Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC, E-18080 Granada, Spain1
Centro de Investigación y Formación Hortícola.E-04700 El Ejido, Almería, Spain2
Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, UK3
Author for correspondence: Eulogio J. Bedmar. Tel: +34 958 121011. Fax: +34 958 129600. e-mail: ejbedmar{at}eez.csic.es
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ABSTRACT |
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Keywords: Rhizobium, nitrate, denitrification, nitric oxide reduction, transcription, regulation
Abbreviations: FNR, fumarate and nitrate reductase regulator; SNP, sodium nitroprusside
b The GenBank accession number for the B. japonicum norCBQD genes reported in this paper is AJ132911.
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INTRODUCTION |
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Species of the family Rhizobiaceae are Gram-negative soil bacteria with the unique ability to establish a N2-fixing symbiosis with legumes (Amarger, 2001 ). Denitrification within this family is rare, and only a few species have been reported to grow under oxygen-limited conditions in the presence of nitrate or nitrite as a terminal electron acceptor (OHara & Daniel, 1985
). Among the Rhizobiaceae, cells of Bradyrhizobium japonicum have been shown to assimilate and denitrify 15
simultaneously to
and 15N2, when cultured anaerobically with nitrate as the only source of nitrogen (Vairinhos et al., 1989
).
The coding properties and the genetic organization of the B. japonicum USDA110 norCBQD genes and a phenotypic analysis of norC and norB mutants are described in this paper. Sequence analysis suggests that the nor gene cluster consists of four ORFs, the first two of which are the nor structural genes. The protein encoded by this cluster is closely related to the respiratory nitric oxide reductases from other denitrifiers. Regulatory studies indicate that a PnorClacZ fusion is induced under microaerobic conditions, but that nitrate is required for maximal expression. norCBQD is a regulated gene cluster in B. japonicum, and its expression seems to be dependent on the fixLJfixK2 regulatory cascade.
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METHODS |
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DNA manipulation and sequencing.
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 ). A B. japonicum USDA110 cosmid library was screened by DNADNA hybridization with the 2·1 kb SalI fragment in plasmid pPNIR3E, which carries a major internal portion of the Par. denitrificans norC and norB genes (de Boer et al., 1996
). Hybridizations were performed with digoxigenin-dUTP-labelled probes (Roche) and the chemiluminescence method was used to detect hybridization bands. A cosmid was thus identified containing B. japonicum cloned DNA, and 4·3 and 4·1 kb EcoRI fragments showing homology with the probe were subcloned in plasmid pBluescript KS+ (Stratagene), resulting in plasmids pJNOR43 and pJNOR41, respectively (Fig. 1
). DNA was sequenced on both strands by using pBluescript-specific primers and the Sanger dideoxy chain termination method. The sequencing reactions were analysed in a DNA sequencer (model 373 Strecht and dye primers from Applied Biosystems). To fill gaps, specific synthetic oligonucleotides complementary to the internal sequences were used as primers. Computer-assisted DNA and protein sequence analyses were performed by using the Genetics Computer Groups (University of Wisconsin) software packages. Homology searches were performed by using the National Center for Biotechnology Information BLAST network server (http://www.ncbi.nlm.nih.gov/BLAST/).
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Construction of norC and norB mutants.
Selected genes were mutated by performing gene-directed mutagenesis using plasmid pUC4-KIXX-aphII-PSP (Kündig et al., 1993 ). Construction of a norC mutant was accomplished by cloning the 1·8 kb SmaI fragment of pUC4-KIXX-aphII-PSP, which contains the aphII gene encoding kanamycin resistance, into the MluNI site in the 4·3 EcoRI fragment of plasmid pJNOR43 harbouring the entire B. japonicum USDA110 norC and the 5' end of the norB genes (Fig. 1
), to give plasmid pJNOR43M1. The 6·1 kb EcoRI fragment from pJNOR43M1 was further subcloned into the EcoRI site of pSUP202 (Simon et al., 1983
) to generate plasmid pJNOR43M2 (data not shown). To construct a norB mutant, the 1·8 kb SmaI fragment of pUC4-KIXX-aphII-PSP was first cloned into the NcoI site of pJNOR41 (Fig. 1
) to yield plasmid pJNOR41V1. Finally, the EcoRI fragment from pJNOR41V1 was subcloned into the EcoRI site of pSUP202 producing plasmid pJNOR41V2 (data not shown). Plasmids pJNOR43M2 and pJNOR41V2 were transferred via conjugation into B. japonicum USDA110 using E. coli S17-1 as donor. Transconjugants were selected for kanamycin resistance, and further screened for tetracycline sensitivity to exclude cointegrates. The correct genomic structure of all mutant strains was confirmed by Southern blot analysis of genomic DNAs. Mutant derivatives norC GRC131 and norB GRB993 were obtained that have been used in this study.
Construction of a PnorClacZ fusion.
To construct a transcriptional fusion of the nor promoter region to the reporter gene lacZ, PnorClacZ, the 4·3 kb EcoRI fragment from pJNOR43 containing the norC promoter region was subcloned into the EcoRI site of pMP220 (Spaink et al., 1987 ) yielding plasmid pNORLZ (Fig. 1
). The correct orientation of the construction was confirmed with BamHI/MluNI digests. To monitor norCB expression, pNORLZ was used to transform E. coli S17-1, and then transferred via conjugation into B. japonicum strains USDA110, 7403, 7360 and 9043.
Analytical methods.
For determination of ß-galactosidase activity, cells were grown aerobically in YEM medium, collected by centrifugation (7650 g, 10 min at 4 °C), washed twice with liquid YEM, and finally incubated aerobically and microaerobically in the same medium supplemented with KNO3, KNO2 or sodium nitroprusside (SNP), an NO+-generating agent (Bates et al., 1991 ). Before addition of N oxides, cultures were incubated for 24 h to permit the cells to lower the O2 concentration. Initial OD600 of the cultures was about 0·2. Cells were incubated until the OD600 was higher than 0·4. ß-Galactosidase activities were determined with permeabilized cells from at least three independently grown cultures as previously described (Miller, 1972
). Cells removed from stoppered flasks were not kept microaerobic but were used immediately for assays. All media and materials used for incubation were sterilized at 120 kPa and 110 °C for 30 min before use.
Complementation tests with B. japonicum 9043.
To demonstrate a role for the FixK2 protein in the regulation of nitric oxide reductase expression, complementation experiments were carried out with the fixK2 mutant strain B. japonicum 9043. For homologous complementation experiments, plasmids pNORLZ and pBBRK2 were introduced by conjugation into strain 9043, and ß-galactosidase transcriptional assays were done. Plasmid pBBRK2 was constructed by cloning the 1·85 kb BamHISalI fragment (fixK2) from pRJ9044 (Nellen-Anthamatten et al., 1998) into BamHI/SalI digested pUC18 (Yanisch-Perron et al., 1985 ). Then, the 1·85 kb KpnISalI fragment (fixK2) from pUC18 was cloned into KpnI/SalI digested pBBR1MCS-2 (Kovach et al., 1994
) resulting in plasmid pBBRK2.
Analysis of haem proteins.
Cell fractionation, protein gel electrophoresis and haem staining were performed as indicated earlier (Fischer et al., 2001 ). Essentially, cells of B. japonicum were disrupted by passage through an ice-cold French pressure cell at about 120 MPa. Unbroken cells were removed by centrifugation (10000 g for 10 min at 4 °C). Membranes were prepared by further centrifugation of the supernatant at 140000 g for 2 h at 4 °C. The membrane pellet was washed once with 50 mM sodium phosphate buffer (pH 7·0), resuspended in loading buffer and electrophoresed on a SDS-12% polyacrylamide gel at 4 °C. Proteins were transferred to a nitrocellulose filter and stained for haem-dependent peroxidase activity by chemiluminescence as described previously (Vargas et al., 1993
). Protein concentration was estimated by using the Bio-Rad assay with BSA as the standard.
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RESULTS AND DISCUSSION |
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Mutational analysis of norB and norC
To confirm the function of the norC and norB gene products in nitrate metabolism, each gene was inactivated by performing mutagenesis using plasmid pUC4-KIXX-aphII-PSP. In contrast to B. japonicum USDA110, cells of mutant strains norC GRC131 and norB GRB993 did not grow microaerobically with either nitrate or nitrite as a terminal electron acceptor. Proteins from the membrane fraction of wild-type and mutant strains were separated by SDS-PAGE and stained for covalently bound haem proteins. Six stained bands of 32, 28, 25, 24, 20 and 16 kDa were detected (Fig. 2, lane 1). The proteins of 28 and 20 kDa have been identified previously as the B. japonicum aerobic, membrane-bound c-type cytochromes c1 (28 kDa) and CycM (20 kDa) (Thöny-Meyer et al., 1989
; Bott et al., 1991
). As described by Preisig et al. (1993)
, there is even a seventh protein of 28 kDa co-migrating with cytochrome c1. This 28 kDa protein and the 32 kDa c-type cytochrome have been identified as the B. japonicum FixP and FixO proteins, respectively, of the cbb3-type high-affinity cytochrome oxidase encoded by the fixNOQP operon (Preisig et al., 1996
). The identity of the 16, 24 and 25 kDa proteins is not known. A haem-stainable band of approximately 16 kDa, which is very prominent in microaerobic wild-type membranes, was absent in the norC mutant GRC131 (Fig. 2
, lanes 1 and 3, respectively), which identifies this protein as the NorC component of B. japonicum USDA110 nitric oxide reductase enzyme. The norB mutant GRB993 showed the same pattern of haem-stained proteins as the wild-type (Fig. 2
, lane 2). The concentration of NorC in the norB mutant was lower than in the parental strain (Fig. 2
, lanes 1 and 2, respectively). Whether NorC is required for assembly or stability of NorB is not known.
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Expression of the nor genes
To study the regulation of nor expression, a PnorClacZ fusion was constructed and transferred by conjugation into the wild-type B. japonicum USDA110 and the mutant strains fixL 7403, fixJ 7360 and fixK2 9043. Since the mutant strains did not grow microaerobically with nitrate, cells of the parental and mutant strains were grown first in YEM medium and further incubated aerobically and microaerobically (1% O2) in the same medium supplemented or not with nitrate (Table 1). After induction under aerobic conditions, cells of USDA110 with the PnorClacZ fusion had basal levels of ß-galactosidase activity. When the cells were incubated under 1% O2 in a medium unamended with nitrate, values of ß-galactosidase activity were threefold higher than in cells grown in air, and there was an 18-fold increase in ß-galactosidase activity after incubation of the cells under oxygen-limiting conditions in the presence of nitrate. These results agree with those obtained from the primer-extension studies showing that the level of the transcript detected in cells incubated microaerobically with nitrate was higher than in cells incubated microaerobically without nitrate (Fig. 3a
).
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Besides microaerobiosis, nitrate, or a derived N oxide, was required for maximal expression of ß-galactosidase activity. Values of activity in cells incubated with nitrate as an oxidant were about 115000 Miller units (mg protein)-1 (Table 1), approximately 2·3-fold higher than those found when either nitrite or SNP was used as the final electron acceptor (data not shown). NO, or a chemical species related to NO, has been shown to be a signal molecule for the coordinated activation of the nor and nir operons in Rho. sphaeroides (Kwiatkowski & Shapleigh, 1996
; Tosques et al., 1996
), Par. denitrificans (van Spanning et al., 1999
), Pse. aeruginosa (Arai et al., 1999
) and Pse. stutzeri (Vollack & Zumft, 2001
). Because nitrite and NO are cytotoxic compounds, 100 µM KNO2 and 10 µM SNP were first determined in control experiments to elicit maximal expression of ß-galactosidase activity (data not shown). Thus, lower levels of ß-galactosidase activity in cells with nitrite or SNP cannot be attributed to the presence of those N oxides. The smaller effect of nitrite and SNP, compared to nitrate, may reflect differences either in the amount of NO that is produced from the different compounds or in the concentration of NO that is present inside the cells. Although the effector molecule used for activation of the B. japonicum USDA110 nor genes cannot be identified from the present results, NO could be a signal candidate. This suggestion is based on results showing the formation of nitrosyl adducts of FixL (Gilles-Gonzalez et al., 1995
; Rodgers et al., 2000
), which indicates that NO may be a physiologically relevant ligand for FixL. Transcriptional activation of nor genes is under the control of NNR (nitrite and nitric oxide reductase regulator) and FnrP in Par. denitrificans (van Spanning et al., 1995
), NnrR in Rho. sphaeroides (Tosques et al., 1996
), DNR (dissimilatory nitrate respiration regulator) in Pse. aeruginosa (Arai et al., 1997
) and DnrD in Pse. sutzeri (Vollack et al., 1999
). Yet the existence of an N oxide sensitive regulator has not been reported in B. japonicum.
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ACKNOWLEDGEMENTS |
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Received 3 June 2002;
revised 15 July 2002;
accepted 22 July 2002.