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Journal of Bacteriology, October 2005, p. 7018-7026, Vol. 187, No. 20
0021-9193/05/$08.00+0     doi:10.1128/JB.187.20.7018-7026.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Gene Products of the hupGHIJ Operon Are Involved in Maturation of the Iron-Sulfur Subunit of the [NiFe] Hydrogenase from Rhizobium leguminosarum bv. viciae

Hamid Manyani,1,{dagger} Luis Rey,1 José M. Palacios,1 Juan Imperial,1,2 and Tomás Ruiz-Argüeso1*

Laboratorio de Microbiología, Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid,1 Consejo Superior de Investigaciones Científicas, Ciudad Universitaria s/n, 28040 Madrid, Spain2

Received 25 May 2005/ Accepted 27 July 2005

In the present study, we investigate the functions of the hupGHIJ operon in the synthesis of an active [NiFe] hydrogenase in the legume endosymbiont Rhizobium leguminosarum bv. viciae. These genes are clustered with 14 other genes including the hydrogenase structural genes hupSL. A set of isogenic mutants with in-frame deletions ({Delta}hupG, {Delta}hupH, {Delta}hupI, and {Delta}hupJ) was generated and tested for hydrogenase activity in cultures grown at different oxygen concentrations (0.2 to 2.0%) and in symbiosis with peas. In free-living cultures, deletions in these genes severely reduced hydrogenase activity. The {Delta}hupH mutant was totally devoid of hydrogenase activity at any of the O2 concentration tested, whereas the requirement of hupGIJ for hydrogenase activity varied with the O2 concentration, being more crucial at higher pO2. Pea bacteroids from the mutant strains affected in hupH, hupI, and hupJ exhibited reduced (20 to 50%) rates of hydrogenase activity compared to the wild type, whereas rates were not affected in the {Delta}hupG mutant. Immunoblot experiments with HupL- and HupS-specific antisera showed that free-living cultures from {Delta}hupH, {Delta}hupI, and {Delta}hupJ mutants synthesized a fully processed mature HupL protein and accumulated an unprocessed form of HupS (pre-HupS). Both the mature HupL and the pre-HupS forms were located in the cytoplasmic fraction of cultures from the {Delta}hupH mutant. Affinity chromatography experiments revealed that cytoplasmic pre-HupS binds to the HupH protein before the pre-HupS-HupL complex is formed. From these results we propose that hupGHIJ gene products are involved in the maturation of the HupS hydrogenase subunit.

* Corresponding author. Mailing address: Laboratorio de Microbiología, Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain. Phone: 34-91-3365752. Fax: 34-91-3365757. E-mail: t.ruizargueso{at}upm.es.

{dagger} Present address: BIOMEDAL, Avenida Américo Vespucio 5-E, 1a planta, Mod. 12, Parque Científico y Tecnológico Cartuja 93, 41092 Sevilla, Spain.

Journal of Bacteriology, October 2005, p. 7018-7026, Vol. 187, No. 20
0021-9193/05/$08.00+0     doi:10.1128/JB.187.20.7018-7026.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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