Recovery of Vibrio cholerae polarized cellular organization after exit from a non-proliferating spheroplast state.

Vibrio cholerae, the causative agent of cholera epidemics, is a rod-shaped bacterium with a highly polarized cellular organization. It can survive harmful growth conditions by entering a non-proliferating spheroplast state, which involves loss of the cell envelope and polarity. How polarized rod organization cells are formed when the spheroplasts exit the non-proliferating state remains largely uncharacterized. To address this question, we investigated how L-arabinose-induced V. cholerae spheroplasts return to growth. We found that de novo morphogenesis started with the elimination of an excess of periplasm, which was immediately followed by cell elongation and the formation of cell branches with a diameter similar to that of normal V. cholerae cells. Periplasm elimination was driven by bifunctional peptidoglycan synthases involved in cell-wall maintenance, the aPBPs. Elongation and branching relied on the MreB-associated monofunctional peptidoglycan synthase PBP2. The cell division monofunctional peptidoglycan synthase FtsI was not involved in any of these processes. However, the FtsK cell division protein specifically targeted the sites of vesicle extrusion. Genetic material was amplified by synchronous waves of DNA replication as periplasmic elimination began. The HubP polarity factor targeted the tip of the branches as they began to form. However, HubP-mediated polarization was not involved in the efficiency of the recovery process. Finally, our results suggest that the positioning of HubP and the activities of the replication terminus organizer of the two V. cholerae chromosomes, MatP, are independent of cell division. Taken together, these results confirm the interest of L-arabinose-induced V. cholerae spheroplasts to study how cell shape is generated and shed light on the de novo establishment of the intracellular organization and cell polarization in V. cholerae.

The SMC-like RecN protein is at the crossroads of several genotoxic stress responses in Escherichia coli.

Introduction: DNA damage repair (DDR) is an essential process for living organisms and contributes to genome maintenance and evolution. DDR involves different pathways including Homologous recombination (HR), Nucleotide Excision Repair (NER) and Base excision repair (BER) for example. The activity of each pathway is revealed with particular drug inducing lesions, but the repair of most DNA lesions depends on concomitant or subsequent action of the multiple pathways. Methods: In the present study, we used two genotoxic antibiotics, mitomycin C (MMC) and Bleomycin (BLM), to decipher the interplays between these different pathways in E. coli. We combined genomic methods (TIS and Hi-SC2) and imaging assays with genetic dissections. Results: We demonstrate that only a small set of DDR proteins are common to the repair of the lesions induced by these two drugs. Among them, RecN, an SMC-like protein, plays an important role by controlling sister chromatids dynamics and genome morphology at different steps of the repair processes. We further demonstrate that RecN influence on sister chromatids dynamics is not equivalent during the processing of the lesions induced by the two drugs. We observed that RecN activity and stability requires a pre-processing of the MMC-induced lesions by the NER but not for BLM-induced lesions. Discussion: Those results show that RecN plays a major role in rescuing toxic intermediates generated by the BER pathway in addition to its well-known importance to the repair of double strand breaks by HR.

The Xer activation factor of TLCΦ expands the possibilities for Xer recombination.

The chromosome dimer resolution machinery of bacteria is generally composed of two tyrosine recombinases, XerC and XerD. They resolve chromosome dimers by adding a crossover between sister copies of a specific site, dif. The reaction depends on a cell division protein, FtsK, which activates XerD by protein-protein interactions. The toxin-linked cryptic satellite phage (TLCΦ) of Vibrio cholerae, which participates in the emergence of cholera epidemic strains, carries a dif-like attachment site (attP). TLCΦ exploits the Xer machinery to integrate into the dif site of its host chromosomes. The TLCΦ integration reaction escapes the control of FtsK because TLCΦ encodes for its own XerD-activation factor, XafT. Additionally, TLCΦ attP is a poor substrate for XerD binding, in apparent contradiction with the high integration efficiency of the phage. Here, we present a sequencing-based methodology to analyse the integration and excision efficiency of thousands of synthetic mini-TLCΦ plasmids with differing attP sites in vivo. This methodology is applicable to the fine-grained analyses of DNA transactions on a wider scale. In addition, we compared the efficiency with which XafT and the XerD-activation domain of FtsK drive recombination reactions in vitro. Our results suggest that XafT not only activates XerD-catalysis but also helps form and/or stabilize synaptic complexes between imperfect Xer recombination sites.

Vibrio cholerae Chromosome Partitioning without Polar Anchoring by HubP.

Partition systems are widespread among bacterial chromosomes. They are composed of two effectors, ParA and ParB, and cis acting sites, parS, located close to the replication origin of the chromosome (oriC). ParABS participate in chromosome segregation, at least in part because they serve to properly position sister copies of oriC. A fourth element, located at cell poles, is also involved in some cases, such as HubP for the ParABS1 system of Vibrio cholerae chromosome 1 (ch1). The polar anchoring of oriC of ch1 (oriC1) is lost when HubP or ParABS1 are inactivated. Here, we report that in the absence of HubP, ParABS1 actively maintains oriC1 at mid-cell, leading to the subcellular separation of the two ch1 replication arms. We further show that parS1 sites ectopically inserted in chromosome 2 (ch2) stabilize the inheritance of this replicon in the absence of its endogenous partition system, even without HubP. We also observe the positioning interference between oriC1 and oriC of ch2 regions when their positionings are both driven by ParABS1. Altogether, these data indicate that ParABS1 remains functional in the absence of HubP, which raises questions about the role of the polar anchoring of oriC1 in the cell cycle.

Plesiomonas shigelloides, an Atypical Enterobacterales with a Vibrio-Related Secondary Chromosome.

About 10% of bacteria have a multichromosome genome with a primary replicon of bacterial origin, called the chromosome, and other replicons of plasmid origin, the chromids. Studies on multichromosome bacteria revealed potential points of coordination between the replication/segregation of chromids and the progression of the cell cycle. For example, replication of the chromid of Vibrionales (called Chr2) is initiated upon duplication of a sequence carried by the primary chromosome (called Chr1), in such a way that replication of both replicons is completed synchronously. Also, Chr2 uses the Chr1 as a scaffold for its partition in the daughter cells. How many of the features detected so far are required for the proper integration of a secondary chromosome in the cell cycle? How many more features remain to be discovered? We hypothesized that critical features for the integration of the replication/segregation of a given chromid within the cell cycle program would be conserved independently of the species in which the chromid has settled. Hence, we searched for a chromid related to that found in Vibrionales outside of this order. We identified one in Plesiomonas shigelloides, an aquatic and pathogenic enterobacterium that diverged early within the clade of Enterobacterales. Our results suggest that the chromids present in P. shigelloides and Vibrionales derive from a common ancestor. We initiated in silico genomic and proteomic comparative analyses of P. shigelloides, Vibrionales, and Enterobacterales that enabled us to establish a list of features likely involved in the maintenance of the chromid within the host cell cycle.

L-arabinose induces the formation of viable non-proliferating spheroplasts in Vibrio cholerae.

Vibrio cholerae, the agent of the deadly human disease cholera, propagates as a curved rod-shaped bacterium in warm waters. It is sensitive to cold, but persists in cold waters under the form of viable but non-dividing coccoidal shaped cells. Additionally, V. cholerae is able to form non-proliferating spherical cells in response to cell wall damage. It was recently reported that L-arabinose, a component of the hemicellulose and pectin of terrestrial plants, stops the growth of V. cholerae. Here, we show that L-arabinose induces the formation of spheroplasts that lose the ability to divide and stop growing in volume over time. However, they remain viable and upon removal of L-arabinose they start expanding in volume, form branched structures and give rise to cells with a normal morphology after a few divisions. We further show that WigKR, a histidine kinase/response regulator pair implicated in the induction of a high expression of cell wall synthetic genes, prevents the lysis of the spheroplasts during growth restart. Finally, we show that the physiological perturbations result from the import and catabolic processing of L-arabinose by the V. cholerae homolog of the E. coli galactose transport and catabolic system. Taken together, our results suggest that the formation of non-growing spherical cells is a common response of Vibrios exposed to detrimental conditions. They also permit to define conditions preventing any physiological perturbation of V. cholerae when using L-arabinose to induce gene expression from the tightly regulated promoter of the Escherichia coli araBAD operon.Importance Vibrios among other bacteria form transient cell wall deficient forms as a response to different stresses and revert to proliferating rods when permissive conditions have been restored. Such cellular forms have been associated to antimicrobial tolerance, chronic infections and environmental dispersion.The effect of L-Ara on V. cholerae could provide an easily tractable model to study the ability of Vibrios to form viable reversible spheroplasts. Indeed, the quick transition to spheroplasts and reversion to proliferating rods by addition or removal of L-Ara is ideal to understand the genetic program governing this physiological state and the spatial rearrangements of the cellular machineries during cell shape transitions.

Protocol for High-Throughput Analysis of Sister-Chromatids Contacts.

Sister chromatid interactions are a key step to ensure the successful segregation of sister chromatids after replication. Our knowledge about this phenomenon is mostly based on microscopy approaches, which have some constraints such as resolution limit and the impossibility of studying several genomic positions at the same time. Here, we present a protocol for Hi-SC2, a high-throughput sequencing-based method, to monitor sister chromatid contacts after replication at high resolution throughout the genome, which we applied to study cohesion in Vibrio cholerae. For complete details on the use and execution of this protocol, please refer to Espinosa et al. (2020).

High-Resolution Whole-Genome Analysis of Sister-Chromatid Contacts.

Sister-chromatid cohesion describes the orderly association of newly replicated DNA molecules behind replication forks. It plays an essential role in the maintenance and faithful transmission of genetic information. Cohesion is created by DNA topological links and proteinaceous bridges, whose formation and deposition could be potentially affected by many processes. Current knowledge on cohesion has been mainly gained by fluorescence microscopy observation. However, the resolution limit of microscopy and the restricted number of genomic positions that can be simultaneously visualized considerably hampered progress. Here, we present a high-throughput methodology to monitor sister-chromatid contacts (Hi-SC2). Using the multi-chromosomal Vibrio cholerae bacterium as a model, we show that Hi-SC2 permits to monitor local variations in sister-chromatid cohesion at a high resolution over a whole genome.

The TLCΦ satellite phage harbors a Xer recombination activation factor.

The circular chromosomes of bacteria can be concatenated into dimers by homologous recombination. Dimers are solved by the addition of a cross-over at a specific chromosomal site, dif, by 2 related tyrosine recombinases, XerC and XerD. Each enzyme catalyzes the exchange of a specific pair of strands. Some plasmids exploit the Xer machinery for concatemer resolution. Other mobile elements exploit it to integrate into the genome of their host. Chromosome dimer resolution is initiated by XerD. The reaction is under the control of a cell-division protein, FtsK, which activates XerD by a direct contact. Most mobile elements exploit FtsK-independent Xer recombination reactions initiated by XerC. The only notable exception is the toxin-linked cryptic satellite phage of Vibrio cholerae, TLCΦ, which integrates into and excises from the dif site of the primary chromosome of its host by a reaction initiated by XerD. However, the reaction remains independent of FtsK. Here, we show that TLCΦ carries a Xer recombination activation factor, XafT. We demonstrate in vitro that XafT activates XerD catalysis. Correspondingly, we found that XafT specifically interacts with XerD. We further show that integrative mobile elements exploiting Xer (IMEXs) encoding a XafT-like protein are widespread in gamma- and beta-proteobacteria, including human, animal, and plant pathogens.

Replication termination without a replication fork trap.

Bacterial chromosomes harbour a unique origin of bidirectional replication, oriC. They are almost always circular, with replication terminating in a region diametrically opposite to oriC, the terminus. The oriC-terminus organisation is reflected by the orientation of the genes and by the disposition of DNA-binding protein motifs implicated in the coordination of chromosome replication and segregation with cell division. Correspondingly, the E. coli and B. subtilis model bacteria possess a replication fork trap system, Tus/ter and RTP/ter, respectively, which enforces replication termination in the terminus region. Here, we show that tus and rtp are restricted to four clades of bacteria, suggesting that tus was recently domesticated from a plasmid gene. We further demonstrate that there is no replication fork system in Vibrio cholerae, a bacterium closely related to E. coli. Marker frequency analysis showed that replication forks originating from ectopic origins were not blocked in the terminus region of either of the two V. cholerae chromosomes, but progressed normally until they encountered an opposite fork. As expected, termination synchrony of the two chromosomes is disrupted by these ectopic origins. Finally, we show that premature completion of the primary chromosome replication did not modify the choreography of segregation of its terminus region.

Broken replication forks trigger heritable DNA breaks in the terminus of a circular chromosome.

It was recently reported that the recBC mutants of Escherichia coli, deficient for DNA double-strand break (DSB) repair, have a decreased copy number of their terminus region. We previously showed that this deficit resulted from DNA loss after post-replicative breakage of one of the two sister-chromosome termini at cell division. A viable cell and a dead cell devoid of terminus region were thus produced and, intriguingly, the reaction was transmitted to the following generations. Using genome marker frequency profiling and observation by microscopy of specific DNA loci within the terminus, we reveal here the origin of this phenomenon. We observed that terminus DNA loss was reduced in a recA mutant by the double-strand DNA degradation activity of RecBCD. The terminus-less cell produced at the first cell division was less prone to divide than the one produced at the next generation. DNA loss was not heritable if the chromosome was linearized in the terminus and occurred at chromosome termini that were unable to segregate after replication. We propose that in a recB mutant replication fork breakage results in the persistence of a linear DNA tail attached to a circular chromosome. Segregation of the linear and circular parts of this "σ-replicating chromosome" causes terminus DNA breakage during cell division. One daughter cell inherits a truncated linear chromosome and is not viable. The other inherits a circular chromosome attached to a linear tail ending in the chromosome terminus. Replication extends this tail, while degradation of its extremity results in terminus DNA loss. Repeated generation and segregation of new σ-replicating chromosomes explains the heritability of post-replicative breakage. Our results allow us to determine that in E. coli at each generation, 18% of cells are subject to replication fork breakage at dispersed, potentially random, chromosomal locations.

Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme.

Marker frequency analysis of the Escherichia coli recB mutant chromosome has revealed a deficit of DNA in a specific zone of the terminus, centred on the dif/TerC region. Using fluorescence microscopy of a marked chromosomal site, we show that the dif region is lost after replication completion, at the time of cell division, in one daughter cell only, and that the phenomenon is transmitted to progeny. Analysis by marker frequency and microscopy shows that the position of DNA loss is not defined by the replication fork merging point since it still occurs in the dif/TerC region when the replication fork trap is displaced in strains harbouring ectopic Ter sites. Terminus DNA loss in the recB mutant is also independent of dimer resolution by XerCD at dif and of Topo IV action close to dif. It occurs in the terminus region, at the point of inversion of the GC skew, which is also the point of convergence of specific sequence motifs like KOPS and Chi sites, regardless of whether the convergence of GC skew is at dif (wild-type) or a newly created sequence. In the absence of FtsK-driven DNA translocation, terminus DNA loss is less precisely targeted to the KOPS convergence sequence, but occurs at a similar frequency and follows the same pattern as in FtsK+ cells. Importantly, using ftsIts, ftsAts division mutants and cephalexin treated cells, we show that DNA loss of the dif region in the recB mutant is decreased by the inactivation of cell division. We propose that it results from septum-induced chromosome breakage, and largely contributes to the low viability of the recB mutant.

Coordination between replication, segregation and cell division in multi-chromosomal bacteria: lessons from Vibrio cholera

Bacteria display a highly flexible cell cycle in which cell division can be temporally disconnected from the replication/segregation cycle of their genome. The accuracy of genetic transmission is enforced by restricting the assembly of the cell division apparatus to the low DNA-density zones that develop between the regularly spaced nucleoids originating from the concurrent replication and segregation of genomic DNA. In most bacteria, the process is simplified because the genome is encoded on a single chromosome. This is notably the case in Escherichia coli, the most well studied bacterial model organism. However, ~10% of bacteria have domesticated horizontally acquired mega-plasmids into extra-numerous chromosomes. Most of our current knowledge on the cell cycle regulation of multi-chromosomal species derives from the study of replication, segregation and cell division in Vibrio cholerae, the agent of the deadly epidemic human diarrheal disease cholera. A nicety of this model is that it is closely related to E. coli in the phylogenetic tree of bacteria. Here, we review recent findings on the V. cholerae cell cycle in the context of what was previously known on the E. coli cell cycle (AU)

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WGADseq: Whole Genome Affinity Determination of Protein-DNA Binding Sites.

We present a method through which one may monitor the relative binding affinity of a given protein to DNA motifs on the scale of a whole genome. Briefly, the protein of interest is incubated with fragmented genomic DNA and then affixed to a column. Washes with buffers containing low salt concentrations will remove nonbound DNA fragments, while stepwise washes with increasing salt concentrations will elute more specifically bound fragments. Massive sequencing is used to identify eluted DNA fragments and map them on the genome, which permits us to classify the different binding sites according to their affinity and determine corresponding consensus motifs (if any).

Enhancing multiplex genome editing by natural transformation (MuGENT) via inactivation of ssDNA exonucleases.

Recently, we described a method for multiplex genome editing by natural transformation (MuGENT). Mutant constructs for MuGENT require large arms of homology (>2000 bp) surrounding each genome edit, which necessitates laborious in vitro DNA splicing. In Vibrio cholerae, we uncover that this requirement is due to cytoplasmic ssDNA exonucleases, which inhibit natural transformation. In ssDNA exonuclease mutants, one arm of homology can be reduced to as little as 40 bp while still promoting integration of genome edits at rates of ∼50% without selection in cis. Consequently, editing constructs are generated in a single polymerase chain reaction where one homology arm is oligonucleotide encoded. To further enhance editing efficiencies, we also developed a strain for transient inactivation of the mismatch repair system. As a proof-of-concept, we used these advances to rapidly mutate 10 high-affinity binding sites for the nucleoid occlusion protein SlmA and generated a duodecuple mutant of 12 diguanylate cyclases in V. cholerae. Whole genome sequencing revealed little to no off-target mutations in these strains. Finally, we show that ssDNA exonucleases inhibit natural transformation in Acinetobacter baylyi. Thus, rational removal of ssDNA exonucleases may be broadly applicable for enhancing the efficacy and ease of MuGENT in diverse naturally transformable species.

Fast growth conditions uncouple the final stages of chromosome segregation and cell division in Escherichia coli.

Homologous recombination between the circular chromosomes of bacteria can generate chromosome dimers. They are resolved by a recombination event at a specific site in the replication terminus of chromosomes, dif, by dedicated tyrosine recombinases. The reaction is under the control of a cell division protein, FtsK, which assembles into active DNA pumps at mid-cell during septum formation. Previous studies suggested that activation of Xer recombination at dif was restricted to chromosome dimers in Escherichia coli but not in Vibrio cholerae, suggesting that FtsK mainly acted on chromosome dimers in E. coli but frequently processed monomeric chromosomes in V. cholerae. However, recent microscopic studies suggested that E. coli FtsK served to release the MatP-mediated cohesion and/or cell division apparatus-interaction of sister copies of the dif region independently of chromosome dimer formation. Here, we show that these apparently paradoxical observations are not linked to any difference in the dimer resolution machineries of E. coli and V. cholerae but to differences in the timing of segregation of their chromosomes. V. cholerae harbours two circular chromosomes, chr1 and chr2. We found that whatever the growth conditions, sister copies of the V. cholerae chr1 dif region remain together at mid-cell until the onset of constriction, which permits their processing by FtsK and the activation of dif-recombination. Likewise, sister copies of the dif region of the E. coli chromosome only separate after the onset of constriction in slow growth conditions. However, under fast growth conditions the dif sites separate before constriction, which restricts XerCD-dif activity to resolving chromosome dimers.

Late assembly of the Vibrio cholerae cell division machinery postpones septation to the last 10% of the cell cycle.

Bacterial cell division is a highly regulated process, which involves the formation of a complex apparatus, the divisome, by over a dozen proteins. In the few model bacteria in which the division process was detailed, divisome assembly occurs in two distinct steps: a few proteins, including the FtsZ tubulin-like protein, form a membrane associated contractile ring, the Z-ring, at ~30% of the cell cycle. The Z-ring serves as a scaffold for the recruitment of a second series of proteins, including integral membrane and periplasmic cell wall remodelling enzymes, at ~50% of the cell cycle. Actual septation occupies most of the remaining half of the cell cycle. In contrast, we present evidence suggesting that early pre-divisional Z-rings form between 40 and 50% of the cell cycle and mature into fully assembled divisome at about 80% of the cell cycle in Vibrio cholerae. Thus, actual septation is restricted to a very short amount of time. Our results further suggest that late assembly of the divisome probably helps maintain the asymmetric polar organisation of V. cholerae cells by limiting the accumulation of a cell pole marker, HubP, at the nascent cell poles.

Coordination between replication, segregation and cell division in multi-chromosomal bacteria: lessons from Vibrio cholerae.

Bacteria display a highly flexible cell cycle in which cell division can be temporally disconnected from the replication/segregation cycle of their genome. The accuracy of genetic transmission is enforced by restricting the assembly of the cell division apparatus to the low DNA-density zones that develop between the regularly spaced nucleoids originating from the concurrent replication and segregation of genomic DNA. In most bacteria, the process is simplified because the genome is encoded on a single chromosome. This is notably the case in Escherichia coli, the most well studied bacterial model organism. However, ~10% of bacteria have domesticated horizontally acquired mega-plasmids into extra-numerous chromosomes. Most of our current knowledge on the cell cycle regulation of multi-chromosomal species derives from the study of replication, segregation and cell division in Vibrio cholerae, the agent of the deadly epidemic human diarrheal disease cholera. A nicety of this model is that it is closely related to E. coli in the phylogenetic tree of bacteria. Here, we review recent findings on the V. cholerae cell cycle in the context of what was previously known on the E. coli cell cycle.

CTXϕ: Exploring new alternatives in host factor-mediated filamentous phage replications.

For a long time Ff phages from Escherichia coli provided the majority of the knowledge about the rolling circle replication mechanism of filamentous phages. Host factors involved in coliphages replication have been fully identified. Based on these studies, the function of Rep protein as the accessory helicase directly implicated in filamentous phage replication was considered a paradigm. We recently reported that the replication of some filamentous phages from Vibrio cholerae, including the cholera toxin phage CTXϕ, depended on the accessory helicase UvrD instead of Rep. We also identified HU protein as one of the host factors involved in CTXϕ and VGJϕ replication. The requirement of UvrD and HU for rolling circle replication was previously reported in some family of plasmids but had no precedent in filamentous phages. Here, we enrich the discussion of our results and present new preliminary data highlighting remarkable divergence in the lifestyle of filamentous phages.

Cell division licensing in the multi-chromosomal Vibrio cholerae bacterium.

Cell division must be coordinated with chromosome replication and segregation to ensure the faithful transmission of genetic information during proliferation. In most bacteria, assembly of the division apparatus, the divisome, starts with the polymerization of a tubulin homologue, FtsZ, into a ring-like structure at mid-cell, the Z-ring(1). It typically occurs at half of the cell cycle when most of the replication and segregation cycle of the unique chromosome they generally harbour is achieved(2). The chromosome itself participates in the regulation of cell division, at least in part because it serves as a scaffold to position FtsZ polymerization antagonists(3). However, about 10% of bacteria have more than one chromosome(4), which raises questions about the way they license cell division(3). For instance, the genome of Vibrio cholerae, the agent of cholera, is divided between a 3 Mbp replicon that originates from the chromosome of its mono-chromosomal ancestor, Chr1, and a 1 Mbp plasmid-derived replicon, Chr2 (ref. 5). Here, we show that Chr2 harbours binding motifs for an inhibitor of Z-ring formation, which helps accurately position the V. cholerae divisome at mid-cell and postpones its assembly to the very end of the cell cycle.