Role of LrpC from Bacillus subtilis in DNA transactions during DNA repair and recombination

Bacillus subtilis LrpC is a sequence-independent DNA-binding and DNA-bending protein, which binds both single-stranded (ss) and double-stranded (ds) DNA and facilitates the formation of higher order protein–DNA complexes in vitro. LrpC binds at different sites within the same DNA molecule promoting intramolecular ligation. When bound to separate molecules, it promotes intermolecular ligation, and joint molecule formation between a circular ssDNA and a homologous ssDNA-tailed linear dsDNA. LrpC binding showed a higher affinity for 4-way (Holliday) junctions in their open conformation, when compared with curved dsDNA. Consistent with these biochemical activities, an lrpC null mutant strain rendered cells sensitive to DNA damaging agents such as methyl methanesulfonate and 4-nitroquinoline-1-oxide, and showed a segregation defect. These findings collectively suggest that LrpC may be involved in DNA transactions during DNA repair and recombination

Purification and In Vitro Activity of Mitochondria Targeted Nitrogenase Cofactor Maturase NifB

Active NifB is a milestone in the process of engineering nitrogen fixing plants. NifB is an extremely O2-sensitive S-adenosyl methionine (SAM)?radical enzyme that provides the key metal cluster intermediate (NifB-co) for the biosyntheses of the active-site cofactors of all three types of nitrogenases. NifB and NifB-co are unique to diazotrophic organisms. In this work, we have expressed synthetic codon-optimized versions of NifB from the ?-proteobacterium Azotobacter vinelandii and the thermophilic methanogen Methanocaldococcus infernus in Saccharomyces cerevisiae and in Nicotiana benthamiana. NifB proteins were targeted to the mitochondria, where O2 consumption is high and bacterial-like [Fe-S] cluster assembly operates. In yeast, NifB proteins were co-expressed with NifU, NifS, and FdxN proteins that are involved in NifB [Fe?S] cluster assembly and activity. The synthetic version of thermophilic NifB accumulated in soluble form within the yeast cell, while the A. vinelandii version appeared to form aggregates. Similarly, NifB from M. infernus was expressed at higher levels in leaves of Nicotiana benthamiana and accumulated as a soluble protein while A. vinelandii NifB was mainly associated with the non-soluble cell fraction. Soluble M. infernus NifB was purified from aerobically grown yeast and biochemically characterized. The purified protein was functional in the in vitro FeMo-co synthesis assay. This work presents the first active NifB protein purified from a eukaryotic cell, and highlights the importance of screening nif genes from different organisms in order to sort the best candidates to assemble a functional plant nitrogenase

Estudio de proteínas que intervienen en procesos de recombinación genética en bacterias: LrpC y Hbsu de Bacillus subtilis, y las proteínas Beta y del plásmido pSM19035 de Streptococcus pyogenes

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular, Fecha de lectura 25-04-200

Identification of SUMO target by a novel proteomic approach in plants

33 p.-3 tab.4 fig.Post-translational modifications (PTMs) chemically and physically alter the properties of proteins, including their folding, subcellular localization, stability, activity, and consequently their function.In spite of their relevance, studies on PTMs in plants are still limited. Small Ubiquitin-like Modifier(SUMO) modification regulates several biological processes by affecting protein-protein interactions,or changing the subcellular localizations of the target proteins. Here, we describe a novel proteomic approach to identify SUMO targets that combines 2-D liquid chromatography, immunodetection, and mass spectrometry (MS) analyses. We have applied this approach to identify nuclear SUMO targets in response to heat shock. Using a bacterial SUMOylation system, we validated that some of the targets identified here are, in fact, labeled with SUMO1. Interestingly, we found that GIGANTEA (GI), a photoperiodic-pathway protein, is modified with SUMO in response to heat shock both in vitro and in vivo.This work was supported by the grants S-GEN-0191-2006 (CAM) and BIO2007-62517 (MEC), CSD-2007-00057, and BIO2011-28184-C02-01 to J. C. P., and S-GEN-0191-2006 (CAM), BIO2007-65284 (MEC) and GEN2006-27787-E (MEC) to J. S. G. L. T. was supported by a postdoctoral contract (Comunidad de Madrid).Peer reviewe

Biosynthesis of cofactor-activatable iron-only nitrogenase in Saccharomyces cerevisiae

11 Pág. Centro de Biotecnología y Genómica de Plantas (CBGP)Engineering nitrogenase in eukaryotes is hampered by its genetic complexity and by the oxygen sensitivity of its protein components. Of the three types of nitrogenases, the Fe-only nitrogenase is considered the simplest one because its function depends on fewer gene products than the homologous and more complex Mo and V nitrogenases. Here, we show the expression of stable Fe-only nitrogenase component proteins in the low-oxygen mitochondria matrix of S. cerevisiae. As-isolated Fe protein (AnfH) was active in electron donation to NifDK to reduce acetylene into ethylene. Ancillary proteins NifU, NifS and NifM were not required for Fe protein function. The FeFe protein existed as apo-AnfDK complex with the AnfG subunit either loosely bound or completely unable to interact with it. Apo-AnfDK could be activated for acetylene reduction by the simple addition of FeMo-co in vitro, indicating preexistence of the P-clusters even in the absence of coexpressed NifU and NifS. This work reinforces the use of Fe-only nitrogenase as simple model to engineer nitrogen fixation in yeast and plant mitochondria.This work was supported by the Bill and Melinda Gates Foundation Grant OPP1143172 to LMR.Peer reviewe

The Arabidopsis Cell Cycle F-Box Protein SKP2A Binds to Auxin[C][W]

This work shows that auxin binds to the cell cycle F-box SKP2A to regulate its proteolysis and DPPB and E2FC degradation. Auxin seems to regulate the interaction between SKP2A and DPB. Mutant SKP2A proteins that do not bind auxin are unable to interact with DPB and to promote cell division. These findings provide evidence that SKP2A directly connects auxin and cell division

Use of synthetic biology tools to optimize the production of active nitrogenase Fe protein in chloroplasts of tobacco leaf cells

The generation of nitrogen fixing crops is considered a challenge that could lead to a new agricultural ‘green’ revolution. Here, we report the use of synthetic biology tools to achieve and optimize the production of active nitrogenase Fe protein (NifH) in the chloroplasts of tobacco plants. Azotobacter vinelandii nitrogen fixation genes, nifH, M, U and S, were re‐designed for protein accumulation in tobacco cells. Targeting to the chloroplast was optimized by screening and identifying minimal length transit peptides performing properly for each specific Nif protein. Putative peptidyl‐prolyl cis‐trans isomerase NifM proved necessary for NifH solubility in the stroma. Purified NifU, a protein involved in the biogenesis of NifH [4Fe‐4S] cluster, was found functional in NifH reconstitution assays. Importantly, NifH purified from tobacco chloroplasts was active in the reduction of acetylene to ethylene, with the requirement of nifU and nifS co‐expression. These results support the suitability of chloroplasts to host functional nitrogenase proteins, paving the way for future studies in the engineering of nitrogen fixation in higher plant plastids and describing an optimization pipeline that could also be used in other organisms and in the engineering of new metabolic pathways in plastids