Results and Discussion

The B. japonicum 3Ilbl 10 nirK, norCBQD and nosRZDFXYL genes were identified using heterologous probes from Alcaligenes faecalis (Nishiyama et al. 1993), Paracoccus denitrificans (de Boer et al. 1996) and Pseudomonas stutzeri (Viebrock, Zumft 1987) encoding the Cu-containing respiratory nitrite reductase, nitric oxide reductase and nitrous oxide reductase, respectively. The deduced primary sequence of nirK (EMBL, accession number AJ002516) has greater than 68%

identity with translated sequences of nirK genes from other denitrifiers, including other members of the Rhizobiaceae family such as Sinorhizobium meliloti (http://sequence.toulouse.inra.fr/ rhime/public/Access/RhimeFormRA.html) and Rhizobium hedysary (Toffanin et al. 1996). Alignment of the deduced NorC amino acid sequence (EMBL, accession number AJ132911) revealed significant identity with NorC proteins of other denitrifiers, ranging from 52% with the deduced amino acid sequence of norC from Paracoccus halodenitrificans to 82% with that of S. meliloti. The deduced primary sequence of the structural nosZ gene (EMBL, accession number AJ002531) exhibits 63%, 64% and 77% identity with the products of nosZ from Pseudomonas aeruginosa, Achromobacter cycloclastes and S. meliloti, respectively, which provides a strong argument to consider the B. japonicum nosZ product as a nitrous oxide reductase enzyme (Figure 1).

Pseudomonas stutzeri

R SDFYL Q S TBMCFDLCB D

Paracoccus denitrificans

R SD FEDQBC SECFD

Pseudomonas aeruginosa

R SD DBCQSMCFDLGH

Bradyrhizobium japonicum

R ZDFYLX K CBQD

Figure 1. Demtrification gene cluster of P. stutzeri and P. aeruginosa (Zumft 1997), P. denitrificans (Baker et al. 1998) and B. japonicum.

Cleavage of genomic DNA from B. japonicum strain 3Ilbl 10 by the restriction enzymes Pmel, Pacl and Swal was used together with pulsed-field gel electrophoresis and Southern hybridization to locate the nirK, norCBQD and nosRZDFYLX denitrification genes on the chromosomal map of B. japonicum strain 110spc4 published earlier (Kiindig et al. 1993; Gottfert et al. 1998). Restriction of parental and mutant strains genomic DNAs with Pacl, Pmel and Swal followed by pulsed-field gel electrophoresis resulted in different fragment patterns that allowed determination of the position of the selected genes. Complementary mapping data obtained by hybridization using B. japonicum 3Ilbl 10 nirK, norBQD and nosZD as gene probes revealed that nirK, norCBQD and nosRZDFYLX gents were dispersed over the entire chromosome, being located close to the groEL2, cycH and cycVWXgenes, respectively, on the strain 110spc4 genetic map (Mesa et al. 2001). In P. denitrificans the nir and nor genes are linked on a 17.7- kb fragment (Baker et al. 1998), and genes neccesary for denitrification are concentrated at 20 to 36 minutes on the P. aeruginosa chromosome, where they form two separate loci, the nir-nor and nos gene clusters (Vollack et al. 1998). In P. stutzeri the nos genes are within 14- kb of the nir-nor genes, forming a single denitrification cluster of about 30 kb (Zumft 1997). Organization of the denitrification genes in B. japonicum resembles that of Rhodobacter sphaeroides strains 2.4.1 and IL106 where nirK and the norCB genes are not contiguous on the chromosome (Bartnikas et al. 1997; Tosques et al. 1997), and the nos locus is located on a 115- kb plasmid (Schwintner et al. 1998) (Figure 1).

To study expression of denitrification genes, lacZ fusions were constructed and transferred by conjugation into B. japonicum 3Ilbl 10. After aerobic growth, cells of strain 3Ilbl 10 containing either the nirK-lacZ, norC-lacZ or the nosZ-lacZ fusion had basal levels of P-galactosidase activity, regardless of the presence or the absence of nitrate in the incubation medium (Figure 2a). When the cells were incubated under 1% O2 in the absence of nitrate, values of p-galactosidase activity were 3 to 6 times greater than in cells grown in air, and there was a 12- to 27-fold increase in p-galactosidase activity after incubation of the cells under both oxygen-limiting conditions and the presence of nitrate (Figure 2a).

Bacteroids isolated from nodules of plants inoculated separately with cells of strain 3Ilbl 10 containing the nirK-lacZ, norC-lacZ or the nosZ-lacZ fusion also showed p-galactosidase activity (Figure 2b). In plants that were N2-dependent, P-galactosidase activity was induced 5-10 times above basal levels found in bacteroids formed by strain 3Ilbl 10 transformed with plasmid pMP220 (Figure 2b). Although denitrification activity, measured as N2O production, was induced by addition of 4 mM KNO3 to the mineral nutrient solution, values of bacteroidal p-galactosidase activity were similar to those found in plants not treated with nitrate (Figure 2b). Similar results were obtained in bacteroids isolated from plants that were only ^-dependent and treated with 5, 10 and 20 mM KNO3 five days before measurements. If nitrate does not induce expression of denitrification genes in bacteroids, and nitrogen-fixing B. japonicum bacteroids use the high-affinity cbb^-type oxidase to produce ATP (Preisig et al. 1993; Arslan et al. 2000), could oxygen within the nodules be a limiting factor preventing maximal expression of bacteroidal denitrification genes?

Figure 2. P-Galactosidase activity m free-livmg cells (a) and bacteroids (b) of B. japonicum 3Ilbll0 containing the nirK-lacZ, norC-lacZ or the nosZ-lacZ fusion. Cells were cultured aerobically and microaerobically in a medium supplemented or not with 10 mM KNO3. Plants were grown in the absence and the presence of 4 mM KNO3 for 45 days.

Figure 2. P-Galactosidase activity m free-livmg cells (a) and bacteroids (b) of B. japonicum 3Ilbll0 containing the nirK-lacZ, norC-lacZ or the nosZ-lacZ fusion. Cells were cultured aerobically and microaerobically in a medium supplemented or not with 10 mM KNO3. Plants were grown in the absence and the presence of 4 mM KNO3 for 45 days.

To analyze the symbiotic phenotype of the nirK, norB and nosZ mutants, soybean plants were inoculated separately with the B. japonicum parental strain 3Ilbl 10 and mutant derivatives nirK GRK13, norC GRC131 and nosZ GRZ25 (Table 1). In plants that were only ^-dependent, no differences in nodule number and nodule fresh weight were found among soybeans inoculated with either the wild-type B. japonicum 3Ilbl 10 or each one of the mutant strains; moreover, values of total plant dry weight and nitrogen content were similar, regardless of the bacterial strain used for inoculation. However, the nodule number and the nodule fresh weight of plants inoculated with the nirK and the norC mutants and grown with 4 mM KNO3 nitrate were significantly lower (P < 0.05) than those of soybeans inoculated with the parental strain or the nosZ mutant. Similarly, the depressive effect on nodulation also affected the dry weight and N content of the plants inoculated with strains GRK13 and GRC131, which were significantly lower (P < 0.05) than those of plants inoculated with the nosZ mutant or the parental strain 3Ilbl 10. Whether mutation in the nirK or norC genes affected viability and persistence of the mutants in the rhizosphere or some other stage during nodule formation and development is not known. Denitrifying wild-type cells of Pseudomonas sp. strain RTC01 had greater advantage to colonize the rhizosphere of maize plants than those of an isogenic mutant deficient in the ability to synthesize respiratory nitrite reductase, which indicates that the presence of a functional structural gene confers higher rhizosphere competence to a microorganism (Philippot et al. 1995).

Table 1. Nodule number (NN), nodule fresh weight (NFW, g/plant), plant dry weight (PDW, g/plant), and total N (N, mg/plant) of soybean plants inoculated with B. japonicum 3Ilbl 10 and mutant derivatives nirK GRK13, norC GRC131 and nosZ GRZ25. Values followed by the same letter are not significantly different at P < 0.05 according to the test of Tukey.

Nitrate treatment (mM)

Table 1. Nodule number (NN), nodule fresh weight (NFW, g/plant), plant dry weight (PDW, g/plant), and total N (N, mg/plant) of soybean plants inoculated with B. japonicum 3Ilbl 10 and mutant derivatives nirK GRK13, norC GRC131 and nosZ GRZ25. Values followed by the same letter are not significantly different at P < 0.05 according to the test of Tukey.

Nitrate treatment (mM)

B. japonicum

strain

0

4

NN

NFW

PDW

N

NN

NFW

PDW

N

3Ilbl10

38 a

0.50 a

2.04 a

49.87 a

47 a

0.87 a

6.35 a

143.67 a

GRK13

44 a

0.50 a

1.88 a

49.53 a

35 b

0.57 b

4.70 b

120.72 b

GRC131

40 a

0.43 a

1.79 a

47.64 a

33 b

0.48 b

4.81 b

108.45 b

GRZ25

42 a

0.49 a

1.98 a

52.28 a

48 a

0.75 a

5.88 a

138.37 a

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