Replicate Once Per Cell Cycle:Replication Control of Secondary Chromosomes

Faithful vertical transmission of genetic information, especially of essential core genes, is a prerequisite for bacterial survival. Hence, replication of all the replicons is tightly controlled to ensure that all daughter cells get the same genome copy as their mother cell. Essential core genes are very often carried by the main chromosome. However they can occasionally be found on secondary chromosomes, recently renamed chromids. Chromids have evolved from non-essential megaplasmids, and further acquired essential core genes and a genomic signature closed to that of the main chromosome. All chromids carry a plasmidic replication origin, belonging so far to either the iterons or repABC type. Based on these differences, two categories of chromids have been distinguished. In this review, we focus on the replication initiation controls of these two types of chromids. We show that the sophisticated mechanisms controlling their replication evolved from their plasmid counterparts to allow a timely controlled replication, occurring once per cell cycle

Resolution of multimeric forms of circular plasmids and chromosomes

One of the disadvantages of circular plasmids and chromosomes is their high sensitivity to rearrangements caused by homologous recombination. Odd numbers of crossing-over occurring during or after replication of a circular replicon result in the formation of a dimeric molecule in which the two copies of the replicon are fused. If they are not converted back to monomers, the dimers of replicons may fail to correctly segregate at the time of cell division. Resolution of multimeric forms of circular plasmids and chromosomes is mediated by site-specific recombination, and the enzymes that catalyze this type of reaction fall into two families of proteins: the serine and tyrosine recombinase families. Here we give an overview of the variety of site-specific resolution systems found on circular plasmids and chromosomes

The coordinated replication of Vibrio cholerae ’s two chromosomes required the acquisition of a unique domain by the RctB initiator

International audienceVibrio cholerae, the pathogenic bacterium that causes cholera, has two chromosomes (Chr1, Chr2) that replicate in a well-orchestrated sequence. Chr2 initiation is triggered only after the replication of the crtS site on Chr1. The initiator of Chr2 replication, RctB, displays activities corresponding with its different binding sites: initiator at the iteron sites, repressor at the 39m sites, and trigger at the crtS site. The mechanism by which RctB relays the signal to initiate Chr2 replication from crtS is not well-understood. In this study, we provide new insights into how Chr2 replication initiation is regulated by crtS via RctB. We show that crtS (on Chr1) acts as an anti-inhibitory site by preventing 39m sites (on Chr2) from repressing initiation. The competition between these two sites for RctB binding is explained by the fact that RctB interacts with crtS and 39m via the same DNA-binding surface. We further show that the extreme C-terminal tail of RctB, essential for RctB self-interaction, is crucial for the control exerted by crtS. This subregion of RctB is conserved in all Vibrio, but absent in other Rep-like initiators. Hence, the coordinated replication of both chromosomes likely results from the acquisition of this unique domain by RctB

Vibrio cholerae chromosome 2 copy number is controlled by the methylation-independent binding of its monomeric initiator to the chromosome 1 crtS site

International audienceBacteria contain a primary chromosome and, frequently , either essential secondary chromosomes or dispensable megaplasmids of plasmid origin. Incoming plasmids are often poorly adapted to their hosts and their stabilization requires integration with the host's cellular mechanisms in a process termed domestication. All Vibrio, including pathogenic species, carry a domesticated secondary chromosome (Chr2) where replication is coordinated with that of the primary chromosome (Chr1). Chr2 replication is triggered by the replication of an in-tergenic sequence (crtS) located on Chr1. Yet, the molecular mechanisms by which crtS replication controls the initiation of Chr2 replication are still largely unknown. In this study, we show that crtS not only regulates the timing of Chr2 initiation but also controls Chr2 copy number. We observed and characterized the direct binding of the Chr2 initia-tor (RctB) on crtS. RctB binding to crtS is independent of its methylation state. RctB molecules, which naturally form dimers, preferentially bind to crtS as monomers, with DnaK/J protein chaperones shown to stimulate binding of additional RctB monomers on crtS. In this study, we addressed various hypothesis of how replication of crtS could trigger Chr2 replica-tion and provide new insights into its mode of action

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

Cassette recruitment in the chromosomal Integron of Vibrio cholerae

Posté sur BioRxiv le 24 novembre 2020Integrons 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

Genetic drivers of chromosomal integron stability

The integron is a bacterial recombination system that allows acquisition, stockpiling and expression of promoterless genes embedded in cassettes. Some integrons, like the one found in the second chromosome of Vibrio cholerae, can be particularly massive and contain hundreds of non-expressed cassettes. It is unclear how such genetic structures can be stabilized in bacterial genomes. Here, we reveal that the orientation of integrons toward replication within bacterial chromosomes is essential to their stability. Indeed, we show that upon inversion of the V. cholerae chromosomal integron, its plasticity is dramatically increased. This correlates with a strong growth defect which we show is mostly due to the excision of a particular type of cassettes bearing their own promoter and encoding toxin-antitoxin systems. This so called "abortive excision" of toxin-antitoxin systems can prevent the inversion of chromosomal integrons and the associated extensive loss of cassettes. Our analysis of the available sedentary chromosomal integrons in genome database show a robust correlation between the size of the cassette array and the number of toxin-antitoxin cassettes. This study thus provides a striking example of the relationship between genome organization, genome stability, and an emerging property of toxin-antitoxin systems

Cassette recombination dynamics within chromosomal integrons are regulated by toxin-antitoxin systems

International audienceIntegrons are adaptive bacterial devices that rearrange promoter-less gene cassettes into variable ordered arrays under stress conditions, thereby sampling 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 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