Biomedical subjects

P López-García

Publications and source records attributed to P López-García.

15 recordsLinked to original sources

PubMed2001

Unexpected diversity of small eukaryotes in deep-sea Antarctic plankton.

Phylogenetic information from ribosomal RNA genes directly amplified from the environment changed our view of the biosphere, revealing an extraordinary diversity of previously undetected prokaryotic lineages. Using ribosomal RNA genes from marine picoplankton, several new groups of bacteria and archaea have been identified, some of which are abundant. Little is known, however, about the diversity of the smallest planktonic eukaryotes, and available information in general concerns the phytoplankton of the euphotic region. Here we recover eukaryotes in the size fraction 0.2-5 microm from the aphotic zone (250-3,000 m deep) in the Antarctic polar front. The most diverse and relatively abundant were two new groups of alveolate sequences, related to dinoflagellates that are found at all studied depths. These may be important components of the microbial community in the deep ocean. Their phylogenetic position suggests a radiation early in the evolution of alveolates.

Animals↗

PubMed2001

Diversity of free-living prokaryotes from a deep-sea site at the Antarctic Polar Front.

To contribute to the understanding of deep-sea planktonic communities, we explored the prokaryotic diversity of a 3000 m deep site at the Antarctic Polar Front using molecular methods. Bacterial 16S rDNA-amplified sequences corresponded to the as yet uncultivated groups SAR11, within the alpha-Proteobacteria, and SAR324, within the delta-Proteobacteria, as well as to the gamma-Proteobacteria, Cytophagales, Planctomyces, Gram-positives, and the group of environmental sequences SAR406. Among them, gamma-proteobacterial sequences were the most abundant and diverse. Within Archaea, and using six different primer sets for 16S rDNA amplification, only euryarchaeotal sequences were retrieved. Most of them clustered with the Thermoplasma-related marine groups II and III, but some corresponded to a recently described group of marine sequences emerging at the base of haloarchaea. Our data suggest that gamma-Proteobacteria and Euryarchaeota may be dominant elements in terms of genetic diversity of the two prokaryotic domains in this deep-sea pelagic area.

Journal Article↗

PubMed2001

A novel haloarchaeal-related lineage is widely distributed in deep oceanic regions.

During our study of the 16S rRNA gene sequence-based archaeal diversity of a deep-sea site located at a 3,000 m depth at the Antarctic Polar Front, we detected several phylotypes ascribed to already known Group II and III Euryarchaeota, and a cluster of distinct sequences that branched off at the base of haloarchaea. The position of this lineage (marine Group IV) was very robust using distance (neighbour-joining) and maximum-likelihood methods. Subsequently, we designed specific primers for the detection of this archaeal group in other marine environments using polymerase chain reaction amplification and sequence comparison. Group IV archaea were found in the Antarctic area (across a gradient from the Southern ocean to the South Atlantic), and also in North Atlantic and Mediterranean waters. In all oceanic locations, Group IV archaea were never detected in surface waters, but were vertically distributed in the deepest part of the water column.

Antarctic Regions↗

PubMed2001

New insights into the phylogenetic position of diplonemids: G+C content bias, differences of evolutionary rate and a new environmental sequence.

The phylum Euglenozoa consists of three distinct groups: the euglenoids, diplonemids and kinetoplastids. The phylogenetic position of the diplonemids within this phylum remains unsettled, since both morphological and molecular data produce weak and contradictory results. It is shown here that taxonomic sampling, G+C content bias, mutational saturation and differences of evolutionary rate among lineages are major factors affecting the topology of the small-subunit rRNA euglenozoan tree. When these problems are minimized by using a larger diplonemid sampling (including a sequence of environmental origin) and correcting for G+C bias (by using both paralinear distances or an unbiased dataset), a diplonemids+euglenoids sisterhood is retrieved. Bootstrap support for this relationship is still moderate, but it is retrieved by all analysis methods, overcoming previously reported disagreements. In addition, the inclusion of a large number of euglenoid sequences in the analysis improves some phylogenetic relationships within this group. Some problematic taxa, such as the species Khawkinea quartana, are now placed with high bootstrap support and monophyly is found for two interesting groups (the photosynthetic genera Eutreptia+Eutreptiella and the loricate genera Strombomonas+Trachelomonas), although with weak statistical support.

Animals↗

PubMed2000

Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon.

Despite a rapid increase in the amount of available archaeal sequence information, little is known about the duplication of genetic material in the third domain of life. We identified a single origin of bidirectional replication in Pyrococcus abyssi by means of in silico analyses of cumulative oligomer skew and the identification of an early replicating chromosomal segment. The replication origin in three Pyrococcus species was found to be highly conserved, and several eukaryotic-like DNA replication genes were clustered around it. As in Bacteria, the chromosomal region containing the replication terminus was a hot spot of genome shuffling. Thus, although bacterial and archaeal replication proteins differ profoundly, they are used to replicate chromosomes in a similar manner in both prokaryotic domains.

Archaeal Proteins↗

PubMed2000

DNA topology and the thermal stress response, a tale from mesophiles and hyperthermophiles.

During heat shock and cold shock, plasmid DNA supercoiling changes transiently both in mesophilic bacteria and in hyperthermophilic archaea, despite a different overall topology (negative supercoiling versus relaxation to positive supercoiling). Transient changes in DNA supercoiling might be essential to generate the stress response, but they could also be a consequence of the physical effects of temperature on cellular components. Indeed, both appear intertwined. Comparison of the mechanisms acting in the two biological systems suggests that the dependence on temperature of the activity of different DNA topoisomerases, as well as of protein binding, are key factors for the control of DNA topology during stress, which may in turn be relevant for the expression of stress-induced genes.

Cold Temperature↗

PubMed2000

Plasmid pGS5 from the hyperthermophilic archaeon Archaeoglobus profundus is negatively supercoiled.

We present evidence that, in contrast to plasmids from other hyperthermophilic archaea, which are in the relaxed to positively supercoiled state, plasmid pGS5 (2.8 kb) from Archaeoglobus profundus is negatively supercoiled. This might be due to the presence of a gyrase introducing negative supercoils, since gyrase genes are present in the genome of its close relative A. fulgidus, and suggests that gyrase activity predominates over reverse gyrase whenever the two topoisomerases coexist in cells.

Archaeoglobus↗

PubMed1999

DNA supercoiling and temperature adaptation: A clue to early diversification of life?

Cellular systems to control an appropriate DNA geometry for function probably evolved simultaneously with DNA genomes. Such systems are basically DNA topoisomerases and DNA-binding proteins. Therefore, their distribution in extant organisms may be a source of information on early evolution and the nature of the last common ancestor (cenancestor). Most living beings need the strand-opening potential of negative DNA supercoiling to allow transcription and other DNA-dependent processes. Mesophiles have global negatively supercoiled DNA, essentially due to gyrase (introducing negative supercoils) in bacteria and to DNA wrapping around histone cores in eukaryotes. Mesophilic archaea, halophilic methanogens, and halophiles might use a gyrase, whereas some methanogens might use histone wrapping. The existence of these two distinct mechanisms suggests that mesophily appeared at least twice in evolution. On the other hand, only one system which is based on reverse gyrase (introducing positive supercoils) appears to be required for hyperthermophilic life. Archaeal hyperthermophiles lacking gyrase have relaxed to positively supercoiled DNA, but hyperthermophilic bacteria of the genus Thermotoga, which have both gyrase and reverse gyrase, have negative supercoiling. This suggests that reverse gyrase is necessary at least locally, but whereas these hyperthermophilic bacteria favor general melting potential and stability at critical active regions, hyperthermophilic archaea favor general linking excess and local melting. In this context, the existence of a thermophilic (60-80 degrees C) ancestor endowed with only relaxing topoisomerases is hypothesized. Such temperatures allow a compromise between melting potential and stability, i.e., an appropriate DNA geometry for function. Subsequent duplication and functional specialization of existing DNA topoisomerases would then have facilitated adaptation to hyperthermophily and mesophily in archaea and bacteria, respectively. If reverse gyrase is an ancient character in hyperthermophilic bacteria, the cenancestor would have already been a hyperthermophile. Histone sequence homology and similarities of nucleosome structural dynamics suggest that eukaryotes inherited this system for DNA structural homeostasis from methanogenic euryarchaea. Some mesophilic archaea would have improved their adaptability to mesophily by importing gyrase from bacteria.

Archaea↗

PubMed1999

Control of DNA topology during thermal stress in hyperthermophilic archaea: DNA topoisomerase levels, activities and induced thermotolerance during heat and cold shock in Sulfolobus.

Plasmid topology varies transiently in hyperthermophilic archaea during thermal stress. As in mesophilic bacteria, DNA linking number (Lk) increases during heat shock and decreases during cold shock. Despite this correspondence, plasmid DNA topology and proteins presumably involved in DNA topological control in each case are different. Plasmid DNA in hyperthermophilic archaea is found in a topological form from relaxed to positively supercoiled in contrast to the negatively supercoiled state typical of bacteria, eukaryotes and mesophilic archaea. We have analysed the regulation of DNA topological changes during thermal stress in Sulfolobus islandicus (kingdom Crenarchaeota), which harbours two plasmids, pRN1 and pRN2. In parallel with plasmid topological variations, we analysed levels of reverse gyrase, topoisomerase VI (Topo VI) and the small DNA-binding protein Sis7, as well as topoisomerase activities in crude extracts during heat shock from 80 degrees C to 85-87 degrees C, and cold shock from 80 degrees C to 65 degrees C. Quantitative changes in reverse gyrase, Topo VI and Sis7 were not significant. In support of this, inhibition of protein synthesis in S. islandicus during shocks did not alter plasmid topological dynamics, suggesting that an increase in topoisomerase levels is not needed for control of DNA topology during thermal stress. A reverse gyrase activity was detected in crude extracts, which was strongly dependent on the assay temperature. It was inhibited at 65 degrees C, but was greatly enhanced at 85 degrees C. However, the intrinsic reverse gyrase activity did not vary with heat or cold shock. These results suggest that the control of DNA topology during stress in Sulfolobus relies primarily on the physical effect of temperature on topoisomerase activities and on the geometry of DNA itself. Additionally, we have detected an enhanced thermoresistance of reverse gyrase activities in cultures subject to prolonged heat shock (but not cold shock). This acquired thermotolerance at the enzymatic level is abolished when cultures are treated with puromycin, suggesting a requirement for protein synthesis.

Archaeal Proteins↗

PubMed1998

In vitro DNA binding of the archaeal protein Sso7d induces negative supercoiling at temperatures typical for thermophilic growth.

The topological state of DNA in hyperthermophilic archaea appears to correspond to a linking excess in comparison with DNA in mesophilic organisms. Since DNA binding proteins often contribute to the control of DNA topology by affecting DNA geometry in the presence of DNA topoisomerases, we tested whether the histone-like protein Sso7d from the hyperthermophilic archaeon Sulfolobus solfataricus alters DNA conformation. In ligase-mediated supercoiling assays carried out at 37, 60, 70, 80 and 90 degrees C we found that DNA binding of increasing amounts of Sso7d led to a progressive decrease in plasmid linking number (Lk), producing negative supercoiling. Identical unwinding effects were observed when recombinant non-methylated Sso7d was used. For a given Sso7d concentration the DNA unwinding induced was augmented with increasing temperature. However, after correction for the overwinding effect of high temperature on DNA, plasmids ligated at 60-90 degrees C exhibited similar sigma values at the highest Sso7d concentrations assayed. These results suggest that Sso7d may play a compensatory role in vivo by counteracting the overwinding effect of high temperature on DNA. Additionally, Sso7d unwinding could be involved in the topological changes observed during thermal stress (heat and cold shock), playing an analogous role in crenarchaeal cells to that proposed for HU in bacteria.

Archaeal Proteins↗

PubMed1997

Isolation of new plasmids from hyperthermophilic Archaea of the order Thermococcales.

A collection of 57 strains of hyperthermophilic Archaea from the order Thermococcales was screened for the presence of plasmids; 9 plasmids present in six of these strains were isolated and characterized in terms of size and cross-hybridization. The Notl macrorestriction patterns of genomic DNA of strains harbouring these plasmids were obtained. Pyrococcus abyssi strains GE27 and GE23 as well as Thermococcus sp. GE31 contained a single plasmid of 3.5 kb (pGN27), 16.8 kb (pGN23) and 5.3 kb (pGN31), respectively, whilst the three strains I559, I560 and I690 all contained two plasmids of 3.5 kb (pSN559, pSN560, pSN690) and 24 kb (pLN559, pLN560, pLN690), respectively. Plasmid pGN27 strongly cross-hybridized with the previously described plasmid pGT5 from P. abyssi strain GE5, whilst plasmids pGN23 and pGN31 did not cross-hybridize with each other, nor with any other plasmid. The three small plasmids of strains I559, I560 and I690 cross-hybridized, as well as their three large plasmids. Macrorestriction pattern analysis and the results of plasmid cross-hybridization experiments indicated that these three strains were different but closely related, and likely belonged to the genus Thermococcus. This study shows that plasmids are widespread in hyperthermophilic archaea, and significantly increases the number and diversity of plasmids available for laboratory work.

Blotting, Southern↗

PubMed1997

DNA topology in hyperthermophilic archaea: reference states and their variation with growth phase, growth temperature, and temperature stresses.

In order to address the dynamics of DNA topology in hyperthermophilic archaea, we analysed the topological state of several plasmids recently discovered in Thermococcales and Sulfolobales. All of these plasmids were from relaxed to highly positively supercoiled in vitro, i.e. they exhibited a significant linking excess compared to the negatively supercoiled plasmids from mesophilic organisms (both Archaea and Bacteria). In the two archaeal orders, plasmid linking number (Lk) decreased as growth temperature was lowered from its optimal value, i.e. positively supercoiled plasmids were relaxed whereas relaxed plasmids became negatively supercoiled. Growth temperatures above the optimum correlated with higher positive supercoiling in Sulfolobales (Lk increase) but with relaxation of positive supercoils in Thermococcus sp. GE31. The topological variation of plasmid DNA isolated from cells at different growth phases were found to be species specific in both archaeal orders. In contrast, the direction of topological variation under temperature stress was the same, i.e. a heat shock correlated with an increase in plasmid positive supercoiling, whilst a cold shock induced negative supercoiling. The kinetics of these effects were analysed in Sulfolobales. In both temperature upshift (from 80 to 85 degrees C) and downshift (from 80 to 65 degrees C), a transient sharp variation of Lk occurred first, and then DNA supercoiling progressively reached levels typical of steady-state growth at the final temperature. These results indicate that DNA topology can change with physiological states and environmental modifications in hyperthermophilic archaea.

Archaea↗

PubMed1995

Genomic stability in the archaeae Haloferax volcanii and Haloferax mediterranei.

Through hybridization of available probes, we have added nine genes to the macrorestriction map of the Haloferax mediterranei chromosome and five genes to the contig map of Haloferax volcanii. Additionally, we hybridized 17 of the mapped cosmid clones from H. volcanii to the H. mediterranei genome. The resulting 35-point chromosomal comparison revealed only two inversions and a few translocations. Forces known to promote rearrangement, common in the haloarchaea, have been ineffective in changing global gene order throughout the nearly 10(7) years of these species' divergent evolution.

Biological Evolution↗

PubMed1994

Halobacterial megaplasmids are negatively supercoiled.

Several covalently closed circular halobacterial megaplasmids (up to more than 500 kb) from different strains of Haloferax mediterranei, have been resolved by orthogonal-field alternating gel electrophoresis (OFAGE). These molecules seem to be negatively supercoiled in vivo, as deduced from the effect of intercalating agents affecting their topology and, therefore, their electrophoretic mobility. It has also been demonstrated that the topoisomerase II inhibitor novobiocin affects the native topological state of halobacterial megaplasmids impeding their migration in OFAGE under standard conditions for resolution of large supercoiled molecules.

DNA, Superhelical↗

PubMed1992

Genomic organization of the halophilic archaeon Haloferax mediterranei: physical map of the chromosome.

Pulsed field gel electrophoresis (PFG) has been used to study the genomic organization of the halophilic archaeon Haloferax mediterranei. Analysis of the different genomic elements as well as the restriction patterns obtained with several endonucleases revealed that this microorganism has a circular chromosome of 2.9 Mb and, at least, three extrachromosomal elements of 490, 320 and 130 kb respectively. The complete physical map of the chromosome for the endonucleases PacI and BamHI has been constructed, and several BcII, BgIII and DraI restriction fragments have been aligned on these maps. The localization of heterologous and homologous genes on the physical map, including those for rRNA, lay the ground work for the construction of a genetic map.

Chromosomes, Bacterial↗