1 Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC, E-18080 Granada, Spain
2 Fachbereich Biologie der Philipps-Universität Marburg, Zellbiologie und Angewandte Botanik, Karl-von-Frisch-Str. 8, D-35032 Marburg, Germany
Correspondence
María J. Delgado
mdelgado{at}eez.csic.es
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ABSTRACT |
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The GenBank accession number for the nucleotide sequences of the B. japonicum nap genes is AF314590.
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INTRODUCTION |
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Bradyrhizobium japonicum is a facultatively anaerobic soil bacterium with the capability to reduce simultaneously to
and N2 when cultured anaerobically with nitrate as terminal electron acceptor and sole source of nitrogen (Vairinhos et al., 1989
). In B. japonicum, the denitrification process depends on the nirK (Velasco et al., 2001
), norCBQD (Mesa et al., 2002
) and nosRZDYFLX (Mesa et al., 2001
) gene clusters encoding nitrite reductase, nitric oxide reductase and nitrous oxide reductase, respectively. The coding properties and the genetic organization of the B. japonicum USDA110 napEDABC genes and a phenotypic analysis of a napA mutant are described in this paper. Sequence analysis suggests that the nap cluster consists of five ORFs, the third and fourth of which are the nap structural genes. The protein encoded by this cluster is closely related to the periplasmic nitrate reductases from other bacteria. This study provides both genetic and biochemical evidence for the requirement of the periplasmic nitrate reductase in the first step of denitrification in B. japonicum USDA110.
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METHODS |
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Routine DNA work and sequence analysis.
Chromosomal and plasmid isolations, restriction enzyme digestions, agarose gel electrophoresis, ligations, and E. coli transformations, were performed according to standard protocols (Sambrook et al., 1989). Hybridizations were performed with digoxigenin-dUTP-labelled probes (Roche), and the chemiluminiscence method. Alternatively the direct staining method, using nitro blue tetrazolium chloride (NBT) and X-phosphate as the chromogenic substrates, was applied to detect hybridization bands. In a shotgun cloning experiment PstI fragments of B. japonicum, preselected for their size within the range of 615 kb, were ligated to the mobilizable vector pJQ200 (Quandt & Hynes, 1993
). Plasmid pPM200P9134 was thus isolated containing a 6·3 kb PstI fragment of cloned B. japonicum DNA (data not shown). After restriction analysis and partial DNA sequencing of pPM200P9134, a 1·96 kb ApaIPstI internal fragment of pPM200P9134 was subcloned into pJQ200, yielding plasmid pPM0610 (Fig. 1
). Genomic DNA of strain GRPA1 (see below) (Fig. 1
) was restricted by ApaI and the resulting fragments were ligated to pBluescript KS(+) (Stratagene). Plasmid pBG605 was thus obtained, harbouring a 14 kb fragment from GRPA1 (data not shown). An internal 4·7 kb PstI fragment of pBG605 was subcloned into pJQ200 in both directions yielding plasmids pPM606-1 and pPM606-3, respectively (Fig. 1
). Plasmids pPM0610, pPM606-1 and pPM606-3 were used to create a series of deletion derivatives which were truncated from both ends, and which were used for sequencing (Fig. 1
). DNA was sequenced by using the DYE-namic Thermosequenase cycle sequencing kit and the IRD-800 labelled reverse or forward standard primers (MWG Biotech). Cycle sequencing reactions were carried out as described earlier (Becker et al., 1998
), using a LI-COR 4000 sequencing device which allows visualization of the IRD 800 dye which is bound to the 5'-end of the primer. The dye is infrared detectable at 790 nm after excitation with a laser beam. Sequence analysis was carried out using the computer programs Gene Base (Applied Maths, 1.0) and vector suite NTI (Informax). Homology searches were performed by using the National Center for Biotechnology Information BLAST network server (http://www.ncbi.nlm.nih.gov/blast/). Transmembrane predictions and signal sequence analysis was performed using an on-line program of the Center of Biological Services (http://www.cbs.dtu.dk/services).
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Construction of a napA mutant.
The napA gene was mutated by performing gene-directed mutagenesis by marker exchange. A 1·2 kb BamHIPstI fragment from pPM200P9134 was subloned into pK18mobsac (Schäfer et al., 1994) to obtain plasmid pBG602 (data not shown). Finally, the 2 kb SmaI fragment (
Spc/Sm interposon) of pHP45
(Prentki & Krisch, 1984
) was inserted to replace a central 24 bp NruI fragment within pBG602 (Fig. 1
). The resulting plasmid pBG602
was transferred via conjugation into B. japonicum USDA110 using E. coli S17-1 as donor. Double recombination events were favoured by growth on agar plates containing sucrose. Mutant strains resistant to spectinomycin/streptomycin but sensitive to kanamycin were checked by Southern hybridization experiments (data not shown) for correct replacement of the wild-type fragment by the
interposon. The mutant derivative GRPA1, used in this study, was obtained.
Genetic complementation of the napA mutant.
To obtain a recombinant plasmid containing the complete napEDABC region, the PstIBamHI deletion derivative from pPM606-1 was cloned in pJQ200 to construct plasmid pPM606-1B. pPM606-1
B was then linearized by double digestion with ApaI and PstI and ligated with the 1·96 kb fragment of pPM0610, which was excised by the identical enzymes (Fig. 1
). The resulting recombinant plasmid (5·1 kb ApaIBamHIBamHI fragment carrying napEDABC cloned in pJQ200) was designated pPM0611 (Fig. 1
). Plasmid pPM0611 was integrated by homologous recombination into the chromosome of B. japonicum GRPA1 using E. coli S17-1 as a donor, resulting in strain GRPA1C. Recombinant strains were selected for gentamicin resistance and their correct genomic structure was confirmed by Southern blot analysis of genomic DNA preparations.
Determination of nitrate reductase activity.
Cells of B. japonicum grown aerobically in YEM medium were harvested by centrifugation (8000 g for 10 min at 4 °C), washed twice with YEM, resuspended in 150 ml of the same medium supplemented with 10 mM KNO3, and finally incubated under anaerobic conditions for 96 h. After incubation, the cells were washed with 50 mM Tris/HCl buffer (pH 7·5) until no nitrite was detected, and then resuspended in 1 ml of the same buffer. The reaction mixture contained 50 mM Tris/HCl buffer (pH 7·5), 10 mM KNO3, 200 µM methyl viologen (MV+) or benzyl viologen (BV+), and 50 µl cell suspension (0·30·5 mg protein). The reaction was started by addition of 50 µl freshly prepared sodium dithionite solution (8 mg ml-1) in Tris/HCl buffer. After incubation for 10 min at 30 °C, the reaction was stopped by vigorous shaking until the samples had lost their blue colour. Nitrite was estimated after diazotation by adding the sulfanilamide/naphthylethylene diamine dyhydrochloride reagent (Nicholas & Nason, 1957).
Analysis of NapA and haem-c proteins.
Cells of B. japonicum grown aerobically in YEM medium (500 ml) were harvested by centrifugation as above, washed twice with YEM, resuspended in 1 l of the same medium supplemented with 10 mM KNO3, and finally incubated under anaerobic conditions for 96 h. Preparation of periplasmic proteins was carried out as described previously (McEwan et al., 1984). Essentially, after incubation, cells were resuspended in 10 ml 100 mM Tris/HCl (pH 8·0) containing 0·5 M sucrose, 3·0 mM EDTA and 250 µg lysozyme ml-1, and incubated for 2 h at 30 °C. The cell suspension was then centrifugated at 12 000 g for 15 min at 4 °C, and the resulting supernatant, containing periplasmic proteins, was concentrated to about 200 µl by using Amicon Centriprep 3 and Centricon 3 filters. The concentrated periplasmic fractions were stored at -20 °C until use. Membrane preparations were performed as described earlier (Fischer et al., 2001
). For this purpose, anaerobically incubated cells were washed with 50 mM sodium phosphate buffer (pH 7·0) containing 1 mM MgCl2, 0·1 mM CaCl2 and 0·9 % NaCl, and resuspended in 3 ml of the same buffer containing 1 mM 4-amidinophenylmethanesulfonyl fluoride (APMSF), 20 µg DNase I ml-1 and 20 µg RNase A ml-1 (fractionation buffer). Cells were disrupted by three passages through an ice-cold French pressure cell (SLM-Aminco) at a pressure of about 120 MPa. Unbroken cells were removed by centrifugation (10 000 g for 10 min at 4 °C). Membranes were prepared by further centrifugation of the supernatant at 140 000 g for 2 h at 4 °C. The membrane pellet was washed once with fractionation buffer, resuspended in 200 µl of the same buffer and stored at -20 °C.
Periplasmic and membrane protein aliquots (30 µg) were diluted in sample buffer (124 mM Tris/HCl pH 7·0, 20 % v/v, glycerol, 4·6 % SDS and 50 mM 2-mercaptoethanol), and incubated at room temperature for 10 min for haem-c staining, or at 100 °C for 3 min for immunostaining. Membrane proteins were separated at 4 °C in SDS-12 % polyacrylamide gel electrophoresis (PAGE), transferred to a nitrocellulose filter and stained for haem-dependent peroxidase activity by chemiluminescence as described previously (Vargas et al., 1993). Periplasmic proteins were electrophoresed on a SDS 12 % polyacrylamide gel for immunostaining assays or on an SDS 1520 % gel gradient for haem-c staining assays. NapA was detected by Western blotting with specific rabbit antisera generated against the purified NapA subunit of Paracoccus pantotrophus (Richardson et al., 1998
). Protein concentration was estimated by using the Bio-Rad assay with bovine serum albumin as the standard.
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RESULTS AND DISCUSSION |
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Analysis of the promoter region and Nap activity
Inspection of the DNA sequence (Fig. 2c) revealed a purine-rich ShineDalgarno-like sequence (AGAGAGA) 13 bases upstream of the putative translational start codon of napE. Located 101 bp upstream of the putative initiation codon of napE there is the sequence 5'-TTGAT-N4-ATCAA-3', which has 10 out of 10 matches with the FNR consensus sequence 5'-TTGAT-N4-ATCAA-3' (Spiro, 1994
), and 8 out of 10 matches with the FixK consensus sequence 5'-TTGAT-N4-GTCAA-3' (Fischer, 1994
; Zumft, 1997
) (Fig. 2c
). Among bacteria containing periplasmic nitrate reductases, FNR boxes have only been identified in the promoter regions of Pseudomonas sp. G-179 napE (Bedzyk et al., 1999
) and E. coli napF (Darwin et al., 1998
). Computer searches revealed no other totally conserved recognition motifs in the nap promoter region.
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Mutation of napA and complementation analysis
To confirm the function of the napA gene product in nitrate metabolism, the gene was mutated by marker-exchange mutagenesis (Fig. 1). In contrast to B. japonicum USDA110, cells of the mutant strain GRPA1 were unable to grow anaerobically with nitrate as the final electron acceptor (Fig. 3
). Both strains, however, grew well when the YEM medium was amended with 0·5 mM nitrite (data not shown). Complementation of strain GRPA1 with the chromosomally integrated plasmid pPM0611 containing the wild-type napEDABC genes restored the ability of the cells to grow on nitrate as the alternative electron acceptor (Fig. 3
). Because antibiotics were used to keep the selective pressure on strain GRPA1C, growth of the complemented mutant was most likely delayed as compared with that observed for the parental strain.
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Membranes from strain GRPA1 also lacked the 16 kDa NorC protein (Fig. 4c, lane 2), which suggests that nitrate reduction by NapA is required for NorC expression.
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ACKNOWLEDGEMENTS |
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Received 2 July 2003;
revised 16 September 2003;
accepted 17 September 2003.
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