Replicación del plásmido de amplio espectro de huésped pLS1

288 p.-103 fig.-9 tab.En los cerca de treinta años transcurridos desde el descubrimiento de los plásmidos bacterianos, estos elementos extracromosómicos han contribuido notablemente al desarrollo actual de la Genética, la Biología Molecular y la Biotecnología.Las primeras investigaciones llevadas a cabo en biología plasmídica tuvieron como objetivo estudiar los mecanismos de fertilidad conjugacional y resistencia a antibióticos en bacterias entéricas. Los conocimientos aportados por estas investigaciones, junto con el desarrollo de la tecnología del DNA recombinante y de la transformación bacteriana, propiciaron el uso de plásmidos como vectores de clonaje. Aún en la actualidad, los plásmidos parecen representar para muchos biotecnólogos poco más que vectores para el clonaje y expresión de genes de interés, mientras que para algunos investigadores en Biomedicina solo parecen ser molestos elementos de diseminación de resistencias a antibióticos. En contraste con esta visión minimalista, solo un conocimiento profundo de la biología de los plásmidos ha permitido(y permitirá en el futuro solucionar los problemas surgidos en otras áreas de investigación. Así, cuando se emplean plásmidos recombinantes en procesos industriales, la capacidad para regular el número de copias de un replicón (manteniéndolo inicialmente bajo y amplificándolo después) puede incrementar su estabilidad durante la fase de expresión. Desde el punto de vista médico, el conocimiento de la replicación plasmídica puede tener una importante aplicación en los problemas de antibiótico-resistencias mediadas por plásmidos y su transmisión entre bacterias patógenas. Como en un circuito de autopotenciación, los problemas surgidos en diversas áreas de la Biología aplicada han contribuido, a su vez,a estimular la investigación básica sobre los plásmidos. Tal investigación ha sido fundamental para el conocimiento de procesos biológicos claves, como la conjugación, el control de la expresión génica, la topología del DNA, la transposición, la recombinación, la partición de información genética en el momento de la división celular, y un largo etcétera que incluye,por supuesto,la replicación plasmídica y su control, protagonistas principales a lo largo de este trabajo. En este sentido, los plásmidos son más adecuados que los cromosomas para el estudio de la replicación y su control, ya que al ser dispensables para el huésped permiten mucha más libertad genética y bioquímica al investigador.Como la Naturaleza parece ofrecer un número limitado de soluciones a un determinado problema, algunos de los mecanismos encontrados en los plásmidos pueden tener un significado funcional mucho más amplio.Quizás el descubrimiento más general efectuado durante los estudios sobre el control de la replicación plasmídica es que el RNA puede funcionar como un represor que actúa en trans. Este mecanismo no solo se ha encontrado posteriormente en muchos otros circuitos reguladores naturales, sino que ha proporcionado también un medio para introducir circuitos sintéticos de control en microbios, plantas y animales (Inouye y Dudock,1987).Peer reviewe

Chemical synthesis of a fully active transcriptional repressor protein

5 pages, 5 figures.-- PMID: 8197204 [PubMed].-- PMCID: PMC43955.Plasmid pLS1-encoded 45-amino acid transcriptional repressor CopG (formerly RepA) has been chemically synthesized. A one-step purification of the synthetic protein has been developed, which yields high levels of pure protein with low or no contamination of truncated products. We have compared some properties of the chemical CopG protein with those of the biologically purified CopG. The two proteins were indistinguishable in (i) their ability to generate specific protein-DNA complexes, (ii) their capacity to protect a restriction site included within the CopG DNA target, and (iii) in their in vitro capacity to specifically repress synthesis of copG mRNA.Research was financed by Comisión Interministerial de Ciencia y Tecnologia, Grant B1091-0691 to (M.E.) and Grant SAL90-0828 (to R.E.). The Special Actions Program of the Consejo Superior de Investigaciones Cientificas stimulated and partially supported this research.Peer reviewe

Plasmid replicons from Pseudomonas are natural chimeras of functional, exchangeable modules

14 p.-5 figPlasmids are a main factor for the evolution of bacteria through horizontal gene exchange, including the dissemination of pathogenicity genes, resistance to antibiotics and degradation of pollutants. Their capacity to duplicate is dependent on their replication determinants (replicon), which also define their bacterial host range and the inability to coexist with related replicons. We characterize a second replicon from the virulence plasmid pPsv48C, from Pseudomonas syringae pv. savastanoi, which appears to be a natural chimera between the gene encoding a newly described replication protein and a putative replication control region present in the widespread family of PFP virulence plasmids. We present extensive evidence of this type of chimerism in structurally similar replicons from species of Pseudomonas, including environmental bacteria as well as plant, animal and human pathogens. We establish that these replicons consist of two functional modules corresponding to putative control (REx-C module) and replication (REx-R module) regions. These modules are functionally separable, do not show specificity for each other, and are dynamically exchanged among replicons of four distinct plasmid families. Only the REx-C module displays strong incompatibility, which is overcome by a few nucleotide changes clustered in a stem-and-loop structure of a putative antisense RNA. Additionally, a REx-C module from pPsv48C conferred replication ability to a non-replicative chromosomal DNA region containing features associated to replicons. Thus, the organization of plasmid replicons as independent and exchangeable functional modules is likely facilitating rapid replicon evolution, fostering their diversification and survival, besides allowing the potential co-option of appropriate genes into novel replicons and the artificial construction of new replicon specificities.This work was funded by the Spanish Plan Nacional I+D+i grant AGL2014-53242-C2-2-R, fromthe Ministerio de Economía y Competitividad (MINECO), co-financed by the Fondo Europeo de Desarrollo Regional (FEDER). M.A. was supported by an FPI fellowship (reference BES-2012-054016, Ministerio de Ciencia e Innovación/Ministerio de Economía y Competitividad, Spain).Peer reviewe

Nivel de conocimiento sobre el manejo estomatológico del paciente diabético en estudiantes de Estomatología de la uss-2022

La Diabetes viene a ser uno de los problemas de salud pública con más relevancia actualmente, con grandes repercusiones sociales y económicas, además de comprometer la productividad y la calidad de vida de pacientes con esta patología. El objetivo del presente estudio fue determinar el nivel de conocimiento sobre el manejo estomatológico del paciente diabético en alumnos de la escuela de estomatología de la Universidad Señor de Sipán en el 2022. Método y Diseño esta investigación es cuantitativa, diseño no experimental, ya que para realizar esta investigación no existió manipulación de variables, es transversal ya que el cuestionario fue aplicado en un tiempo determinado, es descriptivo ya que se usó un instrumento para la recolección de datos el cual es un cuestionario dirigido a los alumnos de Estomatología de la Universidad particular Señor de Sipán. Resultados se encontró que el nivel de conocimiento en alumnos de la escuela de Estomatología presentó un 58% nivel medio, mientras que un nivel alto un 24% y un nivel bajo con 18%. Conclusión se determinó que el nivel de conocimiento sobre el manejo estomatológico del paciente diabético en alumnos de la escuela de estomatología de la Universidad Señor de Sipán en el 2022 es medio.Tesi

Construction of a plasmid vector based on the pMV158 replicon for cloning and inducible gene expression in Streptococcus pneumoniae

18 p.-4 fig.We report the construction of a plasmid vector designed for regulated gene expression in Streptococcus pneumoniae. The new vector, pLS1ROM, is based on the replicon of the streptococcal promiscuous rolling circle replication (RCR) plasmid pMV158. We inserted the controllable promoter PM of the S. pneumoniaemalMP operon, followed by a multi-cloning site sequence aimed to facilitate the insertion of target genes. The expression from PM is negatively regulated by the transcriptional repressor MalR, which is released from the DNA operator sequence by growing the cells in maltose-containing media. To get a highly regulated expression of the target gene, MalR was provided in cis by inserting the malR gene under control of the constitutive Ptet promoter, which in pMV158 directs expression of the tetL gene. To test the functionality of the system, we cloned the reporter gene gfp from Aequorea victoria, encoding the green fluorescent protein (GFP). Pneumococcal cells harboring the recombinant plasmid rendered GFP fluorescence in a maltose-dependent mode with undetectable background levels in the absence of the inducer. The new vector, pLS1ROM, exhibits full structural and segregational stability and constitutes a valuable tool for genetic manipulation and regulated gene expression in S. pneumoniaeThis study was supported by the Spanish Ministerio de Ciencia e Innovación (Grants CSD2008-00013, INTERMODS to ME; BFU2007-63575 and BFU2010-19597, PNEUMOTALK to GdS) and the European Union (Grant EU-CP223111, CAREPNEUMO to ME)Peer reviewe

Combining modules for versatile and optimal labeling of lactic acid bacteria: Two pMV158-family promiscuous replicons, a pneumococcal system for constitutive or inducible gene expression, and two fluorescent proteins

Labeling of bacterial cells with fluorescent proteins allows tracking the bacteria in competition and interactomic in vivo and in vitro studies. During the last years, a few plasmid vectors have been developed aimed at the fluorescent labeling of specific members of the lactic acid bacteria (LAB), a heterogeneous group that includes microorganisms used in the food industry, as probiotics, or as live vectors for mucosal vaccines. Successful and versatile labeling of a broad range of LAB not only requires a vector containing a promiscuous replicon and a widely recognized expression system for the constitutive or regulated expression of the fluorescence determinant, but also the knowledge of the main features of the entire plasmid/host/fluorescent protein ensemble. By using the LAB model species Lactococcus lactis, we have compared the utility properties of a set of labeling vectors constructed by combining a promiscuous replicon (pMV158 or pSH71) of the pMV158 plasmid family with the gene encoding either the EGFP or the mCherry fluorescent protein placed under control of promoter PX or PM from the pneumococcal mal gene cluster for maltosaccharide uptake and utilization, respectively. Some vectors carrying PM also harbor the malR gene, whose product represses transcription from this promoter, thus enabling maltose-inducible synthesis of the fluorescent proteins. We have determined the plasmid copy number (PCN) and segregational stability of the different constructs, as well as the effect of these features on the fitness and fluorescence intensity of the lactococcal host. Constructs based on the pSH71 replicon had a high copy number (~115) and were segregationally stable. The copy number of vectors based on the pMV158 replicon was lower (~8-45) and varied substantially depending on the genetic context of the plasmid and on the bacterial growth conditions; as a consequence, inheritance of these vectors was less stable. Synthesis of the fluorescent proteins encoded by these plasmids did not significantly decrease the host fitness. By employing inducible expression vectors, the fluorescent proteins were shown to be very stable in this bacterium. Importantly, conditions for accurate quantification of the emitted fluorescence were established based on the maturation times of the fluorescent proteins.Fil: Garay Novillo, Javier Nicolás. Consejo Superior de Investigaciones Científicas; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: García-Morena, Diego. Consejo Superior de Investigaciones Científicas; EspañaFil: Ruiz-Masó, José Ángel. Consejo Superior de Investigaciones Científicas; EspañaFil: Barra, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: del Solar, Gloria. Consejo Superior de Investigaciones Científicas; Españ

Conformational plasticity of RepB, the replication initiator protein of promiscuous streptococcal plasmid pMV158

DNA replication initiation is a vital and tightly regulated step in all replicons and requires an initiator factor that specifically recognizes the DNA replication origin and starts replication. RepB from the promiscuous streptococcal plasmid pMV158 is a hexameric ring protein evolutionary related to viral initiators. Here we explore the conformational plasticity of the RepB hexamer by i) SAXS, ii) sedimentation experiments, iii) molecular simulations and iv) X-ray crystallography. Combining these techniques, we derive an estimate of the conformational ensemble in solution showing that the C-terminal oligomerisation domains of the protein form a rigid cylindrical scaffold to which the N-terminal DNA-binding/catalytic domains are attached as highly flexible appendages, featuring multiple orientations. In addition, we show that the hinge region connecting both domains plays a pivotal role in the observed plasticity. Sequence comparisons and a literature survey show that this hinge region could exists in other initiators, suggesting that it is a common, crucial structural element for DNA binding and manipulation

Conformational plasticity of RepB, the replication initiator protein of promiscuous streptococcal plasmid pMV158

13 p.-7 fig.-1 tab.DNA replication initiation is a vital and tightly regulated step in all replicons and requires an initiator factor that specifically recognizes the DNA replication origin and starts replication. RepB from the promiscuous streptococcal plasmid pMV158 is a hexameric ring protein evolutionary related to viral initiators. Here we explore the conformational plasticity of the RepB hexamer by i) SAXS, ii)sedimentation experiments, iii) molecular simulations and iv) X-ray crystallography. Combining these techniques, we derive an estimate of the conformational ensemble in solution showing that the C-terminal oligomerisation domains of the protein form a rigid cylindrical scaffold to which the N-terminal DNA-binding/catalytic domains are attached as highly flexible appendages, featuring multiple orientations. In addition, we show that the hinge region connecting both domains plays a pivotal role in the observed plasticity. Sequence comparisons and a literature survey show that this hinge region could exists in other initiators, suggesting that it is a common, crucial structural element for DNA binding and manipulation.This study was supported by the Ministerio de Economía y Competitividad (Grants BFU2008-02372/BMC; BFU2011-22588, BFU2014-53550 and Unidad de Excelencia Maria de Maeztu MDM-2014-0435 to MC; BIO2009-10964 and E-SCIENCE to MO;BFU2010-19597, PNEUMOTALK, and CSD2008-00013, INTERMODS, to GdS; Ramón and Cajal subprogramme RYC-2011-09071 to CM), the Generalitat de Catalunya (Grants 2014-SGR1309 to MC and SGR2009-1348 to MO),Fundación Marcelino Botín (MO) and the European Commission (Cooperation Project SILVER, GA No. 260644 to MC and SCALALIFE Project to MO).Peer reviewe

Interactions between the RepB initiator protein of plasmid pMV158 and two distant DNA regions within the origin of replication

Plasmids replicating by the rolling circle mode usually possess a single site for binding of the initiator protein at the origin of replication. The origin of pMV158 is different in that it possesses two distant binding regions for the initiator RepB. One region was located close to the site where RepB introduces the replication-initiating nick, within the nic locus; the other, the bind locus, is 84 bp downstream from the nick site. Binding of RepB to the bind locus was of higher affinity and stability than to the nic locus. Contacts of RepB with the bind and nic loci were determined through high-resolution footprinting. Upon binding of RepB, the DNA of the bind locus follows a winding path in its contact with the protein, resulting in local distortion and bending of the double-helix. On supercoiled DNA, simultaneous interaction of RepB with both loci favoured extrusion of the hairpin structure harbouring the nick site while causing a strong DNA distortion around the bind locus. This suggests interplay between the two RepB binding sites, which could facilitate loading of the initiator protein to the nic locus and the acquisition of the appropriate configuration of the supercoiled DNA substrate