the entry of single-stranded cassettes during conjugation and natural transformation, to mediate significant cassette insertion. We demonstrate that the attIA insertion is preferential, despite the presence of 180 attC sites in the integron array. Thanks to the presence of a promoter in the attIA site vicinity, all these newly inserted cassettes are expressed and prone to adaptive selection. We also show that the RecA protein is critical for cassette recruitment in V. cholerae SCI but not in MIs. Moreover, a contrario to MIs, the V. cholerae SCI is not active in others bacterial hosts. MIs might have evolved from the SCIs by overcoming host factors, which would explain their large dissemination in bacteria and their role in the antibioresistance expansion.

molecular biology↗