Inferring the phylogeny of archaebacteria: The use of ribosomal sensitivity to protein-synthesis inhibitors

Summary The sensitivities to 37 protein-synthesis inhibitors were determined in cell-free translational systems for 13 Archaebacteria species. Multivariate factor analysis by principal components, cluster analysis by the unweighted pair-group clustering algorithm, and compatibility and parsimony methods of constructing phylogenetic trees were used both to infer the genealogical relationships within archaebacteria and to establish their relations with the eubacterial and eukaryotic kingdoms.     

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.     

The chloroplast generps 4 as a tool for the study ofPoaceae phylogeny

Phylogenetic analyses of 28Poaceae species based on the chloroplastrps 4 gene are presented using parsimony and distance methods. Two monocots from other families were used as outgroups. The chloroplast generps 4 was amplified, cloned, and sequenced for each species. The inferred phylogenetic trees were compared to recent classifications and are shown to fit their general features. There is a dichotomy in our tree between the pooid group and the other grasses. This is in contradiction with other molecular phylogenies, where the bamboos appear first within the family. This result led us to discuss some hypotheses about the relationships of the bambusoids with the other groups of grasses, and also about the relative position of rice and bamboo, which are found close to each other in our trees.     

Insensitivity of archaebacterial ribosomes to protein synthesis inhibitors. Evolutionary implications.

The effect on Sulfolobus solfataricus (an extremely thermoacidophilic archaebacterium) of selected inhibitors affecting reactions of the polypeptide elongation cycle has been tested by using poly(U) and poly(UG) directed cell-free systems. The results reveal a unique pattern of antibiotic sensitivity of Sulfolobus ribosomes with an inhibitory effect observed for only three of 60 compounds tested. Through comparison with suitable eubacterial and eukaryotic cell-free systems the insensitivity of Sulfolobus ribosomes to most inhibitors of protein synthesis appears to reflect a phylogenetic distinction of ribosome structure, rather than the high temperature conditions of the Sulfolobus assay system. In this respect ribosomes of thermoacidophilic archaebacteria differ not only from their eubacterial and eukaryotic counterparts, but also from ribosomes of archaebacteria belonging to the methanogenic-halophilic branch of the 'third' kingdom. The evolutionary implications of these findings are discussed.     

Increased sensitivity to protein synthesis inhibitors in cells lacking tmRNA.

tmRNA (also known as SsrA or 10Sa RNA) is involved in a trans-translation reaction that contributes to the recycling of stalled ribosomes at the 3' end of an mRNA lacking a stop codon or at an internal mRNA cluster of rare codons. Inactivation of the ssrA gene in most bacteria results in viable cells bearing subtle phenotypes, such as temperature-sensitive growth. Herein, we report on the functional characterization of the ssrA gene in the cyanobacterium Synechocystis sp. strain PCC6803. Deletion of the ssrA gene in Synechocystis resulted in viable cells with a growth rate identical to wild-type cells. However, null ssrA cells (deltassrA) were not viable in the presence of the protein synthesis inhibitors chloramphenicol, lincomycin, spiramycin, tylosin, erythromycin, and spectinomycin at low doses that do not significantly affect the growth of wild-type cells. Sensitivity of deltassrA cells similar to wild-type cells was observed with kasugamycin, fusidic acid, thiostrepton, and puromycin. Antibiotics unrelated to protein synthesis, such as ampicillin or rifampicin, had no differential effect on the deltassrA strain. Furthermore, deletion of the ssrA gene is sufficient to impair global protein synthesis when chloramphenicol is added at sublethal concentrations for the wild-type strain. These results indicate that ribosomes stalled by some protein synthesis inhibitors can be recycled by tmRNA. In addition, this suggests that the first elongation cycle with tmRNA, which incorporates a noncoded alanine on the growing peptide chain, may have mechanistic differences with the normal elongation cycles that bypasses the block produced by these specific antibiotics. tmRNA inactivation could be an useful therapeutic target to increase the sensitivity of pathogenic bacteria against antibiotics.     

TEV protease-mediated cleavage in Drosophila as a tool to analyze protein functions in living organisms

Drosophila provides a powerful experimental system to analyze gene functions in a multi-cellular organism. Here we describe an in vivo method that interferes with the integrity of selected proteins through site-specific cleavage in Drosophila. The technique is based on the highly specific seven-amino-acid recognition site of the tobacco etch virus (TEV) protease. We established transgenic fly lines that direct TEV protease expression in various tissues without affecting fly viability. The insertion of the TEV protease recognition site in defined positions of target proteins mediates their sequence-specific cleavage after controlled TEV protease expression in the fly. Thereby, this technique is a powerful tool that allows the in vivo manipulation of selected proteins in a time- and tissue-specific manner.     

Studies of ribosomes of yeast species: Susceptibility to inhibitors of protein synthesis in vivo and in vitro

Summary The susceptibility of cells from various species of Saccharomyces and Kluyveromyces was tested against different types of protein synthesis inhibitors. Minimal inhibitory concentrations were determined for each yeast species and the sensitivity of their ribosomes in cell-free extracts was tested. Two aminoglycosides, paromomycin and hygromycin B were assayed for capacity to stimulate translation errors with ribosomes of yeast species showing different minimal inhibitory concentrations in vivo. In many cases a correlation exists between natural and in vitro resistance suggesting that some natural antibiotic resistances are ribosomal.     

Arabidopsis sensitivity to protein synthesis inhibitors depends on 26S proteasome activity

The 26S proteasome (26SP), the central protease of the ubiquitin-dependent proteolysis pathway, controls the regulated proteolysis of functional proteins and the removal of misfolded and damaged proteins. In Arabidopsis, cellular and stress response phenotypes of a number of mutants with partially impaired 26SP function have been reported. Here, we describe the responses of proteasome mutants to protein synthesis inhibitors. We show that the rpt2a-3, rpn10-1 and rpn12a-1 mutants are hypersensitive to the antibiotic hygromycin B, and tolerant to the translation inhibitor cycloheximide (CHX) and herbicide l-phosphinothricin (PPT). In addition to the novel mechanism for herbicide tolerance, our data suggests that the combination of hygromycin B, CHX and PPT growth-response assays could be used as a facile diagnostic tool to detect altered 26SP function in plant mutants and transgenic lines.     

The use of a chemically defined artificial diet as a tool to study Aedes aegypti physiology

Aedes aegypti mosquitoes obtain from vertebrate blood nutrients that are essential to oogenesis, such as proteins and lipids. As with all insects, mosquitoes do not synthesize cholesterol but take it from the diet. Here, we used a chemically defined artificial diet, hereafter referred to as Substitute Blood Meal (SBM), that was supplemented with cholesterol to test the nutritional role of cholesterol. SBM-fed and blood-fed mosquitoes were compared regarding several aspects of the insect physiology that are influenced by a blood meal, including egg laying, peritrophic matrix formation, gut microbiota proliferation, generation of reactive oxygen species (ROS) and expression of antioxidant genes, such as catalase and ferritin. Our results show that SBM induced a physiological response that was very similar to a regular blood meal. Depending on the nutritional life history of the mosquito since the larval stage, the presence of cholesterol in the diet increased egg development, suggesting that the teneral reserves of cholesterol in the newly hatched female are determinant of reproductive performance. We propose here the use of SBM as a tool to study other aspects of the physiology of mosquitoes, including their interaction with microbiota and pathogens.     

The sensitivity of the pseudomurein-containing genus Methanobacterium to inhibitors of murein synthesis

The sensitivity to inhibitors of various steps of murein synthesis was studied with six strains of methanogenic bacteria. Four of the strains belong to the genus Methanobacterium, which contains pseudomurein in its cell walls. This polymer-as well as murein-is not present in the two control organisms, Methanosarcina barkeri and Methanospirillum hungatii, which were found to be resistant to all inhibitors of murein synthesis. The four strains of Methanobacterium were resistant to the antibiotics fosfomycin, D-cycloserine, vancomycin, penicillin G and cephalosporin C, all of which inhibit the synthesis or function of the peptide subunits of murein. On the other hand, the four strains were susceptible to bacitracin, nisin, gardimycin and enduracidin. It is therefore assumed that the biosynthesis of murein and pseudomurein, respectively, may have some reactions of the so-called lipid cycle and the polymerization of the heteroglycan strands in common.     

The pharate adult clasper as a tool for measuring chitin synthesis and for identifying new chitin synthesis inhibitors

A rapid, reliable, repeatable bioassay for measuring chitin synthesis is described. It utilizes the clasper from male pharate adult European corn borers and measures the incorporation of [14C]N-acetylglucosamine. Chitin synthesis is maximum in claspers taken from animals 5 and 6 days postpupation. The system is very sensitive to inhibition by the phenylbenzoyl ureas and polyoxins and should be useful for identifying potential inhibitory agents.     

The use of metabolomics as a tool to investigate hepatitis C

According to World Health Organization, an estimated 3 % of the global population is suffering from chronic hepatitis C. Furthermore, 60–70 % of chronically-infected patients develop liver diseases, of which 5–20 % of all cases advance to cirrhosis, and 1–5 % die from hepatitis C related hepatocellular carcinoma. This high incidence might be ascribed to the poor performance of the currently available diagnostic, treatment, and vaccination protocols, together with the lack of knowledge of the underlying disease mechanisms. In this review, we discuss the role that the relatively new research field termed metabolomics, alone or as part of an integrated ‘omics’ approach, has played in the investigation of hepatitis C and associated clinical manifestations. We also consider future research possibilities in this field, and the impact that these results might have on the fight against this global health predicament.