Marta Albareda1, Marie-Andrée Mandrand-Berthelot2, Agnes Rodrigue2, Tomas Ruiz-Argüeso1, Juan. Imperial3, Jose Palacios1 and Belen Brito1. 1 CBGP ...
10th International Hydrogenase Conference
Szeged, Hungary
8th July-12th July, 2013
Biosynthesis of hydrogneases Posters
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Poster Number:18. C haracterization of H up E , a nickel transporter for hydrogenase synthesis in the endosymbiotic bacterium Rhizobium leguminosarum bv. viciae.
Marta Albareda1, Marie-Andrée Mandrand-Berthelot2, Agnes Rodrigue2, Tomas Ruiz-Argüeso1, Juan Imperial3, Jose Palacios1 and Belen Brito1 1
CBGP UPM-INIA, Universidad Politécnica de Madrid, Ca mpus de Montegancedo, M40 km 37.7, 28223 Pozuelo de Alarcón, Madrid, Spain. 2 Université Lyon, F-69622 Lyon, Université Lyon 1, Villeurbanne, INS A de Lyon, F-69621 Villeurbanne, and CNRS UMR5240 Microbiologie, Adaptation et Pathogénie, Lyon, F rance 3 Consejo Superior de Investigaciones Científicas (C S IC) CBGP UPMINIA Ca mpus de Montegancedo, M40 km 37.7, 28223 Pozuelo de Alarcón, Madrid, Spain. Rhizobium leguminosarumbv. viciae (Rlv) UPM791 synthesizes a NiFe hydrogenase that allows the recycling of hydrogen produced by nitrogenase. In this bacterium, the synthesis of hydrogenase requires the participation of 18 proteins encoded in the hup/hyp gene cluster hupS LC D E F G HIJKhypAB F C D EX located in the symbiotic plasmid. One of the genes of this cluster, hupE , encodes a protein harboring a canonical signal peptide and six potential transmembrane domains. Previous hydrogenase activity and Ni63 transport assays demonstrated that HupE is a nickel transporter that provides this metal for the synthesis of hydrogenase in Rlv UPM791 free-living cells and lentil bacteroids, and led to the identification of two histidine-rich motifs required for HupE functionality (Brito et al., 2010). A similar protein (UreJ) is present in gene clusters involved in urease synthesis in other bacteria. HupE/UreJ constitutes a family of single permeases widely distributed in bacteria. The characterization of kinetic and functional properties of Rlv UPM791 HupE is being carried out through heterologous expression of HupE in a nik-deficient Escherichia coli background. The specificity of this transporter for Ni2+ is being determined in competition assays with other divalent metal ions. A HupE variant lacking the N-terminal periplasmic region of the mature protein has been constructed to ascertain the potential role of this domain in ion scavenging and concentration around the transporter. The relevance of other residues conserved in members of the HupE/UreJ family is being tested through the analysis of site-specific mutants. The results of the on-going work will be presented and discussed. A cknowledgement This work was supported by Spain´s MICINN project RHIZOMETAL (BIO2010-15301) and by France PICS project (SYMBIONI n°5992). References Brito B, Prieto RI, Cabrera E, Mandrand-Berthelot MA, Imperial J, Ruiz-Argueso T, Palacios JM (2010) Rhizobium leguminosarum hupE encodes a nickel transporter required for hydrogenase activity. J Bacteriol 2010, 192:925-935.
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