Precipitation of greigite and pyrite induced by Thermococcales: an advantage to live in Fe- and S-rich environments?

Thermococcales, a major order of archaea inhabiting the iron- and sulfur-rich anaerobic parts of hydrothermal deep-sea vents, have been shown to rapidly produce abundant quantities of pyrite FeS2 in iron-sulfur-rich fluids at 85°C, suggesting that they may contribute to the formation of 'low temperature' FeS2 in their ecosystem. We show that this process operates in Thermococcus kodakarensis only when zero-valent sulfur is directly available as intracellular sulfur vesicles. Whether in the presence or absence of zero-valent sulfur, significant amounts of Fe3 S4 greigite nanocrystals are formed extracellularly. We also show that mineralization of iron sulfides induces massive cell mortality but that concomitantly with the formation of greigite and/or pyrite, a new generation of cells can grow. This phenomenon is observed for Fe concentrations of 5 mM but not higher suggesting that above a threshold in the iron pulse all cells are lysed. We hypothesize that iron sulfides precipitation on former cell materials might induce the release of nutrients in the mineralization medium further used by a fraction of surviving non-mineralized cells allowing production of new alive cells. This suggests that biologically induced mineralization of iron-sulfides could be part of a survival strategy employed by Thermococcales to cope with mineralizing high-temperature hydrothermal environments.

Efectos del estradiol-drospirenona o estradiol-trimegestona sobre los lípidos y lipoproteínas en menopáusicas / Effects of estradiol-drosperinone or estradiol-trimegestone over lipids and lipoproteins in postmenopausal women

Objetivo Comparar los efectos del estradiol-drospirenona o estradiol-trimegestona sobre los lípidos y lipoproteínas en menopáusicas. Material y métodos Se seleccionaron 66 menopáusicas que fueron tratadas con estradiol-drospirenona (grupo A) o estradiol-trimegestona (grupo B) como terapia hormonal de reemplazo después de 6 meses de tratamiento. Se midieron las concentraciones séricas de colesterol, triglicéridos, lipoproteínas de baja densidad, lipoproteínas de alta densidad y lipoproteína (a) (Lp[a]).Resultados Se produjo una reducción significativa en las concentraciones de colesterol, luego de 6 meses de tratamiento (p<0,05). En las pacientes tratadas con estradiol-drospirenona se observó un ligero aumento de las concentraciones de triglicéridos luego del tratamiento, mientras que las pacientes tratadas con estradiol-trimegestona presentaron una disminución significativa en las concentraciones luego de 6 meses de tratamiento (p<0,05). En cuanto a las concentraciones de Lp(a), el grupo de pacientes tratadas con estradiol-drospirenona presentaron un ligero aumento después del tratamiento, pero éste no fue significativo (p=NS).Conclusión La administración de estradiol-trimegestona produce modificaciones en el perfil lipídico, que se pueden considerar beneficiosas al compararlas con las producidas por el estradiol-drospirenona luego de 6 meses de uso (AU)

Abstract:Objective: To compare the effects of estradiol-drospirenone and estradiol-trimegestone onlipids and lipoproteins in postmenopausal women. Materials and methods: Sixty-six patients who were treated with estradiol-drospirenone (groupA) or estradiol-trimegestone (group B) as hormone replacement therapy for 6 months wereselected. Serum cholesterol, triglycerides, low-density lipoprotein cholesterol, high-densitylipoprotein cholesterol and lipoprotein(a) concentrations were measured. Results: Cholesterol concentrations were significantly reduced after 6 months of treatment(p<0.05). In patients treated with estradiol-drospirenone, triglyceride concentrations wereslightly increased after treatment, while patients treated with estradiol-trimegestone showeda mild decrease in serum concentrations after 6 months of treatment (p<0.05). Patientstreated with estradiol-drospirenone showed a mild, non-significant increase in lipoprotein (a)concentrations after treatment (p=NS).Conclusion: Administration of estradiol-trimegestone produced beneficial modifications in lipidprofile when compared with those produced by estradiol-drospirenone after 6 months of treatment (AU)

Purification and functional analysis of recombinant Acholeplasma laidlawii histone-like HU protein.

HU is a most abundant DNA-binding protein in bacteria. This protein is conserved either in its heterodimeric form or in one of its homodimeric forms in all bacteria, in plant chloroplasts, and in some viruses. HU protein non-specifically binds and bends DNA as a hetero- or homodimer and can participate in DNA supercoiling and DNA condensation. It also takes part in some DNA functions such as replication, recombination, and repair. HU does not recognize any specific sequences but shows some specificity to cruciform DNA and to repair intermediates, e.g., nick, gap, bulge, 3'-overhang, etc. To understand the features of HU binding to DNA and repair intermediates, a fast and easy HU proteins purification procedure is required. Here we report overproduction and purification of the HU homodimers. The method of HU purification allows obtaining a pure recombinant non-tagged protein cloned in Escherichia coli. We applied this method for purification of Acholeplasma laidlawii HU and demonstrated that this protein possesses a DNA-binding activity and is free of contaminating nuclease activity. Besides that we have shown that expression of A. laidlawii ihf_hu gene in a slow-growing hupAB E. coli strain restores the wild-type growth indicating that aclHU can perform the basic functions of E. coli HU in vivo.

Does the parallel evolution pattern between the replication-segregation proteins and HU have a biological significance?

The bacterial chromosome is a highly compacted nucleoproteic structure. Its apparent disordered morphology is difficult to conciliate with newly discovered mechanisms governing the propagation of genetic information between mother and daughter cells. Recent experiments in bacterial genetics, biochemistry and cytology from a number of laboratories are beginning to unravel how at each cell division, DNA replication and segregation proteins interact spatially with specific DNA motifs to orchestrate replication and movement of replication forks and chromosomes. We propose here a method to confirm and perhaps extend these experiments by in silico protein sequence comparisons and phylogeny. This analysis showed a parallel evolution between the histone-like protein HU and key protein factors involved in DNA replication and chromosome segregation.

Overproduction and improved strategies to purify the threenative forms of nuclease-free HU protein.

The heterodimeric HU protein was isolated from Escherichia coli as one of the most abundant DNA binding proteins associated with the bacterial nucleoid. HUalphabeta is composed of two very homologous subunits, but HU can also be present in E. coli under its two homodimeric forms, HUalpha(2) and HUbeta(2). This protein is conserved either in its heterodimeric form or in one of its homodimeric forms in all bacteria, in plant chloroplasts and in some viruses. HU can participate, like the histones, in the maintenance of DNA supercoiling and in DNA condensation. This protein which does not recognize any specific sequence on double-stranded DNA, has been shown to bind specifically to cruciform DNA as does the eukaryotic HMG1 protein and to a series of structures which are found as intermediates of DNA repair, e.g., nick, gap, 3'overhang, etc. The strong binding of HU to these diverse DNA structures could explain, in part at least, its pleiotropic role in the bacterial cell. To understand all the facets of its interactions with nucleic acids, it was necessary to develop a procedure which allowed the purification of the three forms of HU under their native form and without the nuclease activity strongly associated with the protein. We describe here such a procedure as well as demonstrating that the three histidine-tagged HUs we have produced, have conserved the binding characteristics of native HUs. Interestingly, by two complementation tests, we show that the histidine-tagged HUs are fully active in vivo.

Crystallization and preliminary X-ray diffraction analysis of the homodimeric form alpha2 of the HU protein from Escherichia coli.

The homodimeric form alpha(2) of the Escherichia coli DNA-binding protein HU was crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystals belong to space group I222, with unit-cell parameters a = 31.09, b = 55.34, c = 117. 63 A, and contain one monomer per asymmetric unit. A full diffraction data set was collected to 2.3 A resolution on a conventional X-ray source. The molecular-replacement method, using the HU crystallographic model from Bacillus stearothermophilus as a starting point, gave a reliable solution for the rotation and translation functions.

The Escherichia coli ribosomal protein S16 is an endonuclease.

The histone-like protein HU isolated from Escherichia coli exhibited, after several purification steps, a Mg(2+)-dependent nuclease activity. We show here that this activity can be dissociated from HU by a denaturation-renaturation step, and is due to a small fraction of ribosomal protein S16 co-purifying with HU. S16 is an essential component of the 30S ribosomal particles. We have cloned, overproduced, and purified a histidine-tagged S16 and shown that this protein is a DNA-binding protein carrying a Mg(2+)-Mn(2+)-dependent endonuclease activity. This is an unexpected property for a ribosomal protein.

Serratia marcescens contains a heterodimeric HU protein like Escherichia coli and Salmonella typhimurium.

Homologs of the dimeric HU protein of Escherichia coli can be found in every prokaryotic organism that has been analyzed. In this work, we demonstrate that Serratia marcescens synthesizes two distinct HU subunits, like E. coli and Salmonella typhimurium, suggesting that the heterodimeric HU protein could be a common feature of enteric bacteria. A phylogenetic analysis of the HU-type proteins (HU and IHF) is presented, and a scheme for the origin of the hup genes and the onset of HU heterodimericity is suggested.

The CDC6 gene is required for centromeric, episomal, and 2-microns plasmid stability in the yeast Saccharomyces cerevisiae.

Temperature-sensitive Saccharomyces cerevisiae cdc6 mutants, under restrictive conditions, show an increase in recombination frequency, as well as chromosome and circular minichromosome loss. The role of the essential CDC6 gene was tested in trans and in cis to study circular plasmid stability. It was possible to demonstrate that the product of the CDC6 gene, acting in trans, is important for centromeric, episomal, and also 2-microns plasmid maintenance, while the gene sequence itself has no effect in cis on the stability of the plasmids tested. A high percentage of phenotypic revertants for the cdc6 mutation loses 2 microns upon shifting to the restrictive temperature and, under semipermissive conditions, the endogenous plasmid becomes very unstable, favoring a more efficient curing procedure. A positive correlation between centromeric plasmid size and stability was demonstrated even for small circular plasmids.

A segment of the phage HK022 chromosome is a mosaic of other lambdoid chromosomes.

We report the sequence of a region of the PR operon of lambdoid phage HK022 and an analysis of the proteins it encodes. This region has DNA sequence elements and open reading frames that resemble those found in phages lambda, P22, and phi 80. The open reading frames encode homologs of the lambda CII transcription activator, the P22 DNA replication proteins, and a fourth protein of unknown function.

Histones, HMG, HU, IHF: Même combat.

In this review article we present a compilation of the proteins homologous to Escherichia coli HU: the HU-like family. Two of these, HU and IHF from E coli have been extensively characterized genetically and biochemically. Due to their DNA binding activities, these proteins confer a condensed shape to the chromosome and regulate the transcription of selected sets of its genes. The parallel between the dual function of the HU-like proteins and the roles described for eukaryotic histone and HMG proteins is striking, especially in the view that they are evolutionary unrelated.

Xis and Fis proteins prevent site-specific DNA inversion in lysogens of phage HK022.

HK022, a temperate coliphage related to lambda, forms lysogens by inserting its DNA into the bacterial chromosome through site-specific recombination. The Escherichia coli Fis and phage Xis proteins promote excision of HK022 DNA from the bacterial chromosome. These two proteins also act during lysogenization to prevent a prophage rearrangement: lysogens formed in the absence of either Fis or Xis frequently carried a prophage that had suffered a site-specific internal DNA inversion. The inversion is a product of recombination between the phage attachment site and a secondary attachment site located within the HK022 left operon. In the absence of both Fis and Xis, the majority of lysogens carried a prophage with an inversion. Inversion occurs during lysogenization at about the same time as prophage insertion but is rare during lytic phage growth. Phages carrying the inverted segment are viable but have a defect in lysogenization, and we therefore suggest that prevention of this rearrangement is an important biological role of Xis and Fis for HK022. Although Fis and Xis are known to promote excision of lambda prophage, they had no detectable effect on lambda recombination at secondary attachment sites. HK022 cIts lysogens that were blocked in excisive recombination because of mutation in fis or xis typically produced high yields of phage after thermal induction, regardless of whether they carried an inverted prophage. The usual requirement for prophage excision was bypassed in these lysogens because they carried two or more prophages inserted in tandem at the bacterial attachment site; in such lysogens, viable phage particles can be formed by in situ packaging of unexcised chromosomes.

Antitermination of early transcription in phage HK022. Absence of a phage-encoded antitermination factor.

In phage lambda and its relatives most early phage genes are located downstream from transcription termination sites, and full gene expression requires suppression of termination (or antitermination). Phage HK022, a lambda relative, also antiterminates early transcription, but, unlike its relatives, does so in the absence of any active phage gene product. We found no functional equivalent of the lambda N antitermination protein in HK022. In addition, nus mutations, which alter host proteins required for lambda antitermination, have no apparent effect on HK022 early gene expression. We have shown that terminators located several thousand base-pairs from the start point of transcription are suppressed, and that in the left operon suppression requires a short, promoter-proximal segment. A 40 bp region within this segment is repeated in the right operon. The chromosomal locations of these repeated segments resemble those of the nut antitermination sites of other lambdoid phages, but the HK022 sites lack the conserved sequence elements of the nut sites. It appears that HK022 antiterminates early transcription in a novel way.

The early promoters of bacteriophage HK022: contrasts and similarities to other lambdoid phages.

The pL, pR and pM promoters of lambdoid phages direct the transcription of early phage genes and the prophage repressor gene. We have determined the start points of transcription for these three promoters in the lambdoid phage HK022 and have shown that the HK022 repressor represses the early promoters, pL and pR, and activates the repressor promoter, pM. HK022 resembles other phages of the lambda family in these respects, as it does in the functional organization of most of its early genes and sites. One exception is nun, the first gene of the HK022 pL operon, which is expressed in the presence of prophage repressor and thus differs from its lambda counterpart, gene N. We show that transcription of nun in a lysogen does not initiate at pL but instead starts upstream at the pM promoter. This difference in transcription fits the different roles of Nun and N proteins in the physiology of the two phages: Nun protects HK022 lysogens against superinfection with certain other lambdoid phages, while N promotes the transcription of early lambda genes.

Determinants of site-specific recombination in the lambdoid coliphage HK022. An evolutionary change in specificity.

The temperate bacteriophage HK022, like its relative lambda, inserts its chromosome into a specific site in the bacterial chromosome during lysogenization and excises it after induction. However, we find that the recombinational specificities of the two phages differ: they use different bacterial sites, and neither promotes efficient insertion or excision of the other phage chromosome. In order to determine the basis for this difference in specificity, we sequenced the HK022 elements that are involved in insertion and excision, and compared them to the corresponding lambda elements. The location, orientation, size and overall arrangement of the int and xis genes and the phage attachment sites are nearly identical in the two genomes, as is common for other functionally related elements in lambdoid phages. The Xis proteins of the two phages are functionally interchangeable, and their predicted amino acid sequences differ by but one residue. In contrast, the two Int proteins are not functionally interchangeable, and their sequences, although similar, differ at many positions. These sequence differences are not uniformly distributed: the amino-terminal 55 residues are completely conserved, but the remaining 302 show a pattern of differences interspersed with identities and conservative changes. These findings imply that the specificity difference between HK022 and lambda site-specific recombination is a consequence of the inability of the respective Int proteins to recognize pairs of heterologous attachment sites. The two phage attachment sites are remarkably similar, especially the two "arm" segments, which in lambda contain binding sites for Int, Xis and integration host factor. They are less similar in the segment between the two arms, which in lambda contains the points of recombinational strand exchange and a second class of binding site for Int protein (the "core-type" sites). The two bacterial attachment sites are quite different, although both have a short stretch of perfect homology with their respective phage partners at the points of strand exchange. We propose that the two Int proteins recognize similar or identical sites in the arms of their cognate attachment sites, and that differences in binding or action at the core-type sites is responsible for the divergent specificities. Genetic experiments and sequence comparisons suggest that both proteins recognize different but overlapping families of core-type sites, and that divergence in specificity has been achieved by an alternating succession of small, mutually compatible changes in protein and site.

Structure and function of the nun gene and the immunity region of the lambdoid phage HK022.

The immunity region of the lambdoid phage, HK022, has been sequenced. The HK022 repressor gene, its cognate operators and promoters, and several early phage genes can be discerned. The overall design of the immunity region resembles that of other lambdoid phages. The location of the HK022 nun gene, whose product excludes superinfecting lambda by terminating transcription at (or near) the lambda nut sites, is analogous to that of gene N in lambda. nun is preceded by sequences similar to the lambda nut sites and the lambda pL promoter and is followed by several transcription termination signals. Despite these similarities, Nun is required neither for the lytic nor the lysogenic pathway of phage development. Again, unlike N, Nun is expressed in a prophage, perhaps from a promoter other than pL. We suggest that Nun and N have diverged in evolution and now perform different functions for their respective phages. Although Nun and N compete at the lambda nut sites and interact with the same host Nus proteins, they are only distantly related in predicted amino acid sequence. The presence of transcription terminators in the pL operon suggests that the expression of the HK022 early functions, like those of lambda, entails an antitermination mechanism. However, Nun does not appear to be an essential component of this mechanism. Our most economic model is that the HK022 nutL sequence suppresses pL operon terminators in the absence of a phage-encoded antitermination protein. Striking homologies between the HK022 nutL sequence and related sequences in the Escherichia coli rrn operons support this notion. Alternatively, a phage antitermination gene may be located outside the pL operon.

Vectors with restriction-site banks. III. Escherichia coli-Saccharomyces cerevisiae shuttle vectors.

The bank of unique restriction sites present in plasmid pJRD158 has been incorporated into new vectors carrying selective markers and replicons derived from commonly used Escherichia coli-Saccharomyces cerevisiae shuttle vectors pJDB207 and YRp7. The new vectors pMH158 and pJO158 have 21 and 23 unique restriction sites, respectively, and their complete DNA sequences are known.

Expression of chicken egg white lysozyme by Saccharomyces cerevisiae.

An efficient yeast promoter was isolated using a beta-galactosidase (beta Gal) promoter probe vector. This promoter was then used to express chicken egg white lysozyme in yeast using a complete intron-free lysozyme-coding sequence constructed by in vitro recombination between a cDNA clone lacking the 5' end and the corresponding 5' end from a nuclear DNA clone. The resulting lysozyme is efficiently exported into the growth medium suggesting that the chicken signal sequence is recognized by the yeast secretion process.