Identification of key structural determinants of the IntI1 integron integrase that influence attC × attI1 recombination efficiency

The integron platform codes for an integrase (IntI) from the tyrosine family of recombinases that mediates recombination between a proximal double-strand recombination site, attI and a single-strand target recombination site, attC. The attI site is only recognized by its cognate integrase, while the various tested attCs sites are recombined by several different IntI integrases. We have developed a genetic system to enrich and select mutants of IntI1 that provide a higher yield of recombination in order to identify key protein structural elements important for attC × attI1 recombination. We isolated mutants with higher activity on wild type and mutant attC sites. Interestingly, three out of four characterized IntI1 mutants selected on different substrates are mutants of the conserved aspartic acid in position 161. The IntI1 model we made based on the VchIntIA 3D structure suggests that substitution at this position, which plays a central role in multimer assembly, can increase or decrease the stability of the complex and accordingly influence the rate of attI × attC recombination versus attC × attC. These results suggest that there is a balance between the specificity of the protein and the protein/protein interactions in the recombination synapse

DNA Adenine Methylation Is Required to Replicate Both Vibrio cholerae Chromosomes Once per Cell Cycle

DNA adenine methylation is widely used to control many DNA transactions, including replication. In Escherichia coli, methylation serves to silence newly synthesized (hemimethylated) sister origins. SeqA, a protein that binds to hemimethylated DNA, mediates the silencing, and this is necessary to restrict replication to once per cell cycle. The methylation, however, is not essential for replication initiation per se but appeared so when the origins (oriI and oriII) of the two Vibrio cholerae chromosomes were used to drive plasmid replication in E. coli. Here we show that, as in the case of E. coli, methylation is not essential for oriI when it drives chromosomal replication and is needed for once-per-cell-cycle replication in a SeqA-dependent fashion. We found that oriII also needs SeqA for once-per-cell-cycle replication and, additionally, full methylation for efficient initiator binding. The requirement for initiator binding might suffice to make methylation an essential function in V. cholerae. The structure of oriII suggests that it originated from a plasmid, but unlike plasmids, oriII makes use of methylation for once-per-cell-cycle replication, the norm for chromosomal but not plasmid replication

Etude des intégrases d' intégrons (nature des substrats et relations structure / fonction)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Integron cassette insertion: a recombination process involving a folded single strand substrate

Integrons play a major role in the dissemination of antibiotic resistance genes among Gram-negative pathogens. Integron gene cassettes form circular intermediates carrying a recombination site, attC, and insert into an integron platform at a second site, attI, in a reaction catalyzed by an integron-specific integrase IntI. The IntI1 integron integrase preferentially binds to the ‘bottom strand' of single-stranded attC. We have addressed the insertion mechanism in vivo using a recombination assay exploiting plasmid conjugation to exclusively deliver either the top or bottom strand of different integrase recombination substrates. Recombination of a single-stranded attC site with an attI site was 1000-fold higher for one strand than for the other. Conversely, following conjugative transfer of either attI strand, recombination with attC is highly unfavorable. These results and those obtained using mutations within a putative attC stem-and-loop strongly support a novel integron cassette insertion model in which the single bottom attC strand adopts a folded structure generating a double strand recombination site. Thus, recombination would insert a single strand cassette, which must be subsequently processed

The Two Cis-Acting Sites, <i>parS1</i> and <i>oriC1</i>, Contribute to the Longitudinal Organisation of <i>Vibrio cholerae</i> Chromosome I

<div><p>The segregation of bacterial chromosomes follows a precise choreography of spatial organisation. It is initiated by the bipolar migration of the sister copies of the replication origin (<i>ori</i>). Most bacterial chromosomes contain a partition system (Par) with <i>parS</i> sites in close proximity to <i>ori</i> that contribute to the active mobilisation of the <i>ori</i> region towards the old pole. This is thought to result in a longitudinal chromosomal arrangement within the cell. In this study, we followed the duplication frequency and the cellular position of 19 <i>Vibrio cholerae</i> genome loci as a function of cell length. The genome of <i>V. cholerae</i> is divided between two chromosomes, chromosome I and II, which both contain a Par system. The <i>ori</i> region of chromosome I (<i>ori_I</i>) is tethered to the old pole, whereas the <i>ori</i> region of chromosome II is found at midcell. Nevertheless, we found that both chromosomes adopted a longitudinal organisation. Chromosome I extended over the entire cell while chromosome II extended over the younger cell half. We further demonstrate that displacing <i>parS</i> sites away from the <i>ori_I</i> region rotates the bulk of chromosome I. The only exception was the region where replication terminates, which still localised to the septum. However, the longitudinal arrangement of chromosome I persisted in Par mutants and, as was reported earlier, the <i>ori</i> region still localised towards the old pole. Finally, we show that the Par-independent longitudinal organisation and <i>ori_I</i> polarity were perturbed by the introduction of a second origin. Taken together, these results suggest that the Par system is the major contributor to the longitudinal organisation of chromosome I but that the replication program also influences the arrangement of bacterial chromosomes.</p></div

Longitudinal organisation of <i>V. cholerae</i> chromosome II: Sequential duplication and segregation.

<p>(A) Circular <i>V. cholerae</i> chromosome II map indicating the position of the different tags with respect to <i>oriC2</i>, <i>parS2</i> and <i>matS</i> sites and their corresponding colour code. (B) Proportion of 2 foci cells according to the realigned cell length (cell size intervals of 0.1 µm) for the different loci of chromosome II. For each locus, a minimum number of 800 cells were analysed. The first cell size interval where ≥50% of cells contained duplicated <i>L3_I</i> was used to realigned ADV26 to the ADV24 reference strain. CP708, ADV131, ADV30 and ADV131 cell size distributions were aligned with the GDV552 cell sizes based on the cell size interval where ≥25% of cells contained two <i>ter_II</i> loci. GDV552 was aligned to ADV42 using the cell size interval where <i>ter_I</i> is recruited to midcell. ADV123 was aligned with ADV24 using the first cell size interval where ≥50% of cells contained two <i>ori_I</i>. (C) Reconstitution of the segregation choreographies of the 7 chromosome II loci, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004448#pgen-1004448-g001" target="_blank">Figure 1C</a>. (D) Relative distance between any of the chromosome II loci to the <i>ter_II</i> locus, measured in the cells containing only one focus of each locus. (E) Relative distance between any of the chromosome sister loci, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004448#pgen-1004448-g001" target="_blank">Figure 1E</a>.</p

Intracellular AIEC LF82 relies on SOS and stringent responses to survive, multiply and tolerate antibiotics

Adherent Invasive Escherichia coli (AIEC) strains recovered from Crohn's disease lesions survive and multiply within macrophages. A reference strain for this family, AIEC LF82, forms microcolonies within phagolysosomes, an environment that prevents commensal E. coli multiplication. Little is known about the LF82 intracellular growth status, and signals leading to macrophage intra-vacuolar multiplication. We used single-cell analysis, genetic dissection and mathematical models to monitor the growth status and cell cycle regulation of intracellular LF82. We found that within macrophages, bacteria may replicate or undergo non-growing phenotypic switches. This switch results from stringent response firing immediately after uptake by macrophages or at later stages, following genotoxic damage and SOS induction during intracellular replication. Importantly, non-growers resist treatment with various antibiotics. Thus, intracellular challenges induce AIEC LF82 phenotypic heterogeneity and non-growing bacteria that could provide a reservoir for antibiotic-tolerant bacteria responsible for relapsing infections

Longitudinal organisation of <i>V. cholerae</i> chromosome I: Sequential duplication and segregation.

<p>(A) Circular <i>V. cholerae</i> chromosome I and II maps indicating the position of the different tags with respect to <i>oriC1</i>, <i>parS1</i> and <i>matS</i> sites and their corresponding colour code. (B) Proportion of 2 foci cells according to the realigned cell length (cell size intervals of 0.1 µm) for the different loci of the chromosome I. For each locus, a minimum number of 800 cells were analysed. Left panel: left replichore; Right panel: right replichore. The first cell size interval where ≥50% of cells contained two <i>L3_I</i> foci served to align the cell length distributions of ADV20, ADV21, ADV22, ADV23, ADV25, ADV33, ADV42, ADV50 and ADV51 strains, using ADV24 <i>L3_I</i> as reference. The strain EPV213 was aligned against ADV42 using the timing of recruitment of <i>ter</i>_I to midcell. (C) Reconstitution of the segregation choreographies of the 12 chromosome I loci. Left panel: left replichore; Right panel: right replichore. The median, the 25^th and the 75^th percentiles of the relative cell position of each locus are plotted for each cell size interval. The cells falling into the first interval were named newborn cells and the ones falling into the last interval were named dividing cells. 0: new pole; 1: old pole. (D) The relative distance between different chromosome I loci to the L3I locus was measured as a function of the relative cell length in the cells containing only one focus of each locus. The median (horizontal bar), the 25th and the 75th percentiles (open box) and the 5th and the 95th percentiles (error bars) of the distance of a given locus to L3I were indicated at this locus position along a chromosome I linear genetic map. (E) Relative distance between any of the chromosome sister loci, measured in cells with a length >3.4 µm.</p