Post-replicative pairing of sister ter regions in Escherichia coli involves multiple activities of MatP

Funder: Université Toulouse III - Paul Sabatier (University of Toulouse III Paul Sabatier); doi: https://doi.org/10.13039/501100009160Abstract: The ter region of the bacterial chromosome, where replication terminates, is the last to be segregated before cell division in Escherichia coli. Delayed segregation is controlled by the MatP protein, which binds to specific sites (matS) within ter, and interacts with other proteins such as ZapB. Here, we investigate the role of MatP by combining short-time mobility analyses of the ter locus with biochemical approaches. We find that ter mobility is similar to that of a non ter locus, except when sister ter loci are paired after replication. This effect depends on MatP, the persistence of catenanes, and ZapB. We characterise MatP/DNA complexes and conclude that MatP binds DNA as a tetramer, but bridging matS sites in a DNA-rich environment remains infrequent. We propose that tetramerisation of MatP links matS sites with ZapB and/or with non-specific DNA to promote optimal pairing of sister ter regions until cell division

La superhélicité de l'ADN comme cible clef de l'adaptation au cours de 20000 générations d'évolution expérimentale chez Escherichia coli

Molecular mechanisms of bacterial adaptation have been studied by an experimental evolution strategy. Twelve populations have been founded from an ancestor strain of Escherichia coli and daily propagated in a glucose-minimum media for 20,000 generations. During the course of evolution, the fitness of all 12 populations increased by 70%. The aim of this work is to identify beneficial mutations responsible for this increase and to analyze them in order to understand the mechanisms of evolution. Beneficial mutations were found in two genes implicated in DNA supercoiling : fis, encoding a global regulator, and topA, encoding topoisomerase I, the enzyme responsible for DNA relaxation. The evolution is characterized by a high level of phenotypic parallelism : the DNA superhelicity degree increases in almost all populations, and this parallelism is associated to an exceptional level of genetic and molecular parallelism, and achieved through the modification of DNA superhelicity.Les mécanismes de l'adaptation des bactéries à leur environnement ont été étudiés par une stratégie d'évolution expérimentale. Douze populations, fondées à partir d'une cellule ancêtre d'Escherichia coli, ont évolué indépendamment pendant 20 000 générations par transferts journaliers dans un milieu constant. Au cours de cette évolution, la capacité reproductive des cellules augmente de 70% dans les 12 populations. L'objectif du travail est d'identifier et d'analyser les mutations conduisant à cette adaptation afin de comprendre les mécanismes à la base de l'évolution. Des mutations bénéfiques responsables d'une augmentation parallèle et systématique de la superhélicité de l'ADN ont été identifiées dans deux gènes impliqués dans sa régulation : fis, codant un régulateur global de l'expression des gènes, et topA, codant la topoisomérase I responsable du relâchement de l'ADN. L'évolution de ces populations se caractérise par un degré important de parallélisme phénotypique associé à un fort parallélisme génétique : seuls deux gènes impliqués dans la régulation de la topologie de l'ADN sont les cibles de la sélection naturelle. De plus, les mutations apparues au sein du gène fis, bien qu'affectant la transcription, la traduction, ou la fixation à l'ADN de la protéine Fis, mènent toutes au même résultat : une diminution de l'activité de la protéine. Le parallélisme phénotypique observé s'explique donc par un degré exceptionnel de parallélisme génétique et moléculaire

La superhélicité de l'ADN comme cible clef de l'adaptation au cours de 20 000 générations d'évolution expérimentale chez Escherichia coli

Les mécanismes de l'adaptation des bactéries à leur environnement ont été étudiés par une stratégie d'évolution expérimentale. Douze populations, fondées à partir d'une cellule ancêtre d'Escherichia coli, ont évolué indépendamment pendant 20 000 générations par transferts journaliers dans un milieu constant. Au cours de cette évolution, la capacité reproductive des cellules augmente de 70% dans les 12 populations. L'objectif du travail est d'identifier et d'analyser les mutations conduisant à cette adaptation afin de comprendre les mécanismes à la base de l'évolution. Des mutations bénéfiques responsables d'une augmentation parallèle et systématique de la superhélicité de l'ADN ont été identifiées dans deux gènes impliqués dans sa régulation : fis, codant un régulateur global de l'expression des gènes, et topA, codant la topoisomérase I responsable du relâchement de l'ADN. L'évolution de ces populations se caractérise par un degré important de parallélisme phénotypique associé à un fort parallélisme génétique : seuls deux gènes impliqués dans la régulation de la topologie de l'ADN sont les cibles de la sélection naturelle. De plus, les mutations apparues au sein du gène fis, bien qu'affectant la transcription, la traduction, ou la fixation à l'ADN de la protéine Fis, mènent toutes au même résultat : une diminution de l'activité de la protéine. Le parallélisme phénotypique observé s'explique donc par un degré exceptionnel de parallélisme génétique et moléculaire.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

FtsK DNA translocase: the fast motor that knows where it's going

FtsK is a double-stranded DNA translocase, a motor that converts the chemical energy of binding and hydrolysing ATP into movement of a DNA substrate. It moves DNA at an amazing rate—>5000 bp per second—and is powerful enough to remove other proteins from the DNA. In bacteria it is localised to the site of cell division, the septum, where it functions as a DNA pump at the late stages of the cell cycle, to expedite cytokinesis and chromosome segregation. The N terminus of the protein is involved in the cell-cycle-specific localisation and assembly of the cell-division machinery, whereas the C terminus forms the motor. The motor portion of FtsK has been studied by a combination of biochemistry, genetics, X-ray crystallography and single-molecule mechanical assays, and these will be the focus here. The motor can be divided into three subdomains: α, β and γ. The α and β domains multimerise to produce a hexameric ring with a central channel for dsDNA, and contain a RecA-like nucleotide-binding/hydrolysis fold. The motor is given directionality by the regulatory γ domain, which binds to polarised chromosomal sequences—5′-GGGNAGGG-3′, known as KOPS—to ensure that the motor is loaded onto DNA in a specific orientation such that subsequent translocation is always towards the region of the chromosome where replication usually terminates (the terminus), and specifically to the 28 bp dif site, located in this region. Once the FtsK translocase has located the dif site it then interacts with the XerCD site-specific recombinases to activate recombination

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

Long-term experimental evolution in Escherichia coli. XII. DNA topology as a key target of selection.

The genetic bases of adaptation are being investigated in 12 populations of Escherichia coli, founded from a common ancestor and serially propagated for 20,000 generations, during which time they achieved substantial fitness gains. Each day, populations alternated between active growth and nutrient exhaustion. DNA supercoiling in bacteria is influenced by nutritional state, and DNA topology helps coordinate the overall pattern of gene expression in response to environmental changes. We therefore examined whether the genetic controls over supercoiling might have changed during the evolution experiment. Parallel changes in topology occurred in most populations, with the level of DNA supercoiling increasing, usually in the first 2000 generations. Two mutations in the topA and fis genes that control supercoiling were discovered in a population that served as the focus for further investigation. Moving the mutations, alone and in combination, into the ancestral background had an additive effect on supercoiling, and together they reproduced the net change in DNA topology observed in this population. Moreover, both mutations were beneficial in competition experiments. Clonal interference involving other beneficial DNA topology mutations was also detected. These findings define a new class of fitness-enhancing mutations and indicate that the control of DNA supercoiling can be a key target of selection in evolving bacterial populations

Post-replicative pairing of sister ter regions in Escherichia coli involves multiple activities of MatP

Funder: Université Toulouse III - Paul Sabatier (University of Toulouse III Paul Sabatier); doi: https://doi.org/10.13039/501100009160Abstract: The ter region of the bacterial chromosome, where replication terminates, is the last to be segregated before cell division in Escherichia coli. Delayed segregation is controlled by the MatP protein, which binds to specific sites (matS) within ter, and interacts with other proteins such as ZapB. Here, we investigate the role of MatP by combining short-time mobility analyses of the ter locus with biochemical approaches. We find that ter mobility is similar to that of a non ter locus, except when sister ter loci are paired after replication. This effect depends on MatP, the persistence of catenanes, and ZapB. We characterise MatP/DNA complexes and conclude that MatP binds DNA as a tetramer, but bridging matS sites in a DNA-rich environment remains infrequent. We propose that tetramerisation of MatP links matS sites with ZapB and/or with non-specific DNA to promote optimal pairing of sister ter regions until cell division

Altered Regulation of the OmpF Porin by Fis in Escherichia coli during an Evolution Experiment and between B and K-12 Strains ▿

The phenotypic plasticity of global regulatory networks provides bacteria with rapid acclimation to a wide range of environmental conditions, while genetic changes in those networks provide additional flexibility as bacteria evolve across long time scales. We previously identified mutations in the global regulator-encoding gene fis that enhanced organismal fitness during a long-term evolution experiment with Escherichia coli. To gain insight into the effects of these mutations, we produced two-dimensional protein gels with strains carrying different fis alleles, including a beneficial evolved allele and one with an in-frame deletion. We found that Fis controls the expression of the major porin-encoding gene ompF in the E. coli B-derived ancestral strain used in the evolution experiment, a relationship that has not been described before. We further showed that this regulatory connection evolved over two different time scales, perhaps explaining why it was not observed before. On the longer time scale, we showed that this regulation of ompF by Fis is absent from the more widely studied K-12 strain and thus is specific to the B strain. On a shorter time scale, this regulatory linkage was lost during 20,000 generations of experimental evolution of the B strain. Finally, we mapped the Fis binding sites in the ompF regulatory region, and we present a hypothetical model of ompF expression that includes its other known regulators