Biomedical subjects

R Amils

Publications and source records attributed to R Amils.

At least 37 records · Page 2Linked to original sources

PubMed1997

Genomic organization of the acidophilic chemolithoautotrophic bacterium Thiobacillus ferrooxidans ATCC 21834.

The genomic organization of the acidophilic chemolithoautotrophic bacterium Thiobacillus ferrooxidans ATCC 21834 has been studied by pulsed-field gel electrophoresis (PFGE). Analysis of its intact DNA, as well as the restriction patterns obtained with several endonucleases, allowed the characterization of one circular chromosome of 2.9 Mb and one plasmid of 8.6 kb. The first complete and highly resolved physical map (86 restriction sites) of the chromosome of an acidophilic obligate chemolithoautotrophic bacterium has been constructed by using endonucleases PmeI, SwaI, XbaI, and SpeI. The rRNA and str operons have been located on the chromosomal physical map.

Acidithiobacillus thiooxidans↗

PubMed1996

PCR-mediated detection of the chemolithotrophic bacterium Thiobacillus cuprinus using 23S rDNA- and 16S/23S intergenic spacer region-targeted oligonucleotide primers.

Bioleaching is carried out by chemolithotrophic microorganisms, most of them belonging to the genera Thiobacillus and Leptospirillum. The role of the mixotrophic species T. cuprinus in this process is controversial, since its ecological study applying classical detection techniques to natural or industrial environments is very difficult. For this reason, we have developed an alternative method based on PCR-mediated detection using specific oligonucleotide primers that target variable regions of the 23S rRNA coding gene and of the 16S/23S intergenic spacer region. Specificity and sensitivity of PCR amplifications performed with both kinds of primers were studied.

Base Sequence↗

PubMed1996

Total reconstitution of active small ribosomal subunits of the extreme halophilic archaeon Haloferax mediterranei.

The small ribosomal subunit of the halophilic archaeon Haloferax mediterranei has been reconstituted from its dissociated rRNA and protein components. Efficient reconstitution of particles, fully active in poly(U)-dependent polyphenylalanine synthesis, occurs after 2 h of incubation at 36 degrees C in the presence of 1.5 M of (NH4)2SO4, 100 mM of MgAc2, 20 mM Tris-HCl (pH 8.2) and 6 mM 2-mercaptoethanol. Important differences in the optimal ionic conditions for the reconstitution of the 30S and the 50S ribosomal subunits from Haloferax mediterranei have been found. K+ and NH4+ ions have differing abilities to promote the reconstitution of the particles. The assembly of 30S ribosomal subunits of H. mediterranei has a higher tolerance to ionic strength than the assembly of the 50S subunits and it is independent of the Mg2+ concentration present in the system.

Halobacteriaceae↗

PubMed1996

The action of antibiotics on the anaerobic digestion process.

Antibiotics can disturb the production of biogas during anaerobic digestion. This study shows a systematic approach to understanding how the different bacterial populations involved in the final conversion of organic matter into methane are inhibited by 15 antimicrobial agents with different specificities and modes of action. The results obtained show the following trends: (i) some inhibitors, such as the macrolide erythromycin, lack any inhibitory effect on biogas production; (ii) some antibiotics, with different specificities, have partial inhibitory effects on anaerobic digestion and decrease methane production by interfering with the activity of propionic-acid- and butyric-acid-degrading bacteria, (e.g. antibiotics that interfere with cell wall synthesis, RNA polymerase activity and protein synthesis, especially the aminoglycosides); (iii) the protein synthesis inhibitors chlortetracycline (IC50 40 mg l-1) and chloramphenicol (IC50 15-20 mg l-1) are very powerful inhibitors of anaerobic digestion. The majority of the antibiotics tested lacked activity against acetoclastic methanogens, being active only on the acetogenic bacteria. However, chloramphenicol and chlortetracycline could cause the complete inhibition of the acetoclastic methanogenic archaea.

Anaerobiosis↗

PubMed1996

Optimization of the production of a bacteriocin from Haloferax mediterranei Xia3.

The optimal conditions for the production of the halocin H1, a 31 kDa bacteriocin-like molecule produced by the extreme halophilic Archaea Haloferax mediterranei Xia3 active against Gram-negative haloarchaea, was characterized. The physico-chemical conditions required for the optimal production of halocin H1 are similar to those found in the habitat in which the microorganism was isolated: 20% salt concentration and temperature range between 37 and 42 degrees C. Optimal antimicrobial activity was obtained using 0.5% of N-Z amine E as nutrient.

Bacteriocins↗

PubMed1995

High-ionic strength interference of ribosomal inhibition produced by aminoglycoside antibiotics.

A protein synthesis cell-free system capable of performing with similar efficiencies in different ionic conditions has been developed for the halotolerant marine bacterium Vibrio costicola. The system has been used to test the effect of ionic strength on the interference produced by thirty translation inhibitors with different structural, functional, and domain specificities. In general, at high ionic strengths, the inhibition of protein synthesis produced by polycationic antibiotics like the aminoglycosides is much less pronounced than the inhibition obtained at low ionic strengths, while non-aminoglycosidic antibiotics show similar inhibitory activities at both high and low ionic conditions. These results strongly suggest that competition between polycationic antibiotics and cations at high concentrations in the media is responsible for the lack of inhibition by aminoglycoside antibiotics at high ionic strengths, rather than a lack of binding sites.

Aminoglycosides↗

PubMed1995

Absolute requirement of ammonium sulfate for reconstitution of active 70S ribosomes from the extreme halophilic archaeon Haloferax mediterranei.

Active 70S ribosomes from the halophilic archaeon Haloferax mediterranei have been reconstituted from their isolated rRNAs and proteins. The reconstitution procedure consists of a two-step incubation; first with 1 M ammonium sulfate and 100 mM magnesium acetate for 1 h at 42 degrees C, followed by a 90-min incubation at 50 degrees C after increasing the ammonium sulfate to 2 M final concentration. The total reconstitution of halophilic 70S ribosomes is a process with its own identity, which does not correspond to the conditions required for the reconstitution of the isolated subunits. Ammonium sulfate is the only salt capable of promoting the assembly of active ribosomes. The increase of ammonium sulfate salts in the second incubation step is obligatory for the isolation of functional particles.

Ammonium Sulfate↗

PubMed1995

Fine structure of the peptidyl transferase centre on 23 S-like rRNAs deduced from chemical probing of antibiotic-ribosome complexes.

Ribosomal binding sites were investigated for the diverse group of antibiotics: anisomycin, anthelmycin, blasticidin S, bruceantin, carbomycin, chloramphenicol, griseoviridin, narciclasine, T2 toxin, tylosin and virginiamycin M1 all of which are considered to inhibit the peptidyl transferase reaction by different mechanisms. The drugs also exhibit differing degrees of specificity for bacterial, archaeal and eukaryotic ribosomes despite a high level of conservation of sequence and secondary structure at the peptidyl transferase centre of the 23 S-like rRNAs. The drug binding sites were characterized by incubating each antibiotic with ribosomes from a bacterium, an archaeon and a eukaryote and chemically probing the 23 S-like rRNA. The complexity of the changes in reactivity ranged from one or two nucleotides (anthelmycin, narciclasine) to eight or nine (virginiamycin M1) and it was inferred, at least for those drugs producing complex changes, that they induce, and stabilize, a particular functional conformer in the peptidyl transferase centre. The results were correlated with literature data on both ribosomal ligand binding and the putative inhibitory mechanisms of the drugs, and the following inferences are made concerning the fine structure of the peptidyl transferase centre. (1) An irregular secondary structural motif, which includes unpaired A2439 (Escherichia coli numbering), lies close to the catalytic centre; (2) nucleotides A2451 and C2452 contribute to a site for the binding of the side chains of aromatic amino acids; (3) the P-substrate site encompasses U2585, U2506 and, possibly, a site in domain IV (A1787), and (4) the sequence A2058 to A2062 and nucleotide U2609 contribute to, or modulate, the start of the peptide channel. No drug effects were found that could be directly attributed to an A-site and the possibility is raised that, if it exists, it consists mainly of ribosomal proteins. However, two drugs T2 toxin and virginiamycin M1 protected the only nucleotide in the peptidyl transferase loop region (C2394) associated with the E-site. Finally, it is proposed that the putative sub-sites are physically separated, that some drugs bind to more than one of them, and that they are conformationally interdependent.

Anti-Bacterial Agents↗

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

Location of the streptomycin ribosomal binding site explains its pleiotropic effects on protein biosynthesis.

Photoaffinity-labeling experiments using three nitroguaiacol ether streptomycin derivatives with spacers of different lengths between the antibiotic and the photoreactive moiety (8, 12 and 17 A) allow us to: (1) unambiguously locate the boundaries of the antibiotic binding site; and (2) test the topographical consistency of the photolabeling results. The streptomycin binding site is located in the interface between the ribosomal subunits, close to proteins S5 in the 30 S and to L11 in the 50 S ribosomal subunits. This location explains most of the antibiotic's pleiotropic effects on protein biosynthesis, especially those related to the tRNA selection mechanism, and it also correlates with the location of the ribosomal components involved in the different streptomycin phenotypes.

Affinity Labels↗

PubMed1994

Using protein synthesis inhibitors to establish the phylogenetic relationships of the Sulfolobales order.

The sensitivity of the cell-free protein synthesis systems from Acidanus brierleyi, Acidianus infernus, and Metallosphaera sedula, members of the archaeal order Sulfolobales, to 40 antibiotics with different specificities has been studied. The sensitivity patterns were compared to those of Sulfolobus solfataricus and other archaeal, bacterial, and eukaryotic systems. The comparative analysis shows that ribosomes from the sulfolobales are the most refractory to inhibitors of protein synthesis described so far. The sensitivity results have been used to ascertain in phylogenetic relationships among the members of the order Sulfolobales. The evolutionary significance of these results are analyzed in the context of the phylogenetic position of this group of extreme thermophilic microorganisms.

Anti-Bacterial Agents↗

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↗

PubMed1994

Hypersensitivity of Rhodobacter sphaeroides ribosomes to protein synthesis inhibitors: structural and functional implications.

The elongation cycle of protein synthesis systems of purple nonsulfur photosynthetic bacteria Rhodobacter sphaeroides, grown both phototrophically and chemotrophically, was studied using 33 inhibitors with different chemical structures and functional and domain specificities. No functional differences between phototrophic and chemotrophic ribosomal systems were detected. Rhodobacter sphaeroides ribosomes exhibited strong hypersensitivity to nine functional inhibitors when compared with Escherichia coli ribosomes. Most of the R. sphaeroides ribosomal hypersensitivities corresponded to peptidyltransferase inhibitors, implying that this important functional neighborhood must be somehow different in the two organisms.

Ammonium Chloride↗

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↗

PubMed1990

In vitro reassembly of active large ribosomal subunits of the halophilic archaebacterium Haloferax mediterranei.

The large ribosomal subunits of the halophilic archaebacterium Haloferax mediterranei have been reconstituted in vitro from the dissociated RNA and protein components. Efficient reassembly of particles fully active in poly(U)-directed polyphenylalanine synthesis requires a 2-h incubation at 42 degrees C in the presence of no less than 2.5 M concentrations of monovalent cations and of 60 mM magnesium. K+ and NH4+ ions are equally effective in promoting subunit reconstitution; however, maximal efficiency is attained when they are combined in a 1:2 molar ratio. The reassembly process requires no heat activation step, as under the appropriate ionic conditions it takes place spontaneously within the temperature range optimal for growth of H. mediterranei cells (40-45 degrees C).

Archaea↗

PubMed1990

Synthesis of active nitroguaiacol ether derivatives of streptomycin.

The synthesis, purification, and biological properties of nitroguaiacol ether derivatives of streptomycin and their corresponding radioactive reduced products were examined. These derivatives are biologically active against gram-positive and gram-negative eubacteria and they are also photoreactive because of the presence of the nitroguaiacol group in the molecule. We demonstrated that these derivatives can be used as streptomycin analogs in photoaffinity labeling of the macromolecular structures related to the mode of action of the antibiotic.

Bacillus subtilis↗

PubMed1989

Structural and functional exchangeability of 5 S RNA species from the eubacterium E.coli and the thermoacidophilic archaebacterium Sulfolobus solfataricus.

The role of 5 S RNA within the large ribosomal subunit of the extremely thermophilic archaebacterium Sulfolobus solfataricus has been analysed by means of in vitro reconstitution procedures. It is shown that Sulfolobus 50 S subunits reconstituted in the absence of 5 S RNA are inactive in protein synthesis and lack 2-3 ribosomal proteins. Furthermore, it has been determined that in the course of the in vitro assembly process Sulfolobus 5 S RNA can be replaced by the correspondent RNA species of E.coli; Sulfolobus reconstituted particles containing the eubacterial 5 S molecule are stable and active in polypeptide synthesis at high temperatures.

Archaea↗

PubMed1989

The use of functional analysis of the ribosome as a tool to determine archaebacterial phylogeny.

Forty different antibiotics with diverse kingdom and functional specificities were used to measure the functional characteristics of the archaebacterial translation apparatus. The resulting inhibitory curves, which are characteristic of the cell-free system analyzed, were transformed into quantitative values that were used to cluster the different archaebacteria analyzed. This cluster resembles the phylogenetic tree generated by 16S rRNA sequence comparisons. These results strongly suggest that functional analysis of an appropriate evolutionary clock, such as the ribosome, is of intrinsic phylogenetic value. More importantly, they indicate that the study of the nexus between genotypic and phenotypic (functional) information may shed considerable light on the evolution of the protein synthetic machinery.

Archaea↗