Affiliation:
1. Laboratorio de Microbiología, Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid
2. Consejo Superior de Investigaciones Científicas, Ciudad Universitaria s/n, 28040 Madrid, Spain
Abstract
ABSTRACT
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 (Δ
hupG
, Δ
hupH
, Δ
hupI
, and Δ
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 Δ
hupH
mutant was totally devoid of hydrogenase activity at any of the O
2
concentration tested, whereas the requirement of
hupGIJ
for hydrogenase activity varied with the O
2
concentration, being more crucial at higher pO
2
. 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
ΔhupG
mutant. Immunoblot experiments with HupL- and HupS-specific antisera showed that free-living cultures from
ΔhupH, ΔhupI
, and
ΔhupJ
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
ΔhupH
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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
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