|
|
Main Issues and Achievements of the Laboratory |
||||||
|
|
Last revision on 09/04/97
pour la Version Française
|
|
|
|
|
We are
working on DNA topology and DNA topoisomerases in
Archaea and Hyperthermophilic (HT) Bacteria. We
have shown that HT Archaeal plasmids are either
relaxed or positively supercoiled, meaning that
their DNA have an excess of topological links as
compared to mesophilic plasmids. Moreover, we have
discovered that HT Archaea possess a completely new
family of type II topoisomerases, which led us to
identify in silico a protein which
could be responsible for the initiation of
crossing-over in Eucaryotes. We are interested in
HT plasmids as markers for DNA topology studies,
templates for in vitro HT replication
systems, and cloning vectors
|
This issue is
the basis of our commitment to an European
biotechnology project (BIOTECH) in collaboration
with the laboratories of Drs. D. Prieur in Roscoff
(France) and R. Garrett in Copenhagen (Denmark):
the aim is to setup the conditions for the culture
of HT Archaea in liquid and solid media, on Petri
dishes; to setup and validate efficient
transformation methods, as well as to isolate
mutants resistant to growth inhibitors, in order to
obtain genetic markers which will be ultimately
inserted in cloning/expression vectors for
hyperthermophiles. We have already discovered
several new plasmids in HT Archaea of the
Thermococcales genus. One of them has been fully
sequenced and his replication mechanism has been
identified as being of the rolling circle type.
|
We have
identified duplicated (paralogous) gene families in
E. coli . This work and other
phylogenetic studies undertaken in our laboratory
have led us to settle the importance of gene
duplications before the Eucaryotes/Procaryotes
separation. Finally, we have shown that published
results, dealing with rooting of the universal tree
in the bacterial branch, are questionable.
|
DNA World at High Temperature
DNA under HT conditions in vitro (P. Forterre, E. Marguet).
We have shown that a closed circular DNA is not denatured under HT conditions, at last until 107°C ; however, it is rapidly degraded by a mechanism involving depurination followed by the phosphodiester bond breakage (15). This thermodegradation is independent of DNA topology and is inhibited by high concentrations of monovalent salts. (15).Thus, Reverse Gyrase from HT do not seem to be necessary for DNA protection against overall denaturation or degradation.In vivo DNA in HT Archaea and extreme halophiles(F. Charbonnier, F. Mojica, P. Lopez-Garcia, E. Marguet, D. Gadelle, coll. with D. Prieur, W. Zillig and F. Rodriguez -Valera).
We have isolated pGT5, the first plasmid from a HT Archaea, Pyrococcus abyssi (3) . Moreover we have shown that the plasmid is relaxed at optimal growth temperature (95°C) (4).This conclusion can be applied to other HT plasmids (13), as isolation of new plasmids from Thermococcales and Sulfolobales, shows that they are all either relaxed, or positively supercoiled. (25). On the contrary, plasmids isolated from halophilic archaea are negatively supercoiled (13,16,17). Thus, DNA isolated from HT archaea exhibits a linking number (Lk) excess compared to mesophilic DNA. This excess (presumably due to Reverse Gyrase) could maintain DNA in a structural conformation close to it's state in mesophiles. Accordingly, we have shown that the linking number increases during heat shock and decreases during cold shock in HT. Furthermore, Lk raises upon temperature increase in halophilic and HT archaea (17) as well as in mesophiles. Gyrase and reverse gyrase activities could have appeared during procaryotic evolution in order to allow their adaptation to a very broad temperature range (0 - 110°C) (25).HT DNA topoisomerases (A. Bergerat, O. Guipaud, D. Gadelle, B. Labedan, coll. with M. Duguet and F. Robb).
We are collaborating with M. Duguet's laboratory for several years now on Topoisomerases I, and more specifically on HT Reverse Gyrase. (7, 8). As this lab is carrying out a thorough study of topoisomerases I, we have concentrated our efforts on topo II. Do HT bacteria bearing reverse gyrase also possess a gyrase? We have cloned genes coding for 2 subunits of topo II from HT bacteria T. maritima . Subunits corresponding to GyrB (gyrase) and/or ParE (topo IV) have been fully sequenced, allowing us to draw a distance tree based on the 25 already known GyrB/ParE sequences. As opposed to the 16s rRNA tree, T. maritima doesn't branch deeply on the tree, but rather near B. subtilis , a result also derived from other proteic phylogenetic trees. Poor resolution of branches doesn't allow us to tell whether T. maritima 's topo II is a gyrase or a topo IV. Purification of the enzyme is under way in our laboratory. We have purified to homogeneity and characterized topoisomerases II from HT archaea Sulfolobus shibatae (18) and P. furiosus . Both enzymes are heterotetramers constituted by 45 and 60 kd proteins for A and B subunits respectively, and resistant to usual topo II inhibitors. ATP is binding to B subunit. Peptide sequences have been obtained from S. shibatae A and B subunits, allowing us to clone and fully sequence the genes, which are contiguous and correspond well to purified proteins.Studies on pGT5 Plasmid (S. Marsin, N. Rollet, Y. Zivanovic, coll. with D. Prieur).
pGT5 plasmid (3.4 kb) has been completely sequenced (27). The sequence shows evidences for a rolling circle type of replication: a gene coding for an endonuclease-ligase (Rep) related protein, found in the pC194 plasmid family, as well as two potential, single-stranded(sso) and double-stranded (dso), replication origins (ori). The putative sso resembles ori sequences recognized by bacterial primosome. We have demonstrated the existence of a single stranded replicative intermediate of pGT5 in P. abyssi , which constitutes a signature of the rolling circle mechanism (27).First archaeal genes related to DNA repair, chromosome structure and cell division [ A. Bouyoub, E. Gerard (coll. with G. Barbier et J. Querellou), C. Elie].
We have searched for DNA polymerases genes in three Thermococcales strains (IFREMER collection). A DNA polymerase gene, from the GE8 strain, containing an intein has been isolated. An gene involved in the SOS response, anE. coli dinF homologue, has been identified in strain 700 (24). In bacteria, recA or lexA can be found upstream of the dinF gene. We have cloned and sequenced a gene upstream of dinF in strain 700, but it appears to be homologous to the E. coli minD gene, involved in cellular division. In the course of this work, we have also isolated the purA gene from strain 707, which is now tested as a potential genetic marker (see below). Still during our DNA polymerase search, we have discovered in Sulfolobus acidocaldarius an homologue of eucaryotic ATPases belonging to the SMC family (Structural Maintenance of the Chromosome) and of yeast RAD50 gene. These are motor, dumbbell shaped, proteins bearing the coiled-coiled motif, which play a key role in chromosome condensation or recombinaison.
Genetic Tools for Hyperthermophilic Archaea
This work is headed by Y. Zivanovic in coll. with Prieur and Garrett in the frame of an european program. We have shown that pGT5 bearing an insert in between the two main open reading frames of the plasmid can be transferred and replicated in Pyrococcus cells (26). However, it is unstable at the moment, which could be due to it's replication mode and/or to the lack of efficient selection marker in this type of construct.
Discovery of new plasmids in Thermococcales, characterization of new strains (N. Rollet, P. Lopez-Garcia).
In about twenty Thermococcales strains already studied, six different plasmids have been discovered. Their sizes range from 3,5 to 25 kb. Some of them have been detected by PFGE analysis of total genomes. Moreover, genomic sizes of several HT archaea have been determined (1.7 to 3.9 Mb).
Isolation of the purA gene from Pyrococcus and potential purA mutants . (Y. Zivanovic )
Culture conditions for anaerobic HT archaea have been established in the laboratory (e.g. setup of an anaerobic chamber). We have isolated, by colony plating at 95°C, several P. abyssi mutants resistant to 6-methyl-purine in presence of hypoxanthine, potentially defective in the purA gene. Mutant and wild type purA genes have been cloned and are currently under way of sequencing. Various constructs have been made by merging archaeal pGT5 and bacterial Litmus plasmids along with resistant purA genes.
Origin and Evolution of Genomes
Identification of duplicated (paralogous) genes families in E. coli.(B. Labedan, coll. with M. Riley).
Analysis of the full set of known E. coli genes has permitted to show that 52% of genes are paralogous (originating from the same ancestor by duplication) (22, 23, 54). Various levels of paralogy have been identified, allowing us to define ancestry relationships among groups of genes. 747 known E. coli sequences could originate from only 92 ancestral sequences (23,54).
Evidences for gene duplications before the eucaryotes/procaryotes divergence. (N. Benachenhou, P. Forterre, B. Labedan).
Phylogenetic analysis of GDHs [we have cloned the S. shibatae GDH gene (14)], DNA polymerases (2, 11), DNA topoisomerases I and II (11), OTCases and ATCases (coll. with Glansdorff) has demonstrated that phylogenetic trees thus obtained are not species trees. Their topology imply that some gene duplication took place before the eucaryotes/ procaryotes divergence (9). This result has led us to criticize rooting of the universal tree of life in the bacterial branch, a work based on ATPases analysis (6, 10), this tree being itself spoilt by paralogy. The case of an horizontal gene transfer has been studied in coll. with J.Guespin (19).These analysis could be linked in some instances [GDH (14), purA] with the search for HT signatures. In particular, numerous deletions in the purA protein from Pyrococcus could lead to a more compact structure of the protein.
Origin of HT, use of the clastidic method to solve the universal tree rooting problem. (P. Forterre, C. Elie).
Molecular analysis of HT, in particular the study of the reverse gyrase gene, has led us to question the existence of a link between the hot origin of life and the outbreak of HT (20, 28). We have proposed a novel hypothesis to explain why the common ancestor of archaea and bacteria was an HT (21, iv) : the outbreak of the "procaryotic phenotype" by thermoreduction. According to this hypothesis, rooting of the universal tree falls in the eucaryotic branch. However, a bacterial rooting of the tree has been proposed based on elongation factors and ILeu tRNA synthetases phylogenetic analysis. Nevertheless, by applying a strict cladistic analysis to these protein sequence alignments, we have shown that the bacterial rooting of the tree is not robust. (6). In order to determine whether bacteria and archaea were specifically linked, we tried to establish whether they possess the same type of replication origin (ori). We sequenced 6 kb of Haloferax volcanii chromosome upstream of the gyrB gene (this region is conserved among bacteria and contain very often dnaA and oriC). We have identified an orf which shows week similarity with dnaN, a gene close to OriC in many bacteria, but we didn't find any homologue to dnaA.