Our work is focused on the study of genetics, physiology and biochemistry of traits involved in the establishment and functioning of the Rhizobium-legume symbiotic association. Our present work falls into three main research lines:

1.- Molecular biology of Rhizobium hydrogenases.

Hydrogenases from diazotrophic bacteria contain Ni and Fe at their active site. These enzymes oxidize hydrogen generated by nitrogenase during the nitrogen fixation process. Our interest is focused on the elucidation of the NiFe hydrogenase biosynthetic mechanism and on the development of the biotechnological potential of hydrogen recycling for the improvement of nitrogen fixation by legumes. The Rhizobium leguminosarum hydrogen uptake (hup) system has been genetically modified to allow transfer of hydrogenase activity to other strains with the final aim of generating rhizobial inoculants with added agricultural value and low environmental impact. We are characterizing different factors affecting the expression of the hydrogen recycling capability. These factors include the analysis of bacterial gene promoter activity, mechanisms of enzyme biosynthesis and the effect of legume host on induction of hydrogenase activity.

ii) Quorum sensing. Bacteria are able to detect high cell densities, and also to modify the expression pattern of the cell in response to it, through the accumulation of chemical signals (autoinducers), that in the case of Gram-negative bacteria are acyl-homoserine lactones (AHLs). The ability to provide a coordinate response of the whole bacterial population is a key aspect of bacterial adaptation to rhizosphere conditions. This knowledge should allow us to design molecular strategies for the generation of more competitive rhizobial inoculants.

We are studying endosymbiotic bacteria able to nodulate the recently described species Lupinus mariae-josephii, endemic from a restricted area in Eastern Spain. This lupin species is unique on its adaptation to lime-alkaline soils, where most species Lupinus species cannot be cultivated. Bacteria isolated from nodules of L. mariae-josephii are highly specific, and genetic analysis has revealed that they might constitute a novel phylogenetic clade within the Bradyrhizobium genus.

Neighbour-joining phylogenetic tree of Bradyrhizobium strains based on concatenated glnII+atpD+recA genes

Relevant results

Hydrogenase expression. An important aspect of hydrogenase biosynthesis is the regulation of expression of hydrogenase and hydrogenase biosynthetic genes. We had previously described the NifA-dependent regulation of the major hydrogenase promoter (P1) in R. leguminosarum UPM791. We have now described a novel mechanism in which enhancer sequences function in NifA-dependent activation of the P1 promoter in an independent but cooperative mode. The similarities and differences between cis elements of hup and nif/fix promoters suggest that the structure of the P1 promoter has adapted to activation by NifA in order to coexpress hydrogenase and nitrogenase activities in legume nodules (Martínez et al., 2008).

Analysis of NifA-dependent P1 promoter expression. Relevant regions of the hupSL-lacZ fusions are represented on the left. The relative location and the bases of each of the three identified HSSs in the P1promoter are shown on top. (see Martínez et al., 2008 for details).

Hydrogenase biosynthesis. A critical step on hydrogenase biosynthetic process is the synthesis and assembly of the NiFe active centre of the enzyme. We have contributed to the elucidation of the function of R. leguminosarum HupF and HupK proteins as metallochaperone and scaffolding protein, respectively, in the incorporation of active metal centre into the hydrogenase large subunit (Albareda et al, manuscript in preparation).

Expression of hydrogenase genes in Rhizobium leguminosarum UPM791 bacteroids from pea and lentil nodules. (A) expresión of plasmid-borne gene fusions to phupSL (pHL55) or phypB (pHL14). B, C: In situ ß-galactosidase staining of nodules produced in pea and lentil plants, respectively (see Brito et al., 2008, for details).

Role of the legume host. The legume host affects the expression of Rhizobium leguminosarum hydrogenase activity in root nodules. High levels of symbiotic hydrogenase activity were detected in R. leguminosarum bacteroids from different hosts, with the exception of lentil (Lens culinaris). Results of the comparative analysis of transcription and protein expression in pea and lentil hosts indicate that hydrogenase expression is affected by the legume host at the level of both transcription of hydrogenase structural genes and biosynthesis or stability of nickel-related proteins HypB and HupL, and suggest the existence of a plant-dependent mechanism that affects hydrogenase activity during the symbiosis by limiting nickel availability to the bacteroid (Brito et al., 2008).

Recent Publications

Sánchez-Cañizares, C.; Rey, L.; Durán, D.; Temprano, F.; Sánchez-Jiménez, P.; Navarro, A.; Polajnar, M.; Imperial, J.; Ruiz-Argüeso, T. 2011. "Endosymbiotic bacteria nodulating a new endemic lupine Lupinus mariae-josephi from alkaline soils in Eastern Spain represent a new lineage within the Bradyrhizobium genus". Systematic and applied microbiology. 34(3):207-215

Brito, B.; Prieto, R.I.; Cabrera, E.; Mandrand-Berthelot, M.A.; Imperial, J.; Ruiz-Argueso, T.; Palacios, J.M. 2010. "Rhizobium leguminosarum hupE encodes a nickel transporter required for hydrogenase activity". Journal of Bacteriology. 192(4):925-935

Cacho, C.; Brito, B.; Palacios, J.M.; Perez-Conde, C.; Camara, C. 2010. "Speciation of nickel by HPLC-UV/MS in pea nodules". Talanta. 83(1):78-83

Hidalgo, A.; Margaret, I.; Crespo-Rivas, J.C.; Parada, M.; Murdoch, P.; Lopez, A.; Buendia-Claveria, A.M.; Moreno, J.; Albareda, M.; Gil-Serrano, A.M.; Rodriguez-Carvajal, M.A.; Palacios, J.M.; Ruiz-Sainz, J.E.; Vinardell, J.M. 2010. "The rkpU gene of Sinorhizobium fredii HH103 is required for bacterial K-antigen polysaccharide production and for efficient nodulation with soybean but not with cowpea". Microbiology-Sgm. 156:3398-3411

Rodriguez-Sanz, M.; Antunez-Lamas, M.; Rojas, C.; Lopez-Solanilla, E.; Palacios, J.M.; Rodriguez-Palenzuela, P.; Rey, L. 2010. "The Tat pathway of plant pathogen Dickeya dadantii 3937 contributes to virulence and fitness". FEMS microbiology letters. 302(2):151-158

Brito, B.; Toffanin, A.; Prieto, R.I.; Imperial, J.; Ruiz-Argueso, T.; Palacios, J.M. 2008. "Host-dependent expression of Rhizobium leguminosarum bv. viciae hydrogenase is controlled at transcriptional and post-transcriptional levels in legume nodules". Molecular plant-microbe interactions : MPMI. 21(5):597-604

Martinez, M.; Colombo, M.V.; Palacios, J.M.; Imperial, J.; Ruiz-Argueso, T. 2008. "Novel arrangement of enhancer sequences for NifA-dependent activation of the hydrogenase gene promoter in Rhizobium leguminosarum bv. viciae". Journal of Bacteriology. 190(9):3185-3191