Functional dissection of the conjugative coupling protein TrwB

de Paz H.D., Larrea D., Zunzunegui S., Dehio C., de la Cruz F., Llosa M

Abstract

The conjugative coupling protein TrwB is responsible for connecting the relaxosome to the type IV secretion system during conjugative DNA transfer of plasmid R388. It is directly involved in transport of the relaxase TrwC, and it displays an ATPase activity probably involved in DNA pumping. We designed a conjugation assay in which the frequency of DNA transfer is directly proportional to the amount of TrwB. A collection of point mutants was constructed in the TrwB cytoplasmic domain on the basis of the crystal structure of TrwBN70, targeting the nucleotide triphosphate (NTP)-binding region, the cytoplasmic surface, or the internal channel in the hexamer. An additional set of transfer-deficient mutants was obtained by random mutagenesis. Most mutants were impaired in both DNA and protein transport. We found that the integrity of the nucleotide binding domain is absolutely required for TrwB function, which is also involved in monomer-monomer interactions. Polar residues surrounding the entrance and inside the internal channel were important for TrwB function and may be involved in interactions with the relaxosomal components. Finally, the N-terminal transmembrane domain of TrwB was subjected to random mutagenesis followed by a two-hybrid screen for mutants showing enhanced protein-protein interactions with the related TrwE protein of Bartonella tribocorum. Several point mutants were obtained with mutations in the transmembranal helices: specifically, one proline from each protein may be the key residue involved in the interaction of the coupling protein with the type IV secretion apparatus.

http://dx.doi.org/10.1002/bies.200900164

The Conjugative DNA Translocase TrwB Is a Structure-specific DNA-binding Protein

Matillla Inmaculada., Alfonso Carlos., Rivas Germán., Bolt Edward L., de la Cruz Fernando., Cabezon Elena.

Abstract

TrwB is a DNA-dependent ATPase involved in DNA transport during bacterial conjugation. The protein presents structural similarity to hexameric molecular motors such as F₁-ATPase, FtsK, or ring helicases, suggesting that TrwB also operates as a motor, using energy released from ATP hydrolysis to pump single-stranded DNA through its central channel. In this work, we have carried out an extensive analysis with various DNA substrates to determine the preferred substrate for TrwB. Oligonucleotides with G-rich sequences forming G4 DNA structures were the optimal substrates for TrwB ATPase activity. The protein bound with 100-fold higher affinity to G4 DNA than to single-stranded DNA of the same sequence. Moreover, TrwB formed oligomeric protein complexes only with oligonucleotides presenting such a G-quadruplex DNA structure, consistent with stoichiometry of six TrwB monomers to G4 DNA, as demonstrated by gel filtration chromatography and analytical ultracentrifugation experiments. A protein-DNA complex was also formed with unstructured oligonucleotides, but the molecular mass corresponded to one monomer protein bound to one oligonucleotide molecule. Sequences capable of forming G-quadruplex structures are widespread through genomes and are thought to play a biological function in transcriptional regulation. They form stable structures that can obstruct DNA replication, requiring the action of specific helicases to resolve them. Nevertheless, TrwB displayed no G4 DNA unwinding activity. These observations are discussed in terms of a possible role for TrwB in recognizing G-quadruplex structures as loading sites on the DNA. 

http://dx.doi.org/10.1074/jbc.M109.084137

Cantabria Campus Internacional

Cantabria has made the strategic decision to turn its Community into a “region of knowledge” to broaden its horizons of economic, social and cultural development in a context of growing and accelerated globalisation for the different fields of human activity.

The University of Cantabria (UC), which has encouraged and coordinated this collective project, together with the support of the Menéndez Pelayo International University (UIMP), puts forward the design of an International Campus of Excellence, capable of completely fitting in with the University Strategy 2015 to fulfil the objectives of quality, integration and optimisation necessary in higher education in our country.

This project is not presented as an exclusive aim of the University of Cantabria (UC) and the Menéndez Pelayo International University (UIMP), but rather as a Regional Project in which the University plays a central role, social options have been totally involved in supporting a Knowledge society as a distinctive sign of community identity.

Cantabria wants to specialise in Knowledge through planned and agreed transformation which represents the “Cantabria International Campus (Cantabria Campus Internacional)” and which has solid precedents. In the last four years and in collaboration with the Autonomous Government (through the pluri-annual Contract-Programme), the Menéndez Pelayo International University and practically all institutional, business and social agents, the University of Cantabria has been promoting a model of Integral Campus which has spread teaching, research and transmission potential, which now allows it to consider the practical and contrasted viability of the International Campus of Excellence which is presented.

The Cantabria International Campus means the maturing of a project which aims to seek excellence in education and the employability of its graduates; in research; in connection with local, national and global society; in the transfer of knowledge to private and public sectors; and in establishing cooperation networks with universities, scientific centres and companies, Spanish as well as foreign ones.

A University Project which is simultaneously a Community Project is presented; a project that is necessary for the sustainable development of Cantabria, pioneer because of its global commitment in the aggregation of management agents of knowledge, integrating public bodies as well as private ones, an innovator in processes and management through new organisational structures, which look for solutions and results through modernisation, internationalisation and excellence. The institutional commitment of all sectors becomes a reality with the solemn signing of the Agreement in the Parliament of Cantabria, an institution which represents the whole society.

Strategic ObjectivesThe idea of the Cantabria International Campus, which is summarised on the following page, allows it to establish those objectives which will permit it to reach the established vision, starting out from the strategic plans. In the following chart, these objectives are associated with the strategic plans, complying with the generically required objectives, as commented in the stated conditions of the examination.

PLANS AND STRATEGIC OBJECTIVES
HUMAN RESOURCES

To favour staff talent (capability, competitiveness) through educational activities and using necessary resources

INFRASTRUCTURES AND EQUIPMENT

To physically transform the university campus into a high-value architectural and environmental surrounding, adapted to academic needs and services of Cantabria International Campus, integrated in a functional way with its surroundings.

MANAGEMENT

To extend quality management in order to provide efficient Campus services.

FUNCTIONAL

PLANS AND STRATEGIC OBJECTIVES
EDUCATION AND TRAINING

To place education (training) at a level of international excellence through plans of improvement which favour solid education, as well as generating and attracting talent.

RESEARCH

To place basic and applied research at a level of international excellence through support and improvement actions which favour acquiring knowledge and attracting and stimulating talent.

TRANSFER

To favour economic development and development in values in society through an efficient transfer of knowledge acquired from research results.

GUIDELINES BY SECTOR

PLANS AND STRATEGIC OBJECTIVES
STRATEGIC AREAS

To consolidate areas in excellence with a highly-added value and international renown, capable of attracting intellectual and material resources and of creating local development.

http://www.cantabriacampusinternacional.com/en/

Intergenomics Group - Prof. Fernando de la Cruz Laboratory

Cristina Garmendia: El instituto de Biomedicina y Biotecnología de Cantabria es un ejemplo en España

Garmendia cree que el Instituto de Biomedicina es un ejemplo en España 4-5-2010

La investigación se centrará en infecciones, cáncer, enfermedades del sistema nervioso y biotecnología en biocombustibles

«Símbolo de los nuevos referentes que necesita nuestro país en materia de investigación». Así definió ayer la ministra de Ciencia y Tecnología, Cristina Garmendia, al Instituto de Biomedicina y Biotecnología de Cantabria (Ibbtec) cuya sede estará concluida en agosto de 2011. Un proyecto que situará a la región en la vanguardia de la investigación en oncología, inmunología y enfermedades infecciosas, con 25 grupos de trabajo y 200 empleados que aspiran no sólo a nuevos descubrimientos sanitarios sino a su aplicación a la industria. Quiere ser foco de atracción para empresas de base biológica, especialmente las de los sectores farmacéutico, biotecnológico y agroalimentario.
El Ibbtec nació en 2007 y aunque aún no tiene su sede concluida ya cuenta con trece grupos de investigación trabajando en dependencias de la Universidad de Cantabria. Se trata de un centro mixto, de titularidad compartida por el Gobierno de Cantabria, la Universidad y el Centro Superior de Investigaciones Científicas (CSIC).
Garmendia visitó ayer la marcha de las obras -cuya primera piedra colocó hace un año- acompañada del presidente regional, Miguel Ángel Revilla; de la vicepresidenta, Dolores Gorostiaga; y los consejeros de Industria y Sanidad, Juan José Sota y Luis Truan, respectivamente, además del rector de la Universidad de Cantabria, Federico Gutiérrez Solana; el presidente de CSIC, Rafael Rodrigo, y el alcalde de Santander, Íñigo de la Serna, entre otras autoridades.
Ángel Pazos, director del Ibbtec, fue el encargado de explicar sus retos y objetivos, y entre éstos apostó por conjugar dos, el biomédico e investigador, aprovechando la ubicación del centro junto al Hospital Valdecilla y la Facultad de Medicina; y el desarrollo de la aplicación biotecnológica, no sólo en el campo de la medicina sino en lo que denominó, la 'biotecnología blanca', uno de cuyos ejemplos pueden ser los biocombustibles.
Más apoyo institucional
Pazos advirtió que los próximos años serán críticos para determinar el potencial del Instituto que, dijo, «necesitará un fuerte apoyo institucional para su éxito. Estamos sólo al principio y vienen 'malos tiempos para la lírica' pero necesitamos mucho apoyo».
La ministra destacó que para poder competir en el ámbito internacional, los centros tienen que estar especializados y deben contar con la cooperación entre instituciones y abogó por proyectos que garanticen que el conocimiento llegue al tejido productivo. Afirmó que todos los institutos que trabajen en la excelencia científica tendrán el apoyo del Ministerio y felicitó al Gobierno de Cantabria por su especialización y su visión de trabajo en dos ámbitos claves para la economía española: el conocimiento y las energías renovables.
En este sentido, Revilla resaltó que el Ibbtec y el Instituto de Hidráulica Ambiental, ubicado junto a él y cuyas obras también visitaron, son «dos hitos pioneros en España, de vanguardia nacional e internacional. Yo creo que son el camino».
El Ibbtec centrará su actividad en dos líneas de conocimiento: la señalización celular, para profundizar en la comunicación entre las células y su alteración cuando se produce una enfermedad, los estudios moleculares sobre los mecanismos del cáncer, los genes que influyen o la identificación de 'dianas' para posibles tratamientos oncológicos; y la microbiología molecular y celular.
Ésta última está orientada al estudio de las características de los microorganismos y su posible uso como herramientas biotecnológicas, con fines tanto médicos como industriales. El estudio de microorganismos y su capacidad de infectar, y los mecanismos de resistencia a los antibióticos serán otros de los estudios.
De los 13 millones de euros en inversión que supone la puesta en marcha del Ibbtec, el CSIC asume alrededor de 7 millones, destinados a la construcción del edificio, mientras que el Gobierno de Cantabria y la UC correrán con los gastos de equipamiento, con una aportación inicial de tres millones cada uno.

http://www.eldiariomontanes.es/pg060110/portada.html

Numbers on the edges: A simplified and scalable method for quantifying the Gene Regulation Function

1. Raul Fernandez-Lopez, 
2. Irene del Campo, 
3. Raúl Ruiz, 
4. Val Lanza, 
5. Luis Vielva, 
 Fernando de la Cruz
6. 1. Abstract

      
      The gene regulation function (GRF) provides an operational description of a promoter behavior as a function of the concentration of one of its transcriptional regulators. Behind this apparently trivial definition lies a central concept in biological control: the GRF provides the input/output relationship of each edge in a transcriptional network, independently from the molecular interactions involved. Here we discuss how existing methods allow direct measurement of the GRF, and how several trade-offs between scalability and accuracy have hindered its application to relatively large networks. We discuss the theoretical and technical requirements for obtaining the GRF. Based on these requirements, we introduce a simplified and easily scalable method that is able to capture the significant parameters of the GRF. The GRF is able to predict the behavior of a simple genetic circuit, illustrating how addressing the quantitative nature of gene regulation substantially increases our comprehension on the mechanisms of gene control.

      
      

      
      

      http://dx.doi.org/10.1002/bies.200900164

1. 
   

Relaxase DNA binding and cleavage are two distinguishable steps in conjugative DNA processing that involve different sequence elements of the nic site

Lucas M., González-Pérez B., Cabezas M., Moncalian G., Rivas G., de la Cruz F.

J. Biol. Chem. 2010, 285 (12), 8918-8926

TrwC, the relaxase of plasmid R388, catalyzes a series of concerted DNA cleavage and strand transfer reactions on a specific site (nic) of its origin of transfer (oriT). nic contains the cleavage site and an adjacent inverted repeat (IR₂). Mutation analysis in the nic region indicated that recognition of the IR₂ proximal arm and the nucleotides located between IR₂ and the cleavage site were essential for supercoiled DNA processing, as judged either by in vitro nic cleavage or by mobilization of a plasmid containing oriT. Formation of the IR₂ cruciform and recognition of the distal IR₂ arm and loop were not necessary for these reactions to take place. On the other hand, IR₂ was not involved in TrwC single-stranded DNA processing in vitro. For single-stranded DNA nic cleavage, TrwC recognized a sequence embracing six nucleotides upstream of the cleavage site and two nucleotides downstream. This suggests that TrwC DNA binding and cleavage are two distinguishable steps in conjugative DNA processing and that different sequence elements are recognized by TrwC in each step. IR₂-proximal arm recognition was crucial for the initial supercoiled DNA binding. Subsequent recognition of the adjacent single-stranded DNA binding site was required to position the cleavage site in the active center of the protein so that the nic cleavage reaction could take place.

Aislan la proteína que produce la resistencia de las bacterias a los antibióticos

Un trabajo de investigación de la Facultad de Ciencia y Tecnología de la Universidad del País Vasco (UPV) ha logrado aislar la proteína 'acopladora TrwB', el canal de intercambio de información usado por las bacterias, lo que permitirá desarrollar estrategias contra la resistencia de las bacterias a los antibióticos.


Según ha informado en una nota de prensa la UPV, la tesis que ha permitido aislar esta proteína ha sido realizada por Ana Julia Vecino en el departamento de Bioquímica y Biología Molecular de la citada facultad y en la Unidad de Biofísica, del Centro Mixto CSIC-UPV.

En dicha tesis se ha podido estudiar las características de TrwB en un ambiente similar al natural y obtener información sobre la proteína muy aproximada al funcionamiento de la misma en la bacteria.

"El trabajo aporta información relevante que nos permite estar más cerca de conocer el mecanismo de la conjugación bacteriana y por consiguiente de poder desarrollar estrategias contra la diseminación de la resistencia a antibióticos entre bacterias", ha afirmado Itzi Alkorta, directora de la tesis junto al profesor Fernando de la Cruz.


La conjunción bacteriana es un proceso mediante el cual una bacteria es capaz de transmitir una molécula de DNA (pásmido conjugativo) a otra, mediante un proceso que requiere el contacto físico entre ambas. Este proceso, se explica desde la UPV, permite que las bacterias intercambien información genética útil para hacer frente a su entorno y adaptarse al medio.

Entre la información compartida por las bacterias se encuentra aquella que les permite desarrollar la resistencia a distintos antibióticos y por ello, la conjugación bacteriana es el principal responsable de la diseminación de la resistencia a antibióticos que presentas las bacterias y que en las últimas décadas se ha convertido en uno de los mayores problemas de salud pública.

El trabajo de investigación ha sido subvencionado con una beca predoctoral del Gobierno Vasco y por programas de investigación de la Diputación de Vizcaya y el Ministerio de Educación.

http://noticias.terra.es/2010/genteycultura/0104/actualidad/aislan-la-proteina-que-produce-la-resistencia-de-las-bacterias-a-los-antibioticos.aspx

Aislan la proteína que produce la resistencia de las bacterias a los antibióticos

Un trabajo de investigación de la Facultad de Ciencia y Tecnología de la Universidad del País Vasco (UPV) ha logrado aislar la proteína 'acopladora TrwB', el canal de intercambio de información usado por las bacterias, lo que permitirá desarrollar estrategias contra la resistencia de las bacterias a los antibióticos.

Según ha informado en una nota de prensa la UPV, la tesis que ha permitido aislar esta proteína ha sido realizada por Ana Julia Vecino en el departamento de Bioquímica y Biología Molecular de la citada facultad y en la Unidad de Biofísica, del Centro Mixto CSIC-UPV.

En dicha tesis se ha podido estudiar las características de TrwB en un ambiente similar al natural y obtener información sobre la proteína muy aproximada al funcionamiento de la misma en la bacteria.

'El trabajo aporta información relevante que nos permite estar más cerca de conocer el mecanismo de la conjugación bacteriana y por consiguiente de poder desarrollar estrategias contra la diseminación de la resistencia a antibióticos entre bacterias', ha afirmado Itzi Alkorta, directora de la tesis junto al profesor Fernando de la Cruz.

La conjunción bacteriana es un proceso mediante el cual una bacteria es capaz de transmitir una molécula de DNA (pásmido conjugativo) a otra, mediante un proceso que requiere el contacto físico entre ambas. Este proceso, se explica desde la UPV, permite que las bacterias intercambien información genética útil para hacer frente a su entorno y adaptarse al medio.

Entre la información compartida por las bacterias se encuentra aquella que les permite desarrollar la resistencia a distintos antibióticos y por ello, la conjugación bacteriana es el principal responsable de la diseminación de la resistencia a antibióticos que presentas las bacterias y que en las últimas décadas se ha convertido en uno de los mayores problemas de salud pública.

El trabajo de investigación ha sido subvencionado con una beca predoctoral del Gobierno Vasco y por programas de investigación de la Diputación de Vizcaya y el Ministerio de Educación.

http://noticias.terra.es/2010/genteycultura/0104/actualidad/aislan-la-proteina-que-produce-la-resistencia-de-las-bacterias-a-los-antibioticos.aspx

Conjugative DNA metabolism in Gram-negative bacteria

Fernando De La Cruz, Laura S. Frost, Richard J. Meyer, Ellen L. Zechner
FEMS Microbiology Reviews.Volume 34, Issue 1, pages 18–40, January 2010

Bacterial conjugation in Gram-negative bacteria is triggered by a signal that connects the relaxosome to the coupling protein (T4CP) and transferosome, a type IV secretion system. The relaxosome, a nucleoprotein complex formed at the origin of transfer (oriT), consists of a relaxase, directed to the nic site by auxiliary DNA-binding proteins. The nic site undergoes cleavage and religation during vegetative growth, but this is converted to a cleavage and unwinding reaction when a competent mating pair has formed. Here, we review the biochemistry of relaxosomes and ponder some of the remaining questions about the nature of the signal that begins the process.

Workshop in Biomedicine 2010

Coordinator: Prof. Fernando de la Cruz (delacruz@unican.es)
Dates: January 25 to February 5, 2010
Time: 10h to 13h
Place: Aula 11, Faculty of Medicine, University of Cantabria

Invited speakers:
25 January
“Genetics of complex diseases”
1. The Wnt pathway in skeletal disorders. Dr. José Antonio Riancho (Hospital Universitario Marqués de Valdecilla, Santander)
2. Regulatory SNPs and the Genetics of lipid disorders. Dr. José Carlos Rodríguez-Rey (UC

26 January“The plasticity of microbial genomes”
1. The organization and dynamics of the Vibrio cholerae genome
2. Integrons as agents of genome plasticity
Dr. Didier Mazel (Institut Pasteur, Paris)

27 January“New strategies for combating bacterial antibiotic resistance”
1. Antibiotic resistance as a critical problem in public health
2. The biological clues of evolution and spread of antibiotic resistance as targets for intervention.
3. Eco-Evo strategies in the control of antibiotic resistance.
Dr. Fernando Baquero (Hospital Ramón y Cajal, Madrid)

28 January
“Cancer”
1. Tyrosine kinase receptors as therapeutic targets in cancer. Dr. Atanasio Pandiella (CIC, CSIC, Salamanca)
2. Desorganization of cellular differentiation by oncogenic transcription factors. Dr. Javier León (UC)

29 January
(Sto Tomás de Aquino) NO class!

1 February
“The dilemma of sexuality and vegetative reproduction: the case of mucoral fungi”
Dr. Enrique Cerdá-Olmedo (Dept. of Genetics, University of Sevilla)

2 February“Molecular and systemic perspectives on the origins of life. Recent advances in support of the compartment-first scenario”
1. Origin and definition of life
2. Prebiotic membranes
Dr. Kepa Ruiz Mirazo (UPV, San Sebastián)

3 February “The physics behind systems biology”
1. Computational modelling of gene regulatory networks
2. The gene regulation function
Dr. Luis Vielva (UC)

4 February“Bacterial conjugation”
1. DNA processing during conjugation. Dr. Gabriel Moncalian (UC)
2. Type IV protein secretion systems. Dr. Iñaki Arechaga (UC)

5 February“Genomics of Complex Diseases”
1. A historic account of the human genome project
2. Methods of high-thorughput sequencing
3. SNPs in human disease
Dr. Jesús Sainz (IBBTEC, CSIC Santander).