Temperature increase prevails over acidification in gene expression modulation of amastigote differentiation in Leishmania infantum

<p>Abstract</p> <p>Background</p> <p>The extracellular promastigote and the intracellular amastigote stages alternate in the digenetic life cycle of the trypanosomatid parasite <it>Leishmania</it>. Amastigotes develop inside parasitophorous vacuoles of mammalian phagocytes, where they tolerate extreme environmental conditions. Temperature increase and pH decrease are crucial factors in the multifactorial differentiation process of promastigotes to amastigotes. Although expression profiling approaches for axenic, cell culture- and lesion-derived amastigotes have already been reported, the specific influence of temperature increase and acidification of the environment on developmental regulation of genes has not been previously studied. For the first time, we have used custom <it>L. infantum </it>genomic DNA microarrays to compare the isolated and the combined effects of both factors on the transcriptome.</p> <p>Results</p> <p>Immunofluorescence analysis of promastigote-specific glycoprotein gp46 and expression modulation analysis of the amastigote-specific A2 gene have revealed that concomitant exposure to temperature increase and acidification leads to amastigote-like forms. The temperature-induced gene expression profile in the absence of pH variation resembles the profile obtained under combined exposure to both factors unlike that obtained for exposure to acidification alone. In fact, the subsequent fold change-based global iterative hierarchical clustering analysis supports these findings.</p> <p>Conclusions</p> <p>The specific influence of temperature and pH on the differential regulation of genes described in this study and the evidence provided by clustering analysis is consistent with the predominant role of temperature increase over extracellular pH decrease in the amastigote differentiation process, which provides new insights into <it>Leishmania </it>physiology.</p

A Bacterial Shortcut to Amyloidosis

21 p.-10 fig.The synthetic bacterial prionoid RepA-WH1 causes a vertically transmissible amyloid proteinopathy in Escherichia coli that inhibits growth and eventually kills the cells. Recent in vitro studies show that RepA-WH1 builds pores through model lipid membranes, suggesting a possible mechanism for bacterial cell death. By comparing acutely (A31V) and mildly (ΔN37) cytotoxic mutant variants of the protein, we report here that RepA-WH1(A31V) expression decreases the intracellular osmotic pressure and compromise bacterial viability under either aerobic or anaerobic conditions. Both are effects expected from threatening membrane integrity and are in agreement with findings on the impairment by RepA-WH1(A31V) of the proton motive force (PMF)-dependent transport of ions (Fe3+) and ATP1 synthesis. Systems approaches reveal that, in aerobiosis, the PMF-independent respiratory dehydrogenase NdhII is induced in response to the reduction in intracellular levels of iron. While NdhII is known to generate H2O2 as a by-product of the autoxidation of its FAD cofactor, key proteins in the defense against oxidative stress (OxyR, KatE), together with other stress-resistance factors, are sequestered by co-aggregation with the RepA-WH1(A31V) amyloid. Our findings suggest a route for RepA-WH1 toxicity in bacteria: a primary hit of damage to the membrane, compromising bionergetics, triggers a stroke of oxidative stress, which is exacerbated due to the aggregation-dependent inactivation of enzymes and transcription factors that enable the cellular response to such injury. The proteinopathy caused by the prion-like protein RepA-WH1 in bacteria recapitulates some of the core hallmarks of human amyloid diseases.This work has been supported by grants from Spanish AEI / EU-FEDER (BIO2012-30852, BIO2015- 68730-R and CSD2009-00088) and CSIC (i-LINK0889) to R.G. We acknowledge support of the publication fee by the CSIC Open Access Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Estudio de las barreras para la replicación en el DNA ribosómico de Schizosaccharomyces pombe

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 21-05-2003Polar replication fork barriers (RFBs) near the 3'-end of the rRNA transcriptional unit are a conserved feature of rDNA replication in eukaryotes. In the mouse, in vivo studies indicate that the cis-acting Sal boxes required for rRNA transcription termination are also involved in replication fork blockage. On the contrary, in the budding yeast Saccbarconyls cerevisiae, the rRNA transcription termination factors are not required for RFBs. Here we characterized the rDNA RFBs in the fission yeast Schizosaczhar072)113 park. S. pon-he rDNA contains three closelyspaced polar replication barriers narned RFB1-3, in the 3' to 5' order. The transcription termination protein rebl and its two 17-bp binding sequences, present at the 3' end of the coding region, were required for fork arrest at RFB2 and RFB3. This is the first description of a DNA bin. ding protein involved in in ziw replication stalling at the rDNA. On the other hand, fork arrest at RFB1 was independent of the aboye transcription termination factors. Therefore, RFB2 and RFB3 resemble the barriers present in the mouse rDNA, whereas RFB1 is similar to the budding yeast RFBs. These results suggest that during evolution, ás- and trans-acting factors required for rRNA transcription termination became also involved in replication fork blockage. S. pombe would be a transient species where both mechanisms coexist

The Acquisition of Colistin Resistance Is Associated to the Amplification of a Large Chromosomal Region in Klebsiella pneumoniae kp52145

The appearance of carbapenem-resistant Klebsiella pneumoniae has increased the use of colistin as a last-resort antibiotic for treating infections by this pathogen. A consequence of its use has been the spread of colistin-resistant strains, in several cases carrying colistin resistance genes. In addition, when susceptible strains are confronted with colistin during treatment, mutation is a major cause of the acquisition of resistance. To analyze the mechanisms of resistance that might be selected during colistin treatment, an experimental evolution assay for 30 days using as a model the clinical K. pneumoniae kp52145 isolate in the presence of increasing amounts of colistin was performed. All evolved populations presented a decreased susceptibility to colistin, without showing cross-resistance to antibiotics belonging to other structural families. We did not find any common mutation in the evolved mutants, neither in already known genes, previously known to be associated with the resistance phenotype, nor in new ones. The only common genetic change observed in the strains that evolved in the presence of colistin was the amplification of a 34 Kb sequence, homologous to a prophage (Enterobacteria phage Fels-2). Our data support that gene amplification can be a driving force in the acquisition of colistin resistance by K. pneumoniae.Work in our laboratory is supported by grants from the Instituto de Salud Carlos III (Spanish Network for Research on Infectious Diseases [RD16/0016/0011]), from the Spanish Ministry of Economy, Industry and Competitivity (BIO2017-83128-R) and from the Autonomous Community of Madrid (B2017/BMD-3691).Peer reviewe

High-order interactions distort the functional landscape of microbial consortia

© 2019 Sanchez-Gorostiaga et al.Understanding the link between community composition and function is a major challenge in microbial population biology, with implications for the management of natural microbiomes and the design of synthetic consortia. Specifically, it is poorly understood whether community functions can be quantitatively predicted from traits of species in monoculture. Inspired by the study of complex genetic interactions, we have examined how the amylolytic rate of combinatorial assemblages of six starch-degrading soil bacteria depend on the separate functional contributions from each species and their interactions. Filtering our results through the theory of biochemical kinetics, we show that this simple function is additive in the absence of interactions among community members. For about half of the combinatorially assembled consortia, the amylolytic function is dominated by pairwise and higher-order interactions. For the other half, the function is additive despite the presence of strong competitive interactions. We explain the mechanistic basis of these findings and propose a quantitative framework that allows us to separate the effect of behavioral and population dynamics interactions. Our results suggest that the functional robustness of a consortium to pairwise and higher-order interactions critically affects our ability to predict and bottom-up engineer ecosystem function in complex communities.This work was also supported by a young investigator award from the Human Frontier Science Program (RGY0077/2016) and by the National Institutes of Health through grant 1R35 GM133467-01 to AS. In addition, JFP was supported by grant FIS2016-78781-R from the Spanish Ministerio de Economía y Competitividad

Transcription Termination Factor reb1p Causes Two Replication Fork Barriers at Its Cognate Sites in Fission Yeast Ribosomal DNA In Vivo

Polar replication fork barriers (RFBs) near the 3′ end of the rRNA transcriptional unit are a conserved feature of ribosomal DNA (rDNA) replication in eukaryotes. In the mouse, in vivo studies indicate that the cis-acting Sal boxes required for rRNA transcription termination are also involved in replication fork blockage. On the contrary, in the budding yeast Saccharomyces cerevisiae, the rRNA transcription termination factors are not required for RFBs. Here we characterized the rDNA RFBs in the fission yeast Schizosaccharomyces pombe. S. pombe rDNA contains three closely spaced polar replication barriers named RFB1, RFB2, and RFB3 in the 3′ to 5′ order. The transcription termination protein reb1 and its two binding sites, present at the 3′ end of the coding region, were required for fork arrest at RFB2 and RFB3 in vivo. On the other hand, fork arrest at the strongest RFB1 barrier was independent of the above transcription termination factors. Therefore, RFB2 and RFB3 resemble the barriers present in the mouse rDNA, whereas RFB1 is similar to the budding yeast RFBs. These results suggest that during evolution, cis- and trans-acting factors required for rRNA transcription termination became involved in replication fork blockage also. S. pombe is suggested to be a transitional species in which both mechanisms coexist

The Mating Type Switch-Activating Protein Sap1 Is Required for Replication Fork Arrest at the rRNA Genes of Fission Yeast

Schizosaccharomyces pombe rRNA genes contain three replication fork barriers (RFB1-3) located in the nontranscribed spacer. RFB2 and RFB3 require binding of the transcription terminator factor Reb1p to two identical recognition sequences that colocalize with these barriers. RFB1, which is the strongest of the three barriers, functions in a Reb1p-independent manner, and cognate DNA-binding proteins for this barrier have not been identified yet. Here we functionally define RFB1 within a 78-bp sequence located near the 3′ end of the rRNA coding region. A protein that specifically binds to this sequence was purified by affinity chromatography and identified as Sap1p by mass spectrometry. Specific binding to RFB1 was confirmed by using Sap1p expressed in Escherichia coli. Sap1p is essential for viability and is required for efficient mating-type switching. Mutations in RFB1 that precluded formation of the Sap1p-RFB1 complex systematically abolished replication barrier function, indicating that Sap1p is required for replication fork blockage at RFB1

Structural analysis of the interactions between hsp70 chaperones and the yeast DNA replication protein Orc4p.

You need the Java runtime environment to see the additional information in this article as Protein Structures.Hsp70 chaperones, besides their role in assisting protein folding, are key modulators of protein disaggregation, being consistently found as components of most macromolecular assemblies isolated in proteome-wide affinity purifications. A wealth of structural information has been recently acquired on Hsp70s complexed with Hsp40 and NEF co-factors and with small hydrophobic target peptides. However, knowledge of how Hsp70s recognize large protein substrates is still limited. Earlier, we reported that homologue Hsp70 chaperones (DnaK in Escherichia coli and Ssa1-4p/Ssb1-2p in Saccharomyces cerevisiae) bind strongly, both in vitro and in vivo, to the AAA+ domain in the Orc4p subunit of yeast origin recognition complex (ORC). ScORC is the paradigm for eukaryotic DNA replication initiators and consists of six distinct protein subunits (ScOrc1p–ScOrc 6p). Here, we report that a hydrophobic sequence (IL4) in the initiator specific motif (ISM) in Orc4p is the main target for DnaK/Hsp70. The three-dimensional electron microscopy reconstruction of a stable Orc4p2–DnaK complex suggests that the C-terminal substrate-binding domain in the chaperone clamps the AAA+ IL4 motif in one Orc4p molecule, with the substrate-binding domain lid subdomain wedging apart the other Orc4p subunit. Pairwise co-expression in E. coli shows that Orc4p interacts with Orc1/2/5p. Mutation of IL4 selectively disrupts Orc4p interaction with Orc2p. Allelic substitution of ORC4 by mutants in each residue of IL4 results in lethal (I184A) or thermosensitive (L185A and L186A) initiation-defective phenotypes in vivo. The interplay between Hsp70 chaperones and the Orc4p-IL4 motif might have an adaptor role in the sequential, stoichiometric assembly of ScORC subunits.This work has been financed by Spanish MICINN (grants BFU2006-00494 and BIO2009-06952) and CAM (GR/SAL/0651/2004) to R.G. and MICINN (BFU2007-62382) and the EU (“3D repertoire” LSHG-CT-2005-512028) to J.M.V.Peer reviewe

Liability of the Owner of Real Property before Third Persons

Bakalaura darbā apskatīti problēmjautājumi, kas skar nekustamā īpašuma īpašnieka atbildību pret trešajām personām. Gadījumos, kad īpašnieks- valsts, pašvaldība, fiziska vai juridiska persona- nepienācīgas ēkas vai būves uzturēšanas un apsaimniekošanas rezultātā nodara kaitējumu trešajai personai vai tās mantai, viņš nes atbildību par radītajiem zaudējumiem. Saskaņā ar vispārīgo principu īpašumtiesības ir neaizkaramas, taču īpašnieka rīcība nedrīkst nonākt pretrunā ar likumu un aizskart trešo personu tiesības. Pienākums aizsargāt cilvēka dzīvību ir prioritārs par vispārzināmo īpašumtiesību neaizskaramību. Darbā skatītos problēmjautājumus nosacīti var iedalīt trīs veidos. Pirmkārt, tie ir jautājumi par izmaiņām, kas nepieciešamas likumdošanā, lai ēkas, būves un citi nekustamo īpašumu objekti netiktu novesti līdz stāvoklim, kas apdraud ikvienas personas veselību un dzīvību. Otrkārt, jautājums, ko iesākt, ja atbildīgā persona- nekustamā īpašuma īpašnieks- nepietiekoši rūpējas par savu īpašumu un aizskar citu personu tiesības dzīvot drošā un sakoptā vidē. Treškārt, ko iesākt ar nekustamajiem īpašumiem, kas ir daļēji vai pilnīgi sagruvuši un tādejādi rada draudus garāmgājējiem un īpašuma lietotājiem. Darbā sniegti priekšlikumi par grozījumu veikšanu likumos. Pirmkārt, jāizceļ grozījumu nepieciešamību normatīvajos aktos noteiktajā kultūras vēstures pieminekļu atsavināšanas kārtībā, nosakot to atsavināšanu atbilstoši Satversmē paredzētajai kārtībai. Tāpat nepieciešami grozījumi, kas noteiktu naudas uzkrājumu veidošanu nekustamā īpašuma uzturēšanai un kārtējo apsaimniekošanas izdevumu, un periodisku remontu veikšanai. Ieteicams noteikt dzīvokļu īpašnieku kopsapulču obligātumu, kas veicinātu dzīvokļu īpašnieku sadarbību īpašuma apsaimniekošanā un uzturēšanā. Nepieciešama precīzu termiņu noteikšana noteikumos, kas paredz dokumentu, kas saistīti ar būves sakopšanu vai nojaukšanu, jo ilgstoša izskatīšanas procesa pašvaldības institūcijās laikā graustu tehniskais stāvoklis pasliktinās un tie kļūst arvien bīstamāki apkārtējiem. Šie un citi priekšlikumi īpašuma uzturēšanai un trešo personu aizsardzībai sniegti bakalaura darbā.Bachelor work examines themes about real property owners’ liability before third persons. When owner- state, local government, physical or juridical person- do not take care of the building or premises, he is under an obligation to pay damages- to compensate third persons for loss, injury, or harm suffered because of the dangerous condition of property. In compliance with general principle ownership is untouchable, but an action of owner should not contravene the rights of third persons. Even more important than the longstanding principle of protecting property is the duty to protect human life. There are three kind of problematic questions examined in the bachelor work. First of all, question regarding amendments of law, which are necessary to ensure that buildings and premises are kept in safe condition, so they do not constitute a menace to third persons’ life and safety. Second, question, what to do, when liable person does not pay much attention to his property and therefore violates neighbors’ rights to a safe and clean environment. Third, what to do with premises, which are vulnerable to collapse and constitute a menace to every passerby and occupier. There are provided suggestions for amendments of law in the bachelor work. One of the most needed amendments is connected with an alienation procedure of cultural and historical monuments, which should be stated in compliance with Constitution. There are also need for amendments of law, which provide accruals for real property management and running repairs. Similarly the general meeting of the owners of apartments is commendable to be stated as obligatory, so it would stimulate owners to cooperate with each other, when there are questions regarding real property management. There is also a need for precise, fixed terms in a process of examination of documents of local government institutions for a building reconstruction or tearing down, because buildings become more and more dangerous, while documents are long-drawn examined. These and other suggestions for real property management and third person protection are made in this work

Effect of free or SBS bound SlmA on FtsZ-GTP protofilaments as measured by FCS and EM.

<p><i>A</i> and <i>B</i>. Normalized FCS autocorrelation profiles of FtsZ-GTP (12 μM) in the presence of free SlmA (10 μM, <i>A</i>) and SlmA:SBScons (10 μM:2 μM, <i>B</i>). In both panels, curves 2 and 3 indicate SlmA (with or without SBS) added before and after FtsZ polymerization, respectively. Traces corresponding to FtsZ-GTP polymers (curve 1) or FtsZ-GDP (curve 4) are shown for reference. Lines represent best fits of the models indicated in the Materials and Methods section. FtsZ-Alexa 488 (120 nM) was added as a tracer. Measurements were performed in working buffer at 21°C and RS was always added together with GTP. <i>C</i> and <i>D</i>. Representative micrographs of FtsZ-GTP polymers (12 μM) in the absence (<i>C</i>) or presence of the complex formed by SlmA and SBScons (10 and 2 μM, respectively; <i>D</i>). Samples were equilibrated in working buffer and polymerization was triggered by the addition of 1 mM GTP. The bar represents 90 nm.</p