1,721,040 research outputs found
Guidelines for Inferring and Characterizing a Family of Bacterial trans-Acting Small Noncoding RNAs
No full textSo far, every sequenced bacterial transcriptome encompasses hundreds of small regulatory noncoding RNAs (sRNAs). From those sRNAs that have been already characterized, we learned that their regulatory functions could span over almost every bacterial process, mostly acting at the posttranscriptional control of gene expression (Wagner and Romby, Adv Genet 90:133?208, 2015). Canonical molecular mechanisms of sRNA action have been described to rely on both sequence and/or structural traits of the RNA molecule. As for protein-coding genes, the conservation of sRNAs among species suggests conserved and adjusted functions across evolution. Knowing the phylogenetic distribution of an sRNA gene and how its functional traits have evolved may help to get a broad picture of its biological role in each single species. Here, we present a simple computational workflow to identify close and distant sRNA homologs present in sequenced bacterial genomes, which allows defining novel sRNA families. This strategy is based on the use of Covariance Models (CM) and assumes the conservation of sequence and structure of functional sRNA genes throughout evolution. Moreover, by carefully inspecting the conservation of the close genomic context of every member of the RNA family and how the patterns of microsynteny follow the path of species evolution, it is possible to define subgroups of sRNA orthologs, which in turn enables the definition of RNA subfamilies.Fil: Lagares, Antonio. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Valverde, Claudio Fabián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin
An Integrated Affinity Chromatography-Based Approach to Unravel the sRNA Interactome in Nitrogen-Fixing Rhizobia
No full textThe activity, mechanism, and function of bacterial base-pairing small non-coding RNA regulators (sRNAs) are largely shaped by their main interacting cellular partners, i.e., proteins and mRNAs. We describe here an MS2 affinity chromatography–based procedure adapted to unravel the sRNA interactome in nitrogen-fixing legume endosymbiotic bacteria. The method consists of tagging of the bait sRNA at its 5′-end with the MS2 aptamer followed by pulse overexpression and immobilization of the chimeric transcript from cell lysates by an MS2-MBP fusion protein conjugated to an amylose resin. The sRNA-binding proteins and target mRNAs are further profiled by mass spectrometry and RNAseq, respectively.Fil: García Tomsig, Natalia Isabel. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidín; EspañaFil: Lagares, Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnologia. Centro de Bioquimica y Microbiologia de Suelos. Laboratorio de Fisiologia, Genetica de Bacterias Para Plantas.; ArgentinaFil: Becker, Anke. University of Marburg; AlemaniaFil: Valverde, Claudio Fabián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnologia. Centro de Bioquimica y Microbiologia de Suelos. Laboratorio de Fisiologia, Genetica de Bacterias Para Plantas.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Jimenez Zurdo, José Ignacio. Consejo Superior de Investigaciones Científicas. Estación Experimental del Zaidín; Españ
Quantification of Bacterial Polyhydroxybutyrate Content by Flow Cytometry
Full text linkWe describe here a detailed protocol for the quantification of the intracellular content of polyhydroxybutyrate (PHB) in a population of bacterial cells by flow cytometry, which is based on a consensus of several previously reported works.Fil: Lagares, Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes; ArgentinaFil: Valverde, Claudio Fabián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes; Argentin
Structural studies on the O-specific polysaccharide of the lipopolysaccharide from Pseudomonas donghuensis strain SVBP6, with antifungal activity against the phytopathogenic fungus Macrophomina phaseolina
No full textAn O-specific polysaccharide (OPS) was isolated from the lipopolysaccharide (LPS) of Pseudomonas donghuensis SVBP6, a bacterium with a broad-spectrum antifungal activity in vitro, particularly that against Macrophomina phaseolina. This latter is one of the most virulent and dangerous pathogens of plants, including soybean which is an economically important crop in Argentina today. The OPS was studied by sugar analysis and spectroscopy (1D and 2D 1H and 13C NMR) showing the following trisaccharide repeating unit: →6)-ɑ-D-ManpNAc-(1 → 3)-β-L-Rhap-(1 → 4)-β-D-Glcp-(1→. The crude LPS, the purified LPS and the O-chain were assayed for their antifungal activity against M. phaseolina at 25, 50, 100, and 200 μg plug−1. The results showed that the crude LPS best inhibition was at 200 μg plug−1, able to inhibit the fungus growth by about 45%, while purified LPS and the corresponding OPS, in the same condition, reduced fungus growth by 65%, and 75%, respectively. Furthermore, the purified LPS and OPS significantly reduced the growth of M. phaseolina already at 100 μg plug−1 compared to the crude LPS. The structure of the O-chain is unique among the bacterial LPS and this is the first time that both the antifungal activity of a bacterial LPS and its corresponding O-chain were described.Fil: Zdorovenko, Evelina L.. Università degli Studi di Napoli Federico II; ItaliaFil: Dmitrenok, Andrey S.. Università degli Studi di Napoli Federico II; ItaliaFil: Masi, Marco. Università degli Studi di Napoli Federico II; ItaliaFil: Castaldi, Stefany. Università degli Studi di Napoli Federico II; ItaliaFil: Muzio, Federico Matías. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Isticato, Rachele. Università degli Studi di Napoli Federico II; ItaliaFil: Valverde, Claudio Fabián. Universidad Nacional de Quilmes; ArgentinaFil: Knirel, Yuriy A.. Università degli Studi di Napoli Federico II; ItaliaFil: Evidente, Antonio. Università degli Studi di Napoli Federico II; Itali
Artificial sRNAs activating the Gac/Rsm signal transduction pathway in Pseudomonas fluorescens
No full textIn Pseudomonas Xuorescens CHA0, the synthesis of antifungal compounds is post-transcriptionally activated by the Gac/Rsm cascade. The two-component system GacS/GacA promotes transcription of three small regulatory RNAs (i.e., sRNAs), RsmX, RsmY, and RsmZ, which remove the regulatory proteins RsmA and RsmE from the ribosome-binding sites of exoproduct-related mRNAs. The GacS/GacA-dependent accumulation of RsmX/Y/Z and formation of RsmX/Y/Z-RsmA/E complexes relieve mRNA translational repression. Other bacteria as E. coli and Vibrio spp. utilize similar sRNA–protein based systems to adjust mRNA translation (e.g., the E. coli Csr system for carbon storage, motility and bioWlm regulation). The Rsm/Csr sRNAs are remarkably similar in that they contain several stem-loops with an invariant GGA trinucleotide exposed in the hairpin loop that would be the characteristic structuralsequence motifs relevant for sRNA activity and stability. Here it is shown that the dysfunctional Gac/Rsm cascade of P. Xuorescens rsmXYZ mutants could be restored by appropriate transcription levels of artiWcial genes encoding RNAs with unrelated primary sequence but with two or more hairpins displaying the RsmA/E binding motifs. The results support the hypothesis that the molecular mimicry of Rsm/Csr sRNAs is based on proper secondary structures that expose critical binding motifs irrespective of their overall sequence.Fil: Valverde, Claudio Fabián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin
Who's the boss here? The post-transcriptional global regulator Hfq takes over control of secondary metabolite production in the nematode symbiont Photorhabdus luminiscens
Full text linkA Trojan? Dr. Jekyll and Mr. Hyde? This is Photorhabdus luminiscens: an enterobacterial symbiont of nematodes of the Heterorhabditis genus, and, at the same time, an insect killer. It colonizes the nematode gut; the worm gets into a host insect, where it regurgitates the bacteria; P. luminiscens then spreads throughout the insect hemolymph and secrete toxins that kill the host (Clarke, 2014). At this stage, the capacity of P. luminiscens to produce a wide set of secondary metabolites (SM) is turned on to serve a dual task: to keep the decaying insect tissue free of other competitor bacteria, and to serve as food and source of developmental factors for the nematode transition from infective juveniles into hermaphrodites with reproductive ability (Joyce et al., 2011) (Fig. 1). It is therefore a journey with changing environments for P. luminiscens, which has attracted interest not only in terms of the regulatory processes modulating its adaptation to the varying ecological niches to which it is exposed, but also because of the biological properties and diversity of the SM that it can produce. Such prolific secondary metabolism is mainly determined by a number of non-ribosomal peptide synthetase and polyketide synthase gene clusters, whose expression regulatory details have been poorly exploredFil: Valverde, Claudio Fabián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentin
A Single Mutation in the oprF mRNA Leader Confers Strict Translational Control by the Gac/Rsm System in Pseudomonas fluorescens CHA0
Full text linkThe rhizobacterium Pseudomonas fluorescens CHA0 is able to antagonize fungal phytopathogens on a variety of crop plants, mainly due to the production of secondary metabolites that are coordinately upregulated by the global regulatory Gac/Rsm cascade. The two-component system GacS/GacA activates transcription of the three small regulatory RNAs RsmX, RsmY, and RsmZ, which counteract translational repression of target mRNAs by RsmA and RsmE proteins. In a search for novel Gac/Rsm targets based on the minimal sequence on mRNA leaders required for RsmA/RsmE control, the leader region of the major porin OprF emerged as a candidate. Although an isogenic CHA0 oprF mutant showed a reduced ability to attach to cucumber and tomato roots, suggesting a role for OprF in root colonization as a requisite for pathogen antagonism, a translational oprF0-0lacZ fusion was weakly regulated by Gac/Rsm despite its high sequence similarity to the hcnA leader. A single base substitution put the modified oprF 50-UTR under strict control by Gac/Rsm. The results highlight the subtle sequence requirements of Gac/Rsm targets.Fil: Alvarez Crespo, María Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Valverde, Claudio Fabián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentin
A simple laboratory class using a Pseudomonas aeruginosa auxotroph to illustrate UV-mutagenic killing, DNA photorepair and mutagenic DNA repair
Full text linkA simple and cheap laboratory class is proposed to illustrate the lethal effect of UV radiation on bacteria and the operation of different DNA repair mechanisms. The class is divided into two sessions, an initial 3-hour experimental session and a second 2-hour analytical session. The experimental session involves two separate experiments: one dedicated to illustrating the lethal effect of UV radiation and the protective effect of DNA photorepair; the second to explore the operation of DNA repair mechanisms that prioritise survival but introduce mutations. The procedure makes use of a Pseudomonas aeruginosa double auxotroph, which serves to detect UV-induced back-mutations to prototrophy. The proposed scheme is carried out by undergraduate students of the Bacterial Physiology and Genetics course, as part of our Biotechnology curriculum. We think that it will be a valuable tool for microbiology students to increase their understanding of basic genetic concepts.Fil: Sobrero, Patricio Martín. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Valverde, Claudio Fabián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin
A mutation in the glta gene from a native isolate of the Pseudomonas chlororaphis subgroup induces a phenotypic change associated with phenazine production
No full textMembers of the Pseudomonas genus colonise the rhizosphere of different plant species and display plant-probiotic traits. Among our collection of 19 isolates of native pseudomonads, isolate SPAN5 was obtained from a bulk soil sample of grasslands. This isolate, related with the Pseudomonas chlororaphis subgroup, showed several plant-growth promoting activities in vitro. We carried out Tn5 mutagenesis for identifying genes related to the biocontrol. We here report that the mutant clone SPAN5-135, which lost its orange pigmentation, suffered the insertion of Tn5 into the gltA gene encoding a type II citrate synthase (CS). The CS enzyme activity was reduced significantly in SPAN-135, as well as its phenazine production in several media. Also, siderophore and exoprotease secretion were affected. SPAN-135 could not recover the complete inhibition potential of the wild type version against the phytopathogenic fungi tested. This is the first report of the requirement of gltA for phenazine and siderophores production.Fil: Agaras, Betina Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; ArgentinaFil: Valverde, Claudio Fabián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Interacciones Biológicas; Argentin
A novel oligonucleotide pair for genotyping members of the pseudomonas genus by single-round PCR amplification of the gyrb gene
Full text linkPseudomonas is a phylogenetically diverse bacterial genus which is broadly distributed in different ecological niches, and whose taxonomy is continuously under revision. For that purpose, gyrB is one of the housekeeping genes routinely used for multilocus sequence analysis (MLSA). As we noticed that there was not a single primer pair available in the literature suitable for direct sequencing of this gene, we decided to design a unique oligonucleotide pair and to set up a polymerase chain reaction (PCR) protocol to obtain a single amplicon for the entire Pseudomonas genus. Based on the available gyrB sequence from 148 Pseudomonas species, we identified highly conserved regions to design oligonucleotides without fully degenerate positions. We then set up cycling conditions for achieving high specificity and yield of the PCR protocol. Then, we showed that the amplicons produced with this procedure were appropriate for direct sequencing with both primers, obtaining more than 95% of amplicons coverage. Finally, we demonstrated that a PCR-RFLP (restriction fragment length polymorphism) approach served to differentiate among Pseudomonas species, and even between members of the same species.Fil: Agaras, Betina Cecilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Valverde, Claudio Fabián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin
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