Identification and characterization of the two-component NtrY / NtrX regulatory system in Azospirillum brasilense

Two Azospirillum brasilense open reading frames (ORFs) exhibited homology with the two-component NtrY/NtrX regulatory system from Azorhizobium caulinodans. These A. brasilense ORFs, located downstream to the nifR3ntrBC operon, were isolated, sequenced and characterized. The present study suggests that ORF1 and ORF2 correspond to the A. brasilense ntrY and ntrX genes, respectively. The amino acid sequences of A. brasilense NtrY and NtrX proteins showed high similarity to sensor/kinase and regulatory proteins, respectively. Analysis of lacZ transcriptional fusions by the ß-galactosidase assay in Escherichia coli ntrC mutants showed that the NtrY/NtrX proteins failed to activate transcription of the nifA promoter of A. brasilense. The ntrYX operon complemented a nifR3ntrBC deletion mutant of A. brasilense for nitrate-dependent growth, suggesting a possible crosstalk between the NtrY/X and NtrB/C sensor/regulator pairs. Our data support the existence of another two-component regulatory system in A. brasilense, the NtrY/NtrX system, probably involved in the regulation of nitrate assimilation. Correspondence


Introduction
The ntrYX genes were identified and sequenced and their probable physiological function was characterized in Azorhizobium caulinodans ORS571 (1).In this symbiotic nitrogen-fixing bacterium, the NtrY and NtrX proteins constitute a two-component regulatory system apparently involved in nitrogen fixation and metabolism (1).The NtrY protein was homologous with sensor transmembrane proteins while NtrX exhibited a high degree of homology with positive regulatory proteins, such as NtrC.The expression of the ntrYX operon was depressed in an ntrC mutant grown in the presence of nitrate, suggesting a possible interaction between the ntrYX/ntrBC systems in A. caulinodans (1). A. caulinodans ntrC or ntrX mutants were also unable to activate the expression of the nifA gene, implying that NtrC and NtrX proteins could be involved in nifA expression.The authors suggested that the ntrYX and ntrBC genes were involved in nitrogen metabolism in A. caulinodans and that the NtrY/NtrB sensors could cross-talk with the NtrX/NtrC regulators to activate trans-cription initiation from ntr-dependent promoters (1).The ntrYX genes were also found in other microorganisms such as Acetobacter diazotrophicus (Gluconacetobacter diazotrophicus) (2), Caulobacter crescentus (3), Sinorhizobium meliloti (4), Mesorhizobium loti (5), Rickettsia prowazekii (6), Neisseria meningitidis (7) and Zymomonas mobilis (8).The function of these genes in these bacteria remains unknown.
Regulation of nitrogen fixation in Azospirillum brasilense, a free-living bacterium, is still under investigation.Pedrosa and Yates (9) suggested a mechanism of regulation similar to that observed in Klebsiella pneumoniae after they isolated a nifA (FP10) and two ntrC (FP8 and FP9) mutants, which were unable to fix nitrogen.However, Liang et al. (10), sequencing the A. brasilense nifA gene, did not find NtrC-binding sites or a σ 54 -type promoter in the upstream region of this gene.An essential region for nifA promoter activity was identified between nucleotides -67 and -47 from the nifA transcription start site (11).A sequence resembling a σ 70 recognition site occurs in this region and may constitute the nifA gene promoter (11).Oxygen inhibited expression of the nifA gene, but only partial repression by ammonium was observed (10).On the other hand, when oxygen and ammonium were present repression of nifA expression reached high levels (80-90%) (11).In glnB mutants of A. brasilense the NifA protein is synthesized in an inactive form, suggesting the involvement of the PII protein in the regulation of NifA activity by ammonia (12).In the presence of ammonia, NifA is in an inactive form since its N-terminal domain has been suggested to auto-inhibit its activity (13).On the other hand, under ammonium-limiting conditions, the PII protein was necessary to maintain the NifA protein in the active form, apparently by preventing the N-terminal inhibition.The mechanism by which PII prevents N-terminal inhibition under conditions of ammonia limitation is still unknown.
The NtrB/NtrC system in A. brasilense is involved in regulation of nitrate utilization (9,14,15), switch-off of nitrogenase by ammonium (9,16) and (methyl) ammonium transport (17).Deletion or mutation of the ntrBC genes did not abolish nitrogenase activity but reduced it to half of that observed in the wild-type strain (14,15), suggesting the probable involvement of a second system in the regulation of nitrogen fixation.
In the present study, we found the ntrYX genes in A. brasilense located downstream from the nifR3ntrBC operon.The genes were sequenced completely and the translation start codon of the ntrX gene was shown to overlap the 3' end of the ntrY gene.Their gene products were highly homologous to the NtrY and NtrX proteins from other organisms.The ntrYX genes complemented a nifR3ntrBC deletion mutant of A. brasilense for nitrate-dependent growth.

Bacterial strains and plasmids
The bacterial strains and plasmids used in the present study are listed in Table 1.
Plasmid pTH6E3 was constructed by cloning an EcoRI fragment of 6.3 kb containing part of ntrB and the whole of ntrC, ntrY and ntrX genes into pTZ18R.The 5.1kb SalI fragment from pTH6E3 was subsequently cloned into pSUP202, producing pSH5S1.The Km-lacZ cassette from pKOK6.1 was then inserted into the NsiI site of pSH5S1 in both orientations, producing plasmids pLKIII and pLK015 (Figure 1 and Table 1).
To construct pL46 the 4.85-kb BglII/ EcoRI fragment from pTH6E3 was cloned into pLAFR3.18digested with BamHI and EcoRI (Table 1).In this construction, the ntrC gene lacks its N-terminal region and its orientation of transcription is opposite to that of the lacZ promoter, and the ntrYX genes are expressed from their native promoter, as in pTH6E3.

Analytical assays
ß-Galactosidase activity was determined as described by Miller (25) in E. coli cultures grown in liquid NFDM (26).The nitrogen source was NH 4 Cl (20 mM) and serine (100 µg/ml) was added to cultures without ammonium.The NFDM medium was supplemented with 50 µg/ml L-glutamine and 1 mM IPTG. A. brasilense cultures were grown in NFbHPN medium under conditions of ammonium deficiency (5 mM L-glutamate) or excess (20 mM NH 4 Cl).

DNA manipulations and sequencing
Isolation of plasmid DNA, gel electrophoresis and cloning experiments were carried out as described by Sambrook et al. (24).The 4.85-kb BglII/EcoRI fragment derived from pTH6E3, containing the A. brasilense ntrYX genes, was cloned into the vector pSPORT2 producing plasmid pSPL46 (Table 1).The inserted fragment was fully sequenced in both directions.Doublestranded DNA was sequenced with the Thermosequenase kit (Amersham Pharmacia Biotech, Uppsala, Sweden) or the Big Dye kit (Applied Biosystems ABI 310 se-quencer, Foster City, CA, USA).The database was searched using the Blast program (27) and DNA or protein sequences were compared using the Clustal W program (28).The sequences of A. brasilense ntrY and ntrX were deposited at the EMBL-GenBank under the accession number AF426449.

Results and Discussion
Identification and sequencing of the ntrYX genes The 4.85-kb insert of plasmid pSPL46 was fully sequenced and showed the pres-      A σ 70 -type promoter was identified 26 bp upstream from the start codon of NtrY (TTGGCA-N18-TATCAT).Machado et al. (15) sequenced the N-terminal region of NtrY downstream from the ntrC gene and reported constitutive promoter activity located in the intergenic region in an E. coli background.These results suggest that the ntrYX operon is expressed from its own promoter although readthrough from an upstream promoter cannot be ruled out due to the absence of a transcription terminator sequence downstream from the ntrC gene.

Effect of the ntrYX gene products on the expression of an A. brasilense nifA fusion in an E. coli mutant strain
E. coli ET8556 (ntrC -) transformants containing plasmid pCF2 (A.brasilense pnifA:: lacZ fusion with its native promoter) alone or together with plasmid pSPL46 (A.brasilense ntrYX genes expressed from their own promoter) were assayed for ß-galactosidase activity, under conditions of ammonium deficiency or excess.The results showed that the presence of the ntrYX genes had no effect on the expression of the pnifA::lacZ (pCF2) 50.9 ± 6.9 140.6 ± 28.1 FP9.R (pL46) 81.9 ± 6.9 163.3 ± 11.0 Cultures were grown in liquid NFbHPN medium in the presence (20 mM) or absence of NH 4 Cl, for 24 h.L-glutamate (5 mM) was added to cultures grown in the absence of NH 4 Cl.Expression of the nifA::lacZ chromosomal fusion was determined by ß-galactosidase activity.The results are the average of three independent experiments run in duplicate ± SD. (31,32).The C-terminus of the A. brasilense NtrY protein (Figure 3A) showed a high degree of identity (35%) with the conserved C-termini of homologous proteins from A. caulinodans, C. crescentus, M. loti and S. meliloti.
ORF2 encoded a 466-amino acid protein with a high degree of identity (52%) to A. caulinodans NtrX (Figure 3B), and a lower identity (32%) to NtrC proteins of other organisms including A. brasilense.The aspartic acid residue at position 53 in the A. brasilense NtrX is probably the site of phosphorylation, since it is conserved in all NtrX proteins (Figure 3B).This site is equivalent to the phosphorylation site of the NtrC pro- fusion (Figure 4), suggesting that the NtrY/ NtrX proteins are not involved in the expression of the nifA promoter in A. brasilense (Figure 4).Previous results showed that there is an active promoter immediately upstream from the ntrY gene (15).

Effect of ntrYX genes on the expression of chromosomal nifA::lacZ fusions in A. brasilense strains
A. brasilense FP2.R (nifA::lacZ) and FP9.R (ntrC, nifA::lacZ) transconjugants containing pL46 (ntrYX expressed from its own promoter) were grown under conditions of ammonium deficiency or excess, and assayed for ß-galactosidase activity.No effect of the ntrYX genes was observed in either of the Azospirillum strains (Table 2), consistent with the results for the E. coli ET8556 background.The data suggested that the NtrY/NtrX pair has no effect on the expression of the nifA promoter in A. brasilense.The function for this regulatory pair in this organism is different from the proposed involvement in nitrogen metabolism in A. caulinodans (1).Our findings agree with those of Kaminski and Elmerich (33) who disputed the functions ascribed to the NtrY/ NtrX proteins of A. caulinodans by Pawlowski et al. (1).

Attempts to construct ntrY mutants of A. brasilense
Plasmids carrying Km cassette insertions in the ntrY genes in both orientations were constructed (Figure 1; Table 1).The wildtype A. brasilense strain FP2 was transformed with plasmids pLKIII and pLK015 in separate experiments and plated onto a selective medium containing kanamycin plus ammonium chloride (20 mM) to isolate ntrY mutants.Transformants resistant to kanamycin and sensitive to chloramphenicol (MLY9 and MLY84, respectively), indicating that the mutated gene had recombined into the chromosome by double-crossover events, were isolated and analyzed by hybridization.The hybridization results (data not shown) showed that the Km cassette was inserted into the chromosome of these mutants, however, at a site different from the ntrY gene.These results were surprising since this method of mutagenesis had previously yielded ntrBC mutants of A. brasilense (15).The reasons for our failure to obtain A. brasilense ntrY or ntrX mutants are not known.It is possible that NtrY and/or NtrX have pleiotropic effects affecting metabolic pathways involved in cell survival.

The ntrYX genes complement the Nar - phenotype of an A. brasilense nifR3ntrBC mutant
The A. brasilense nifR3ntrBC deletion mutant (HDK1) failed to grow on nitrate as sole nitrogen source and was complemented by the ntrBC genes (15).The same mutant HDK1 was complemented for nitrate-dependent growth by the A. brasilense ntrYX genes carried by plasmid pL46 (Figure 5).Taken together, these results corroborate previous observations that suggested that in A. brasilense the ntrYX pair was interchangeable with the ntrBC pair with respect to nitrate-dependent growth (34).
In this study, we sequenced and identified the ntrYX genes as a second two-component system in A. brasilense.The A. brasi- lense NtrY protein contains probable transmembrane segments located in its N-terminus and may be involved in sensing the extracellular nitrogen concentration.The NtrX protein is suggested to be a transcriptional activator of alternative nitrogen assimilation pathways such as nitrate in A. brasilense.The ntrYX genes, similar to the nifR3ntrBC operon, are apparently not required for the expression of the nifA gene in A. brasilense (11,14,15).

Figure 1 .
Figure 1.Genetic and physical maps of the DNA region containing the ntrBC/ntrYX genes of Azospirillum brasilense.The vectors of plasmid pTH6E3, pSH5S1 and pL46 are pTZ18R, pSUP202 and pLAFR3.18,respectively.Vector maps are not shown.Plasmids pLKIII and pLK015 were used to produce mutants MLY9 and MLY84, respectively, and contained the lacZ::Km cassette inserted into the NsiI site of ntrY in the direction indicated.Restriction enzymes are: E, EcoRI; G, Bgl II; N, Nsi I, and S, SalI.

Figure 2 .AFigure 3 .
Figure 2. A, Hydrophobicity graph of the N-terminal region of the NtrY proteins from Azorhizobium caulinodans (A; EMBL-GeneBank accession number X63841) and from Azospirillum brasilense (B).Arrows indicate the equivalent putative transmembrane regions.The hydropathy profiles were determined according to Kyte and Doolittle (30).

Figure 3 .
Figure 3. Continued on next page

Figure 4 .
Figure 4. Expression of the nifA promoter in the presence of the NtrY/NtrX proteins in an ntrC - Escherichia coli strain ET8556.The cultures were grown for 24 h in NFDM minimal medium.The nitrogen source was NH 4 Cl (20 mM); cultures without ammonium were supplemented with serine (100 µg/ml).The expression of the nifA promoter was determined by ß-galactosidase activity.The results are the average of 3 independent experiments run in 3 replicates and bars indicate the standard deviation.

Figure 5 .
Figure 5. Complementation of the Nar -phenotype of an Azospirillum brasilense nifR3ntrBC mutant.The cultures were incubated for 24 h in liquid NFbHPN medium with 10 mM NaNO 3 as the sole nitrogen source.Nitrate-dependent growth was determined by absorbance of the culture at 600 nm.The results are from a representative experiment.Absorbance at 600 nm 2

Table 2 .
Effect of ntrYX genes on the expression of chromosomal nifA::lacZ fusions in Azospirillum brasilense strains.