Etude du mécanisme de résolution des dimères de chromosomes chez les streptocoques

La majorité des bactéries connues possèdent un unique chromosome circulaire. Lors de la réplication du chromosome, des évènements de recombinaison homologue peuvent lier les deux chromosomes frères en une molécule unique, appelée dimère de chromosome. Le système de recombinaison spécifique de site dédié à résoudre les formes dimériques en monomères est le système Xer. Il est composé chez la bactérie modèle Escherichia coli de deux recombinases à tyrosine, XerC et XerD, qui agissent sur un site chromosomique unique, dif, localisé dans la région du terminus de réplication. Le système de recombinaison Xer est sous le contrôle de la protéine septale FtsK. FtsK est une translocase à ADN qui est dirigée sur le chromosome par des motifs, les KOPS, dont l'orientation s'inverse à dif. De ce fait, elle est toujours dirigée vers le site de recombinaison où elle active la machinerie permettant la résolution des dimères de chromosomes. Des homologues des protéines XerC, XerD et FtsK, ainsi qu'un site dif, sont retrouvés chez un grand nombre de bactéries, suggérant que la machinerie ainsi que le contrôle de la recombinaison sont conservés. Cependant, chez les Streptocoques, la machinerie de recombinaison implique une seule recombinase, XerS, qui agit sur un site atypique, difSL. Ce manuscrit concerne l'étude du mécanisme et du contrôle de la recombinaison Xer chez la bactérie Lactococcus lactis appartenant à la famille des Streptocoques. Dans une première partie, nous mettons en évidence in vitro que l'implication d'une seule recombinase ne modifie pas le mode de fixation et de recombinaison au site, en comparaison au système Xer " classique " d'E. coli. D'autre part, nous montrons in vivo que le système est contrôlé par la protéine FtsK mais que le mode d'activation de la recombinaison est différent de celui décrit chez E. coli. Dans une deuxième partie nous nous intéressons au mode d'orientation de la protéine FtsK de L.lactis. Nous mettons en évidence par une combinaison d'approches in vivo, in vitro et in silico, que la reconnaissance de motifs biaisés sur le chromosome est conservée mais que la taille et la séquence du motif reconnu diffère des systèmes déjà décrits.Most known bacteria harbour a unique circular chromosome. Recombination between sister chromosome during replication may fuse them into a single DNA molecule called a chromosome dimer. Dimers are resolved to monomers by Xer site-specific recombination. In Escherichia coli it consists of two recombinases, XerC and XerD acting at a specific dif site located in the replication terminus. The Xer recombination system is controlled by the septum associated protein, FtsK. FtsK is a DNA translocase oriented by specific motif of the chromosome, named KOPS. The polarity of the KOPS is skewed and revers at dif site, hence FtsK is always directed toward the recombination site. Homolog of XerC, XerD an FtsK proteins, as well as a dif site, are find in most bacteria suggesting that the machinery of recombination and its control are well conserved. However, in the Streptococci family of bacteria, dimer resolution uses a single recombinase, XerS, which acts at an atypical dif site, difSL. This manuscript concerns the study of the mechanism and control of Xer recombination in Lactoccocus lactis, a member of the Streptococci family. In a first part we demonstrate, in vitro, that using a single recombinase does not change the mode of binding and recombination at the specific site, by comparison with the "classical" E. coli Xer recombination system. Furthermore, we show in vivo that the L. lactis Xer system is controlled by FtsK but that activation of recombination is achieved by a different mechanism. In a second part, we investigate the orientation of FtsK translocation. We demonstrate, by combining in silico, in vitro and in vivo approaches, that orientation by skew motifs is conserved in L. lactis but that the sequence and length of the motif used is different from known systems

The K-loop, a general feature of the Pyrococcus C/D guide RNAs, is an RNA structural motif related to the K-turn

The C/D guide RNAs predicted from the genomic sequences of three species of Pyrococcus delineate a family of small non-coding archaeal RNAs involved in the methylation of rRNA and tRNA. The C/D guides assemble into ribonucleoprotein (RNP) that contains the methyltransferase. The protein L7Ae, a key structural component of the RNP, binds to a Kink-turn (K-turn) formed by the C/D motif. The K-turn is a structure that consists of two RNA stems separated by a short asymmetric loop with a characteristic sharp bend (kink) between the two stems. The majority of the pyrococcal C/D guides contain a short 3 nt-spacer between the C′/D′ motifs. We show here that conserved terminal stem–loops formed by the C′/D′ motif of the Pyrococcus C/D RNAs are also L7Ae-binding sites. These stem–loops are related to the K-turn by sequence and structure, but they consist of a single stem closed by a terminal loop. We have named this structure the K-loop. We show that conserved non-canonical base pairs in the stem of the K-loop are necessary for L7Ae binding. For the C/D guides with a 3 nt-spacer we show that the sequence and length is also important. The K-loop could improve the stability of the C/D guide RNAs in Pyrococcal species, which are extreme hyperthermophiles

Co-evolution of segregation guide DNA motifs and the FtsK translocase in bacteria: identification of the atypical Lactococcus lactis KOPS motif

Bacteria use the global bipolarization of their chromosomes into replichores to control the dynamics and segregation of their genome during the cell cycle. This involves the control of protein activities by recognition of specific short DNA motifs whose orientation along the chromosome is highly skewed. The KOPS motifs act in chromosome segregation by orienting the activity of the FtsK DNA translocase towards the terminal replichore junction. KOPS motifs have been identified in γ-Proteobacteria and in Bacillus subtilis as closely related G-rich octamers. We have identified the KOPS motif of Lactococcus lactis, a model bacteria of the Streptococcaceae family harbouring a compact and low GC% genome. This motif, 5′-GAAGAAG-3, was predicted in silico using the occurrence and skew characteristics of known KOPS motifs. We show that it is specifically recognized by L. lactis FtsK in vitro and controls its activity in vivo. L. lactis KOPS is thus an A-rich heptamer motif. Our results show that KOPS-controlled chromosome segregation is conserved in Streptococcaceae but that KOPS may show important variation in sequence and length between bacterial families. This suggests that FtsK adapts to its host genome by selecting motifs with convenient occurrence frequencies and orientation skews to orient its activity

Are two better than one? Analysis of an FtsK/Xer recombination system that uses a single recombinase

Bacteria harbouring circular chromosomes have a Xer site-specific recombination system that resolves chromosome dimers at division. In Escherichia coli, the activity of the XerCD/dif system is controlled and coupled with cell division by the FtsK DNA translocase. Most Xer systems, as XerCD/dif, include two different recombinases. However, some, as the Lactococcus lactis XerS/difSL system, include only one recombinase. We investigated the functional effects of this difference by studying the XerS/difSL system. XerS bound and recombined difSL sites in vitro, both activities displaying asymmetric characteristics. Resolution of chromosome dimers by XerS/difSL required translocation by division septum-borne FtsK. The translocase domain of L. lactis FtsK supported recombination by XerCD/dif, just as E. coli FtsK supports recombination by XerS/difSL. Thus, the FtsK-dependent coupling of chromosome segregation with cell division extends to non-rod-shaped bacteria and outside the phylum Proteobacteria. Both the XerCD/dif and XerS/difSL recombination systems require the control activities of the FtsKγ subdomain. However, FtsKγ activates recombination through different mechanisms in these two Xer systems. We show that FtsKγ alone activates XerCD/dif recombination. In contrast, both FtsKγ and the translocation motor are required to activate XerS/difSL recombination. These findings have implications for the mechanisms by which FtsK activates recombination

The bacterial chromosome: architecture and action of bacterial SMC and SMC-like complexes

Structural Maintenance of Chromosomes (SMC) protein complexes are found in all three domains of life. They are characterized by a distinctive and conserved architecture in which a globular ATPase ‘head’ domain is formed by the N- and C-terminal regions of the SMC protein coming together, with a c. 50-nm-long antiparallel coiled-coil separating the head from a dimerization ‘hinge’. Dimerization gives both V- and O-shaped SMC dimers. The distinctive architecture points to a conserved biochemical mechanism of action. However, the details of this mechanism are incomplete, and the precise ways in which this mechanism leads to the biological functions of these complexes in chromosome organization and processing remain unclear. In this review, we introduce the properties of bacterial SMC complexes, compare them with eukaryotic complexes and discuss how their likely biochemical action relates to their roles in chromosome organization and segregation

Étude du mécanisme de résolution des dimères de chromosomes chez les streptocoques

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Prediction of KOPS motifs involved in segregating of bacterial chromosomes in Lactocoques / Streptocoques

Statistics analysis of bacterial genomes allows prediction of various motifs involved in chromosome organization, whose sequence is not necessarily conserved from one species to another. One of these motifs, KOPS (FtsK Orienting Polar Sequence) [1], is implicated in chromosome dimer resolution. Formation of such dimers, in the absence of a mechanism for resolution, may result in cell death. To overcome this phenomenon, the Escherichia coli FtsK protein translocates DNA to bring a specific region to the septum. Recombination at this region then resolves the chromosome dimer, thereby permitting the normal scission of the mother cell into twodaughter cells. The orientation of translocation is determined in a sequence dependent manner through the recognition by FtsK of the KOPS motif, the sequence of which has been characterized in E. coli, Vibrio cholerae and Bacillus subtilis [1,2,3]. To define the KOPS sequence in Streptococcus and Lactococcus we used these examples and determined the general properties of KOPS. More precisely, we determined the statistical properties related to KOPS location and orientation features needed for recognition by FtsK :local and/or general over-representation, orientation over-skew, absolute orientation skew, etc... We searched for motifs with similar statistical properties in Lactococcus lactis,Streptococcus pneumoniae and Streptococcus agalactiae. The analysis indicated potential KOPS in these bacteria. The L. lactis candidate, whose sequence is different from the known KOPS, was validated experimentally. Thus, KOPS motifs were predicted from exhaustive statistical analysis based on information obtained from known other motifs. It would have been impossible to define them only with experiments using currently available techniques as little is known concerning FtsK/KOPS interactions