A translocation motif in relaxase TrwC specifically affects recruitment by its conjugative type IV secretion system

Type IV secretion system (T4SS) substrates are recruited through a translocation signal that is poorly defined for conjugative relaxases. The relaxase TrwC of plasmid R388 is translocated by its cognate conjugative T4SS, and it can also be translocated by the VirB/D4 T4SS of Bartonella henselae, causing DNA transfer to human cells. In this work, we constructed a series of TrwC variants and assayed them for DNA transfer to bacteria and human cells to compare recruitment requirements by both T4SSs. Comparison with other reported relaxase translocation signals allowed us to determine two putative translocation sequence (TS) motifs, TS1 and TS2. Mutations affecting TS1 drastically affected conjugation frequencies, while mutations affecting either motif had only a mild effect on DNA transfer rates through the VirB/D4 T4SS of B. henselae. These results indicate that a single substrate can be recruited by two different T4SSs through different signals. The C terminus affected DNA transfer rates through both T4SSs tested, but no specific sequence requirement was detected. The addition of a Bartonella intracellular delivery (BID) domain, the translocation signal for the Bartonella VirB/D4 T4SS, increased DNA transfer up to 4% of infected human cells, providing an excellent tool for DNA delivery to specific cell types. We show that the R388 coupling protein TrwB is also required for this high-efficiency TrwC-BID translocation. Other elements apart from the coupling protein may also be involved in substrate recognition by T4SSs

Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase

This is an open-access article distributed under the terms of the Creative Commons Attribution License.[Background]: Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering. [Methodology/Principal Findings]: We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome. [Conclusions/Significance]: The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells.This work was supported by grant BIO2008-00133 from the Spanish Ministry of Science and Innovation to ML. CGP was a recipient of a predoctoral fellowship from the University of Cantabria, Spain.Peer reviewe

Primary skin fibroblasts as a model of Parkinson's disease

Parkinson's disease is the second most frequent neurodegenerative disorder. While most cases occur sporadic mutations in a growing number of genes including Parkin (PARK2) and PINK1 (PARK6) have been associated with the disease. Different animal models and cell models like patient skin fibroblasts and recombinant cell lines can be used as model systems for Parkinson's disease. Skin fibroblasts present a system with defined mutations and the cumulative cellular damage of the patients. PINK1 and Parkin genes show relevant expression levels in human fibroblasts and since both genes participate in stress response pathways, we believe fibroblasts advantageous in order to assess, e.g. the effect of stressors. Furthermore, since a bioenergetic deficit underlies early stage Parkinson's disease, while atrophy underlies later stages, the use of primary cells seems preferable over the use of tumor cell lines. The new option to use fibroblast-derived induced pluripotent stem cells redifferentiated into dopaminergic neurons is an additional benefit. However, the use of fibroblast has also some drawbacks. We have investigated PARK6 fibroblasts and they mirror closely the respiratory alterations, the expression profiles, the mitochondrial dynamics pathology and the vulnerability to proteasomal stress that has been documented in other model systems. Fibroblasts from patients with PARK2, PARK6, idiopathic Parkinson's disease, Alzheimer's disease, and spinocerebellar ataxia type 2 demonstrated a distinct and unique mRNA expression pattern of key genes in neurodegeneration. Thus, primary skin fibroblasts are a useful Parkinson's disease model, able to serve as a complement to animal mutants, transformed cell lines and patient tissues

Design of Novel Relaxase Substrates Based on Rolling Circle Replicases for Bioconjugation to DNA Nanostructures

During bacterial conjugation and rolling circle replication, HUH endonucleases, respectively known as relaxases and replicases, form a covalent bond with ssDNA when they cleave their target sequence (nic site). Both protein families show structural similarity but limited amino acid identity. Moreover, the organization of the inverted repeat (IR) and the loop that shape the nic site differs in both proteins. Arguably, replicases cleave their target site more efficiently, while relaxases exert more biochemical control over the process. Here we show that engineering a relaxase target by mimicking the replicase target, results in enhanced formation of protein-DNA covalent complexes. Three widely different relaxases, which belong to MOBF, MOBQ and MOBP families, can properly cleave DNA sequences with permuted target sequences. Collaterally, the secondary structure that the permuted targets acquired within a supercoiled plasmid DNA resulted in poor conjugation frequencies underlying the importance of relaxase accessory proteins in conjugative DNA processing. Our results reveal that relaxase and replicase targets can be interchangeable in vitro. The new Rep substrates provide new bioconjugation tools for the design of sophisticated DNA-protein nanostructures.This work was financed by grants BFU2014-55534-C2-1-P from the Spanish Ministry of Economy and Competitiveness and 612146/FP7-ICT- 2013 and 282004/FP7-HEALTH.2011.2.3.1-2 from the European Union Seventh Framework Programme to FC and grant BFU2014-55534-C2-2-P from the Spanish Ministry of Economy and Competitiveness to GM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Exchange of functional domains between a bacterial conjugative relaxase and the integrase of the human adeno-associated virus

Endonucleases of the HUH family are specialized in processing single-stranded DNA in a variety of evolutionarily highly conserved biological processes related to mobile genetic elements. They share a structurally defined catalytic domain for site-specific nicking and strand-transfer reactions, which is often linked to the activities of additional functional domains, contributing to their overall versatility. To assess if these HUH domains could be interchanged, we created a chimeric protein from two distantly related HUH endonucleases, containing the N-terminal HUH domain of the bacterial conjugative relaxase TrwC and the C-terminal DNA helicase domain of the human adeno-associated virus (AAV) replicase and site-specific integrase. The purified chimeric protein retained oligomerization properties and DNA helicase activities similar to Rep68, while its DNA binding specificity and cleaving-joining activity at oriT was similar to TrwC. Interestingly, the chimeric protein could catalyse site-specific integration in bacteria with an efficiency comparable to that of TrwC, while the HUH domain of TrwC alone was unable to catalyze this reaction, implying that the Rep68 C-terminal helicase domain is complementing the TrwC HUH domain to achieve site-specific integration into TrwC targets in bacteria. Our results illustrate how HUH domains could have acquired through evolution other domains in order to attain new roles, contributing to the functional flexibility observed in this protein superfamily.This work was supported by the Medical Research Council (MRC) grant MR/N022890/1 to EH and grant 1001764 to RML; National Institutes of Health (NIH) grant RO1-GM09285 to CRE; Spanish Ministry of Economy and competitiveness (MINECO) grant BIO2013-46414-P to ML and AFM is supported by a Doc.Mobility fellowship from the Swiss National Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Unique Helicase Determinants in the Essential Conjugative TraI Factor from Salmonella enterica Serovar Typhimurium Plasmid pCU1

The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. In this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity

Resolviendo la variabilidad espacial y temporal del aploramiento noroeste ibérico

III Simposio Internacional de Ciencias del Mar, Cádiz, 25-28 de enero de 2012N

Tracing sinking organic matter sources in the NW Iberian upwelling system (NE Atlantic Ocean): comparison between elemental, isotopic and molecular indicators

30 pages, 8 figures, 2 tablesWe studied the seasonal fluctuations in the composition of sinking particulate organic matter (POM) in the only upwelling-affected continental margin of Europe, to improve our understanding of the carbon (C) cycle of such systems. The methods employed targeted the elemental, stable isotope (δ13Coc and δ15N) and molecular (Py-GC-MS) composition. The results showed that the sinking POM in this margin is predominantly of marine origin, with δ15N (5.2 ± 0.3 ‰) and C/N (9.7 ± 1.0) values similar to those of marine plankton, and high abundances of pyrolysis products with isoprenoid structures, N-containing products from proteins (indoles, cyanobenzenes, etc.), and N-acetylated polysaccharides (acetamide, acetamidofurans) from chitin and peptidoglycan. Furthermore, the dataset demonstrates seasonal differences in POM composition under different oceanographic scenarios: i) dominance of most N-compounds, fatty acids and isoprenoid products during highly productive upwelling seasons, and ii) an increase in the relative abundance of linear alkanes/alkenes, phenols, lignin and PAH reflecting terrestrial influence during downwelling periods. In addition, the detection of isoprenoid alkylthiophenes traces (compounds formed under reducing conditions in the sediment) point to resuspended sediment in the trap material due to hydrodynamic effects of currents and waves on the seafloor. The results show that analytical pyrolysis is a useful tool for identifying different types of autochthonous and terrestrial POM, and therefore identification of the sources of sinking POM.Peer reviewe

CALIBERIA_Site_CTD _V1.0

This item is made of 2 files, of which 1 is the dataset in TXT format and the other (Readme .txt) include a small description of the computed variables. Dataset contributed to the Projects RAIA, RAIAco, EXCAPA and CALIBERIAThis dataset contains information about the hydrographic cruises carried out off Cape Silleiro (NW Iberia, Atlantic Ocean) between July 2010 and June 2012. These cruises sampled CALIBERIA station, located at 42º05´ N, 9º23´ W (~350 m depth). A CTD (Seabird 911) equipped with fluorescence and oxygen sensors was used for these hydrographic cruisesThis work has been funded by RAIA: ‘Observatorio oceánico del margen Ibérico’ (INTERREG 2009/2011; 0313/RAIA/E) and RAIA.co: ’Observatorio marino del margén ibérico y del litoral’ (INTERREG 2011/2013; 052/RAIA.co/1E) funded by European Union; EXCAPA: Vertical export of biogenic matter in the coastal upwelling system of NW Iberian Peninsula (Xunta de Galicia 10MDS402013PR) and CALIBERIA: Calibration of several proxies along the NW Iberian margin: Improvement of the paleoceanographic proxies (PTDC/MAR/102045/2008, FCT Portugal)Peer reviewe

CALIBERIA_Site _V1.0

This item is made of 2 files, of which 1 is the dataset in TXT format and the other (Readme .txt) include a small description of the computed variables.-- Dataset contributed to the Projects RAIA, RAIAco, EXCAPA and CALIBERIA.-- Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)From July 2010 to June 2012, 21 hydrographic cruises were carried out off Cape Silleiro (NW Iberia, Atlantic Ocean). These cruises sampled CALIBERIA station, located at 42º05´ N, 9º23´ W (~350 m depth). A CTD (Seabird 911) equipped with fluorescence, oxygen, turbidity and transmittance sensors was used for these hydrographic cruises. Additionally, an oceanographic rosette with 12 Niskin bottles was casted during each cruise to collect discrete seawater samples at different depthsThis work has been funded by RAIA: ‘Observatorio oceánico del margen Ibérico’ (INTERREG 2009/2011; 0313/RAIA/E) and RAIA.co: ’Observatorio marino del margén ibérico y del litoral’ (INTERREG 2011/2013; 052/RAIA.co/1E) funded by European Union; EXCAPA: Vertical export of biogenic matter in the coastal upwelling system of NW Iberian Peninsula (Xunta de Galicia 10MDS402013PR) and CALIBERIA: Calibration of several proxies along the NW Iberian margin: Improvement of the paleoceanographic proxies (PTDC/MAR/102045/2008, FCT Portugal)Peer reviewe