Utilization of tmRNA sequences for bacterial identification

In recent years, molecular approaches based on nucleotide sequences of ribosomal RNA (rRNA) have become widely used tools for identification of bacteria [1-4]. The high degree of evolutionary conservation makes 16S and 23S rRNA molecules very suitable for phylogenetic studies above the species level [3-5]. More than 16,000 sequences of 16S rRNA are presently available in public databases [4,6]. The 16S rRNA sequences are commonly used to design fluorescently labeled oligonucleotide probes. Fluorescence in situ hybridization (FISH) with these probes followed by observation with epifluorescence microscopy allows the identification of a specific microorganism in a mixture with other bacteria [2-4]. By shifting probe target sites from conservative to increasingly variable regions of rRNA, it is possible to adjust the probe specificity from kingdom to species level. Nevertheless, 16S rRNA sequences of closely related strains, subspecies, or even of different species are often identical and therefore can not be used as differentiating markers [3]. Another restriction concerns the accessibility of target sites to the probe in FISH experiments. The presence of secondary structures, or protection of rRNA segments by ribosomal proteins in fixed cells can limit the choice of variable regions as in situ targets for oligonucleotide probes [7,8]. One way to overcome the limitations of in situ identification of bacteria is to use molecules other than rRNA for phylogenetic identification of bacteria, for which nucleotide sequences would be sufficiently divergent to design species specific probes, and which would be more accessible to oligonucleotide probes. For this purpose we investigated the possibility of using tmRNA (also known as 10Sa RNA; [9-11]). This molecule was discovered in E. coli and described as small stable RNA, present at ~1,000 copies per cell [9,11]. The high copy number is an important prerequisite for FISH, which works best with naturally amplified target molecules. In E. coli, tmRNA is encoded by the ssrA gene, is 363 nucleotides long and has properties of tRNA and mRNA [12,13]. tmRNA was shown to be involved in the degradation of truncated proteins: the tmRNA associates with ribosomes stalled on mRNAs lacking stop codons, finally resulting in the addition of a C-terminal peptide tag to the truncated protein. The peptide tag directs the abnormal protein to proteolysis [14,15]. 165 tmRNA sequences have so far (August 2001; The tmRNA Website: http://www.indiana.edu/~tmrna/) been determined [16,17]. The tmRNA is likely to be present in all bacteria and has also been found in algae chloroplasts, the cyanelle of Cyanophora paradoxa and the mitochondrion of the flagellate Reclinomonas americana[10,17,18]

Avaliação do uso de materiais porosos na perda de transmissão de painéis duplos

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia Mecânica, Florianópolis, 2016.Painéis multicamadas são amplamente utilizados nas indústrias automotiva e aeroespacial. Sob a perspectiva vibroacústica, esses setores atuam no aprimoramento desses painéis através do aumento da perda de trans-missão sonora com o intuito de proporcionar maior conforto acústico aos seus consumidores. Neste trabalho, o objeto de estudo é um painel duplo composto por duas placas lisas e sem reforços, preenchido com material poroso. O principal objetivo desta dissertação é analisar diferentes materiais porosos na configuração citada. O estudo parte de uma revisão bibliográfica e, em seguida, é realizada a caracterização dos materiais porosos, na qual foi utilizado um método inverso para a obtenção dos parâmetros macroscópicos. A primeira investigação dos materiais porosos na perda de transmissão é realizada com o auxílio do método da matriz de transferência. A partir dessa abordagem, são analisados os modelos de propagação em meios porosos, os parâmetros caracterizadores, as condições de contorno e propagação em meios estratificados. Ao final dessas análises, o método da matriz de transferência é aplicado na obtenção de configurações otimizadas considerando diferentes materiais dispostos em camadas. Para analisar os oito materiais disponíveis em uma condição mais realista, são realizados testes de perda de transmissão em câmaras reverberantes. Os resultados são comparados com a modelagem em SEA (Statistical Energy Analysis), na qual os materiais porosos são representados através de um modelo de fluido equivalente em que a estrutura do material é considerada flexível. Os resultados em SEA apresentam boa concordância com os experimentais autenticando, além do modelo SEA, o modelo de propagação em meios porosos e os parâmetros obtidos pelo método inverso. Observou-se nos resultados experimentais de perda de transmissão uma forte influência dos vazamentos. As análises indicam, ainda, que a resistividade ao fluxo atua como parâmetro de maior sensibilidade na perda de transmissão.Abstract : Multilayered panels are widely used in automotive and aerospace industry. From the vibroacoustic perspective, these industries improve the multilayered panels by increasing the sound transmission loss in order to provide more acoustic comfort to consumers. In this work, the object of study is a double panel composed of two flat plates without reinforcement filled with porous materials. The main objective of this work is to analyze different porous materials in the aforementioned configuration. The study starts of the literature review, it is then performed the characterization of porous materials, in which an inverse method was used to obtain the macroscopic parameters. The first investigation of porous materials in the transmission loss is performed with the transfer matrix method. This approach is applied to evaluate the models of propagation in the porous media, materials properties, boundary conditions and propagation in the stratified media. At the end, the transfer matrix method for optimizing acoustical linings is applied to multilayered panels. In sequence, transmission loss tests are performed in reverberation rooms in order to analyze eight materials in a more realistic condition. The results are compared with statistical energy analysis (SEA) model, in which the porous materials are represented using the equivalent fluid model considering the porous materials as flexible material structure. The SEA results exhibits good agreement with experimental results validating also the propagation model in porous media and the parameters obtained by the inverse method. It was observed in the experimental results of transmission loss a strong influence of the leaks. The analyzis also shows that the flow resistivity is the stronger parameter in determining the transmission loss

Positive role of cell wall anchored proteinase PrtP in adhesion of lactococci

<p>Abstract</p> <p>Background</p> <p>The first step in biofilm formation is bacterial attachment to solid surfaces, which is dependent on the cell surface physico-chemical properties. Cell wall anchored proteins (CWAP) are among the known adhesins that confer the adhesive properties to pathogenic Gram-positive bacteria. To investigate the role of CWAP of non-pathogen Gram-positive bacteria in the initial steps of biofilm formation, we evaluated the physico-chemical properties and adhesion to solid surfaces of <it>Lactococcus lactis</it>. To be able to grow in milk this dairy bacterium expresses a cell wall anchored proteinase PrtP for breakdown of milk caseins.</p> <p>Results</p> <p>The influence of the anchored cell wall proteinase PrtP on microbial surface physico-chemical properties, and consequently on adhesion, was evaluated using lactococci carrying different alleles of <it>prtP</it>. The presence of cell wall anchored proteinase on the surface of lactococcal cells resulted in an increased affinity to solvents with different physico-chemical properties (apolar and Lewis acid-base solvents). These properties were observed regardless of whether the PrtP variant was biologically active or not, and were not observed in strains without PrtP. Anchored PrtP displayed a significant increase in cell adhesion to solid glass and tetrafluoroethylene surfaces.</p> <p>Conclusion</p> <p>Obtained results indicate that exposure of an anchored cell wall proteinase PrtP, and not its proteolytic activity, is responsible for greater cell hydrophobicity and adhesion. The increased bacterial affinity to polar and apolar solvents indicated that exposure of PrtP on lactococcal cell surface could enhance the capacity to exchange attractive van der Waals interactions, and consequently increase their adhesion to different types of solid surfaces and solvents.</p

Surface-Related Features and Virulence Among Acinetobacter baumannii Clinical Isolates Belonging to International Clones I and II

Acinetobacter baumannii currently represents one of the most important nosocomial infection agent due to its multidrug-resistance and a propensity for the epidemic spread. The A. baumannii strains belonging to the international clonal lineages I (IC I) and II (IC II) are associated with the hospital outbreaks and a high virulence. However, the intra and inter lineage-specific features of strains belonging to these most worldwide spread A. baumannii clones are not thoroughly explored. In this study we have investigated a set of cell surface-related features of A. baumannii IC I (n = 20) and IC II (n = 16) lineage strains, representing 30 distinct pulsed-field gel electrophoresis types in the collection of clinical isolates obtained in Lithuanian tertiary care hospitals. We show that A. baumannii IC II strains are non-motile, do not form pellicle and display distinct capsular polysaccharide profile compared with the IC I strains. Moreover, in contrast to the overall highly hydrophobic IC I strains, IC II strains showed a greater variation in cell surface hydrophobicity. Within the IC II lineage, hydrophilic strains demonstrated reduced ability to form biofilm and adhere to the abiotic surfaces, also possessed twofold thicker cell wall and exhibited higher resistance to desiccation. Furthermore, these strains showed increased adherence to the lung epithelial cells and were more virulent in nematode and mouse infection model compared with the hydrophobic IC II strains. According to the polymerase chain reaction-based locus-typing, the reduction in hydrophobicity of IC II strains was not capsule or lipooligosaccharide locus type-dependent. Hence, this study shows that the most widespread A. baumannii clonal lineages I and II markedly differ in the series of cell surface-related phenotypes including the considerable phenotypic diversification of IC II strains at the intra-lineage level. These findings suggest that the genotypically related A. baumannii strains might evolve the features which could provide an advantage at the specific conditions outside or within the host

A moonlighting role for LysM peptidoglycan binding domains underpins Enterococcus faecalis daughter cell separation

Control of cell size and morphology is of paramount importance for bacterial fitness. In the opportunistic pathogen Enterococcus faecalis, the formation of diplococci and short cell chains facilitates innate immune evasion and dissemination in the host. Minimisation of cell chain size relies on the activity of a peptidoglycan hydrolase called AtlA, dedicated to septum cleavage. To prevent autolysis, AtlA activity is tightly controlled, both temporally and spatially. Here, we show that the restricted localization of AtlA at the septum occurs via an unexpected mechanism. We demonstrate that the C-terminal LysM domain that allows the enzyme to bind peptidoglycan is essential to target this enzyme to the septum inside the cell before its translocation across the membrane. We identify a membrane-bound cytoplasmic protein partner (called AdmA) involved in the recruitment of AtlA via its LysM domains. This work reveals a moonlighting role for LysM domains, and a mechanism evolved to restrict the subcellular localization of a potentially lethal autolysin to its site of action

Structural and evolutionary classification of Type II restriction enzymes based on theoretical and experimental analyses

For a very long time, Type II restriction enzymes (REases) have been a paradigm of ORFans: proteins with no detectable similarity to each other and to any other protein in the database, despite common cellular and biochemical function. Crystallographic analyses published until January 2008 provided high-resolution structures for only 28 of 1637 Type II REase sequences available in the Restriction Enzyme database (REBASE). Among these structures, all but two possess catalytic domains with the common PD-(D/E)XK nuclease fold. Two structures are unrelated to the others: R.BfiI exhibits the phospholipase D (PLD) fold, while R.PabI has a new fold termed ‘half-pipe’. Thus far, bioinformatic studies supported by site-directed mutagenesis have extended the number of tentatively assigned REase folds to five (now including also GIY-YIG and HNH folds identified earlier in homing endonucleases) and provided structural predictions for dozens of REase sequences without experimentally solved structures. Here, we present a comprehensive study of all Type II REase sequences available in REBASE together with their homologs detectable in the nonredundant and environmental samples databases at the NCBI. We present the summary and critical evaluation of structural assignments and predictions reported earlier, new classification of all REase sequences into families, domain architecture analysis and new predictions of three-dimensional folds. Among 289 experimentally characterized (not putative) Type II REases, whose apparently full-length sequences are available in REBASE, we assign 199 (69%) to contain the PD-(D/E)XK domain. The HNH domain is the second most common, with 24 (8%) members. When putative REases are taken into account, the fraction of PD-(D/E)XK and HNH folds changes to 48% and 30%, respectively. Fifty-six characterized (and 521 predicted) REases remain unassigned to any of the five REase folds identified so far, and may exhibit new architectures. These enzymes are proposed as the most interesting targets for structure determination by high-resolution experimental methods. Our analysis provides the first comprehensive map of sequence-structure relationships among Type II REases and will help to focus the efforts of structural and functional genomics of this large and biotechnologically important class of enzymes

Kai galima žaisti diskursais: studija apie rusinimo politiką Lietuvoje ir Baltarusijoje pirmajame dešimtmetyje po 1863 m. sukilimo : recenzija

Recenzijoje aptariama Dariaus Staliūno monografija „Rusinimas: Lietuva ir Baltarusija po 1863 metų“ (Vilnius, 2009). Apžvelgiamos knygos leidybinės aplinkybės, temos aktualumas, turinys ir leidžiamasi į polemiką su autoriaus mintimis. Ši monografija – tai 2007 metais anglų kalba išleistos D. Staliūno knygos „Making Russians: Meaning and Practice of Russification in Lithuania and Belarus after 1863“ vertimas į lietuvių kalbą. Monografijoje apžvelgiama ir analizuojama XIX a. 7 deš. – 8 deš. pradžios Rusijos imperijos tautinė politika Šiaurės Vakarų krašte. Pabrėžiamas monografijos teksto originalo ir vertimo bei šios knygos skaitytojų auditorijos santykis. Monografija yra savarankiškas ir originalus tyrimas, grindžiamas pirminių šaltinių analize, kurios tikslas prisidėti prie tarptautinėje bei šiuolaikinės Rusijos istoriografijoje tebediskutuojamo klausimo apie Rusijos imperijos valdžios tautinės politikos apskritai ir Šiaurės Vakarų krašte atskirai pobūdį: ar tokia politika, kuri vadinamajame Šiaurės Vakarų krašte tyrinėjamu laikotarpiu oficialiai vadinta rusinimu, siekta kitataučių asimiliacijos, ar tik politinės ir kultūrinės integracijos. Istorinė medžiaga yra analizuojama per sociologinių sąvokų – asimiliacija, akultūracija ir integracija – prizmę, o tyrimo objektas aprėpia visą vadinamąjį Šiaurės Vakarų kraštą. Valdžios tautinė politika nagrinėjama lenkų, lietuvių, latvių, baltarusių bei žydų, taip pat konfesinių grupių – katalikų, stačiatikių, judėjų ir protestantų – atžvilgiu, tačiau vietomis pritrūksta gilesnių refleksijų, atidesnio santykio su istoriografija

Klasikinė istorijos studija : apie naujausią Vytauto Merkio knygą : recenzija

Darbe recenzuojama Vytauto Merkio knyga "Tautiniai santykiai Vilniaus vyskupijoje 1798 - 1918 m.". Jos tyrimo objektas yra tautiniai santykiai ir etnosocialiniai procesai XIX amžiuje. Tiriama jų raida ir kaita Rytų Lietuvoje. Ši teritorija siejama su Vilniaus vyskupija. Knygos autorius heliocentrinės sistemos modelį pritaikė savo tyrimo koncepcijai, pagal kurią Katalikų Bažnyčia yra šios sistemos centras - "Saulė", o trys katalikiškos tautos: lenkai, liaudinė lietuvių tauta ir tuteišių etnosas yra "planetos", skriejančios aplink ją. Tiriama jų tiesioginė tarpusavio sąveika ir sąveika per tarpininkę - Katalikų Bažnyčią. Šiems santykiams didelės įtakos turėjo imperijos politinė ir etnoreliginė "gravitacija". Po pirmojo pasaulinio karo Rytų Lietuvos regionas atsidūrė ypatingai komplikuotoje padėtyje. Šis darbas - pirmas itin išsamus ir kompleksiškas tyrimas tokia tematika. Knygoje puikiai dera konceptualumas, analitiškumas, faktografija bei įvykių pasakojimas. Išleista monografija tapo istoriografijos klasika, svariu indėliu tiek į Lietuvos, tiek į tarptautinę istoriografiją. Reikšminiai žodžiai: Istoriografija; Vilniaus vyskupija; Vytautas MerkysThe study reviews Vytautas Merkys’s book “Tautiniai Santykiai Vilniaus Vyskupijoje 1798 - 1918 m.”. The object of the study in the book is the national relations and the ethno-social processes in the 19th century, as well as their development in the Eastern Lithuania. The territory is related to the Vilnius bishopric. The author of the book applies the model of the heliocentric system to the concept of his study, according to which, the Catholic Church is the center of the system, i. e. the “Sun” and the three Catholic nations: the Polish, the folk Lithuanians and the local Lithuanian Polish ethnos are the “planets”, revolving around it. The mutual relations between the nations both directly and via an intermediary, i. e. the Catholic Church, are examined. The relations were largely influenced by the political and ethno-religious “gravity” of the Russian Empire. After the WWI the Eastern Lithuanian region found itself in an especially complicated situation. The study is the first comprehensive and complex analysis of the topic. Conceptuality, analysis, factography and narration of events are perfectly matched in the book. The published monograph became a classic study of historiography and a ponderous contribution into both Lithuanian and international historiography