The Azospirillum brasilense rpoN gene is involved in ...

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The Azospirillum brasilense rpoN gene is involved in nitrogen fixation, nitrate assimilation, ammonium uptake, and flagellar biosynthesis Anne Milcamps, Anne Van Dommelen, John Stigter, Jos Vanderleyden, and Frans J. de Bruijn

Abstract: The rpoN (ntrA)gene (encoding sigma 54) of Azospirillum brasilense Sp7 was isolated by using conserved rpoN primers and the polymerase chain reaction, and its nucleotide sequence was determined. The deduced amino acid sequence of the RpoN protein was found to share a high degree of homology with other members of the sigma 54 family. Two additional open reading frames were found in the Azospirillum brasilense rpoN region, with significant similarity to equivalent regions surrounding the rpoN locus in other bacteria. An rpoN mutant of Azospirillum brasilense Sp7 was constructed by gene replacement and found to be defective in nitrogen fixation, nitrate assimilation, and ammonium uptake. Lack of ammonium uptake was also found in previously isolated Azospirillum brasilense ntrB and ntrC mutants, further supporting the role of the ntr system in this process. In addition, the rpoN mutant was found to be nonmotile, suggesting a role of RpoN in Azospirillum brasilense flagellar biosynthesis. Key words: Azospirillum brasilense, sigma factor, nitrogen fixation, ammonium assimilation, motility.

Resume : Le gkne rpoN (ntrA)de la souche Sp7 d'dzospirillum brasilense qui code pour sigma 54 a kt6 isolk 2 l'aide d'amorces spkcifiques pour la rkgion conservke rpoN, et la skquence nuclkotidique a Ctk dCterminCe. Un fort degrC d'homologie a kt6 observk entre la sCquence d'acides aminCs de la protCine RpoN et d'autres membres de la famille sigma 54. Deux cadres de lecture ouvert additionnels ont etk identifiks dans la rCgion rpoN de la bactkrie Azospirillum brasilense qui dkmontraient une similaritk significative B des regions Cquivalentes entourant le locus rpoN chez d'autres bactkries. Un mutant rpoN de la souche Sp7 de la bactkrie Azospirillum brasilense a CtC construit par remplacement de gknes et s'est aver6 incapable de fixer l'azote, d'assimiler le nitrate et d'ingerer l'ammoniaque. L'isolement de mutants ntrB et ntrC de I'Azospirillum hrasilense qui sont incapables d'ingkrer l'arnmoniaque est une indication supplkmentaire de l'importance du systkme ntr dans ce processus. De plus, la perte de motilitk chez le mutant rpoN suggke que RpoN chez Azospirillum brasilense joue un r61e important dans la biosynthkse flagellaire. Mots clts : Azospirillum brasilense, facteur sigma, fixation de l'azote, assimilation de l'ammoniaque, motilitk. [Traduit par la redaction]

Received September 5, 1995. Revision received December 22, 1995. Accepted January 3, 1996.

I

A. Milcamps. MSU-DOE Plant Research Laboratory and National Science Foundation Center for Microbial Ecology, Michigan State University, East Lansing, MI 48824, U.S.A. A. Van Dommelen. FA. Janssens Laboratory of Genetics, Katholieke Universiteit Leuven, De Croylaan 42,3001 Heverlee, Belgium. J. Stigter. MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, U.S.A. J. Vanderleyden. FA. Janssens Laboratory of Genetics, Katholieke Universiteit Leuven, De Croylaan 42, 3001 Heverlee, Belgium. F. J. de Bruijn.l MSU-DOE Plant Research Laboratory, National Science Foundation Center for Microbial Ecology, and Department of Microbiology, Michigan State University, East Lansing, MI 48824, U.S.A. Author to whom all correspondence should be sent to the following address: MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, U.S.A. (e-mail: [email protected]).

Can. J. Microbiol. 42: 467-478 (1996). Printed in Canada / Imprim6 au Canada

Introduction Azospirillum brasilense belongs to a group of plant growth promoting bacteria, capable of fixing nitrogen under microaerobic conditions. The regulation of expression of the Azospirillum brasilense nitrogen fixation (nif) genes has been intensively studied but remains less understood in comparison to that of other diazotrophs such as Klebsiella pneumoniae and several rhizobial species (Merrick 1992; Fisher 1994). As in other diazotrophs, Azospirillum brasilense nifgene expression is controlled by the regulatory gene nifA, and this gene has been cloned and characterized (Liang et al. 1991). While in K. pneumoniae and Azorhizobium caulinodans (Gussin et al. 1986; Pawlowski et al. 1991) n f A expression is regulated by the nitrogen regulation (ntr) system in response to fluctuating concentrations of combined nitrogen, in Azospirillum brasilense ntrC mutants nifA expression is only slightly affected (Liang et al. 1993). However, for this ntrC mutant strain, a process of so-called ammonium switch-off of nitrogenase has been described (Zhang et al. 1993). This mechanism causes


Can. J. Microbiol. Vol. 42, 1996 rapid and reversible inhibition of nitrogenase activity, which is exerted at the posttranslational level via ADP-ribosylation in response to micromolar concentrations of ammonium. Motility of Azospirillum brasilense is mediated via the action of a single polar flagellum and several lateral flagella (Tarrand et al. 1978). The polar flagellum is synthesized constitutively, while the lateral flagella are only produced when the bacterium is grown on semisolid medium (Hall and Krieg 1983). It has been suggested that the polar flagellum may play a role in the attachment of the bacterium to plant roots (Croes et al. 1993). The gene encoding the major protein of the lateral flagella has been identified recently (Moens et al. 1995), but nothing is known about the gene(s) encoding the proteins of the polar flagellum. Sigma factors determine the promoter specificity of the bacterial RNA polymerase enzyme. A number of different sigma factors, involved in the transcription of constitutively expressed genes, and genes that are environmentally regulated have been described (Merrick 1993). In general, two families of sigma factors can be distinguished: the sigma 70 family, containing multiple types of sigma factors and the sigma 54 family, with sigma 54 as its only member. At present, sigma 54 has been identified in a direct or indirect fashion in 20 different bacterial genera. The DNA sequence of the rpoN (ntrA) gene, encoding the sigma 54 factor, has been determined for 17 bacterial species and evidence for its existence in 12 other bacteria has been reported. The rpoN gene was originally identified in enteric bacteria as a component of a global regulation circuit responding to the cellular nitrogen status (Magasanik 1982; de Bruijn and Ausubel 1983; Hunt and Magasanik 1985). In diazotrophs, such as K. pneumoniae, several operons involved in nitrogen metabolism, such as the nitrogen fixation (nif) and glutamine synthetase (glnA) genes, are subject to RpoN control. Since then, multiple and diverse physiological functions in bacteria have been found to be RpoN regulated (Kustu et al. 1989). These include anaerobic fermentation in Escherichia coli (Birkmann et al. 1987), the utilization of xylene in Pseudomonasputida (Inouye et al. 1989),pilin and flagellin formation in Pseudomonas aeruginosa (Totten et al. 1990), and the degradation of phenol in Pseudomonas species (Shingler et al. 1992) and Acinetobacter calcoaceticus (Ehrt et al. 1994). Sigma 54 is neither structurally nor functionally related to members of the sigma 70 family. Genes with promoters recognized by RNA polymerase containing sigma 54 show a conserved sequence at position -241-12 relative to the start of transcription, rather than the canonical -351-10 motif found in genes transcribed by the RNApolymerase - sigma 70 complex (Morett and Buck 1989). In addition, rpoN-dependent promoters are generally subject to positive control, exerted by transcriptional activator proteins whose activity is modulated by physiological signals and which bind at conserved sequences (UAS) located upstream of the promoter consensus motif (Buck et al. 1986). DNA loop formation and binding of the integration host factor (IHF) have been reported to be involved in generating the contact between activator and bound sigma 54 - RNA polymerase (Santero et al. 1992). Merrick and Chambers (1992) and Cannon et al. (1994,1995) describe three domains for RpoN proteins, with functions ranging from promoter recognition to interaction with the activator.

In this paper we describe the isolation and characterization of the Azospirillum brasilense rpoN gene and flanking regions, as well as a phenotypic analysis of an rpoN mutant, constructed using gene replacement. In addition, we report the results of ammonium transport studies in Azospirillum brasilense rpoN, ntrB, and ntrC mutants.

Materials and methods Bacterial strains and plasmids The bacterial strains and plasmids used in this study are described in Table 1.

Media and growth conditions Azospirillum brasilense strains were grown at 2S0Cin L* broth, which is Luria-Bertani medium supplemented with 2.5 rnM CaC12 and 2.5 mM MgS04, or in minimal AB (Vanstockem et al. 1987) liquid or solidified medium. Rhizobium meliloti and Azorhizobium caulinodans strains were grown at 28 and 37OC, respectively, on TY plates (Beringer 1974). LB agar and LB liquid medium (Miller 1972) were used for the growth of E. coli strains at 37OC. Antibiotics were added at the following concentrations: 100 pg ampicillinlml, 10 p g tetracycline/mL, and 25 pg kanamycin/mL for the E. coli strains; and 200 pg kanamycin/mL for the rhizobial species.

Southern blot hybridization Total genomic DNA of Azospirillum brasilense was isolated by the method described by Marmur (1961) and digested with restriction enzymes. The resulting DNA fragments were separated on a 0.7% agarose gel and transferred to a nitrocellulose membrane according to the manufacturer's procedure of membrane transfer and detection methods (Amersham Life Science Inc., Arlington Heights, Ill.). The hybridization probe was generated by random primed incorporation of digoxigenin (DIG) labeled dUTP, using the DIG DNA labeling kit from Boehringer Mannheim (Indianapolis, Ind.). Hybridization was performed at 6S°C, and the filters were washed and processed for signal detection according to the protocol of Boehringer Mannheim.

Colony blot hybridization A genomic DNA library of Azospirillum brasilense Sp7, constructed in the broad host range cosmid vector pLAFRl (Friedman et al. 1982), was transferred to nitrocellulose filters as described by Amersham (Life Science Inc.). The filters were used for hybridization with a DIG-labeled probe, as described for the Southern blot hybridization experiments.

Polymerase chain reaction Three oligonucleotide primers were designed, derived from three conserved regions found in all RpoN proteins. Primer 1 (5'-TCGCGG/ATACmTCGCGACCGTGCGGmC-3') was derived from DNA sequences around the RpoN box (positions 1605-1630 in Fig. 2), primer 2 (5'-CGGGTG/CACCT/GCC/GAACAAA/GTACATG-3') was derived from a conserved motif about 70 amino acids closer to the N terminus (positions 1396-1420), and primer 3 (5'CTGGAGA/TIG/CAACCCGCTGCTG/CGAG-3') was derived from the N-terminal portion of the protein (positions 247-271). Polymerase chain reactions (PCRs) were carried out according to de Bmijn (1992). The products of the PCRs were analyzed by agarose gel electrophoresis on 1% gels and isolated from the gel matrix by centrifugation through a filtration column. The purified PCR products were used for additional PCRs, as probes for DIG hybridizations, and cloned into Blue Script vectors for DNA sequence analysis.

Milcamps et al.

Table 1. Bacterial strains and plasmids. Relevant characteristics

Strain or plasmid


Strains Azospirillum brasilense Sp7 Azospirillum brasilense FAJ301 Azospirillum brasilense FAJ302 Azospirillum brasilense 7148 Azospirillum brasilense 7 194 Rhizobium meliloti 1680 Azorhizobium caulinodans ORS57lN136-lc Escherichia coli DHSa Escherichia coli HB 101 Escherichia coli TH1 Plasmids pUC18 pBluescript I1 pSUP202 pUC4K pFAJ26 pFAJ27 pFAJ28 pFAJ29 pFAJ30 pFAJ30- 1 pFAJ30-2

Source or Ref. Tarrand et al. 1978 This study This study Liang et al. 1993 Liang et al. 1993 Ronson et al. 1987 Stigter et al. 1993

Wild type (ATCC29145) rpoN:: Km cassette, Kmr rpoN::Km cassette, Kmr ntrC::Tn5-148, Kmt ntrB::TnS-194, Kmr rpoN: : Tn.5, Nmr rpoN::TnS, Kmr

Hanahan 1983 Boyer and Roulland-Dussoix 1969 Hunt and Magasanik 1985 Cloning and sequencing vector, Apr Cloning and sequencing vector, Apr ColEl replicon, Apt, Cmr, Tct Kmt, Apt, Km cassette 200 bp Sp7 PCR product cloned into BS, AP' pUC18 with PstI rpoN fragment, Apl pUC18 with SalI rpoN fragment, Apt pLAFRl clone of Sp7, carrying rpoN, Tc'I pSUP202 with SalI rpoN fragment, Tc' pFAJ30 with Km cassette, Tcr, Kml pFAJ30 with Km cassette, Tcr, Kml

DNA sequence analysis The nucleotide sequence of the rpoN region was determined, by analyzing the PstI and SalI fragments of the Azospirillum brasilense rpoN region, cloned in pUC18 (pFAJ27 and pFAJ28, respectively). pFAJ27 was subjected to unidirectional deletions using the Erase-aBase kit from Promega (Madison, Wis.). DNA sequencing reactions were carried out according to the method of Sanger et al. (1977), using a DNAsequencing kit (USB, Cleveland, Ohio; sequenase version 2.0) and the M13 forward and reverse sequencing primers. Some of the sequencing was carried out with the AutoRead Sequencing kit (Pharmacia-LKB, Uppsala, Sweden) and an automated laser fluorescent sequencer (Pharmacia-LKB). Oligonucleotides corresponding to previously determined DNA sequences were synthesized by the Macromolecular Facility at Michigan State University and used as primers to complete the double-stranded DNA sequence analysis. pFAJ28 was used to determine the DNA sequence of the 5' end and upstream region of the Azospirillum brasilense Sp7 rpoN gene. The DNA sequence obtained was analyzed using the programs SeqEd (Applied Biosystems, Foster City, Calif.), Sequencher (Gene Codes Corporation, Ann Arbor, Mich.) and CodonUse (C. Halling, University of Chicago, Chicago, Ill.). Data-base searches were carried out with the program Blastx (Altschul et al. 1990). The alignments of the deduced proteins were obtained using the program Pileup from the GCG package (Genetics Computer Group, Madison, Wis.).

Nucleotide sequence accession number The nucleotide sequence data reported in this paper have been submitted to the EMBL Nucleotide Sequence Database under accession No. X84991.

Phenotypic characterization of the rpoN mutant strains Growth characteristics Wild-type Azospirillum brasilense Sp7 and the Sp7 rpoN mutant strain were analyzed for their growth characteristics in L* medium, in

Yanisch-Perron et al. 1985 Stratagene(La Jolla, Calif.) Simon et al. 1983 Pharmacia-LKB This study This study This study This study This study This study This study

minimal AB medium with fructose (0.5%) or malate (20 mM), and in minimal AB medium with ammonium (20 or 5 mM), nitrate (10 mM), glutamine (5 mM), arginine (1 mg/mL ), proline (1 mg/mL ), or histidine (1 mg/mL) as nitrogen sources. Growth with dicarboxylic acids (malate, 20 mM; succinate, 20 mM; fumarate, 20 mM) as carbon source was also tested. Synthesis of exopolysaccharide (EPS) a n d indole-3-acetic acid

(IAA) EPS synthesis was examined by growth on BIV plates (de Troch et al. 1992) containing 200 mg Calcofluor dye/mL and screening for fluorescence under UV light. The same medium was used to analyze the production of the dark pigment normally produced by Azospirillum brasilense cells after several days of incubation. IAA production was colorirnetrically determined with Salkowsky reagents in the supernatant of cultures of the wild-type and mutant strains, as described by Costacurta et al. (1 994). Uptake of [ 1 4 ~ ] ~ ~ f l ~ 3 + Wild-type Azospirillum brasilense Sp7 and rpoN, ntrB, and ntrC mutant strains were pregrown in minimal AB medium containing 10 mM aspartate and kanamycin (25 pg/mL), when appropriate. When the cultures had reached a cell concentration of approximately 109 cells/mL, the cells were centrifuged and resuspended in the same medium without nitrogen source. After 15 min of incubation at room temperature, [14C]methylammonium (Amersham, Gent, Belgium; 2.11 Gbqlmmol) was added to a final concentration of 8.75 pM. Samples (100 pL) were filtered through a MultiScreen Durapore (type DV, 0.65-pm pore size) filtration plate, placed on a multiscreen vacuum filtration manifold. Filters were dried and the radioactivity was measured using a scintillation counter (Wallac 1410; Pharmacia-LKB). The protein concentration was determined with the bicinchoninic acid assay (Smith et al. 1985), after lysis of the cells in 1 N NaOH at 6S°C. As a negative control, NH4+uptake was measured in wild-type Azospirillum brasilense Sp7 grown under

Can. J. Microbiol. Vol. 42. 1996 ammonium-uptake repressing conditions (20 mM NH4+ ) (Hartmann and Kleiner 1982).


Nitrogen fixation assays (acetylene reduction assay, ARA) Nitrogen fixation conditions were created by inoculating 20 pL washed Azospirillum brasilense cells (exponential phase) into semisolid N-free medium (0.07% agarose) supplemented with 5 mM glutamate. ARA was measured after 12 h of growth and after 6 and 24 h of incubation in the presence of acetylene, using a Hewlett Packard 5890A gas chromatograph, with a plot fused silica column.

Motility assays and visualization offlagella Motility of Azospirillum brasilense wild-type and rpoN mutant strains was examined in liquid grown cultures under a Leitz laborlux S light microscope. For transmission electron microscopic analysis, Azospirillum brasilense cells were carefully scraped from a fresh L* plate and resuspended in 0.9% NaCl sterile water. Aliquots were adsorbed to a Formvar-coated, carbon-stabilized grid for 1 min and negatively stained with 1% phosphotungstate solution for 1 min. The grids were examined with a Philips EM400 electron microscope.

Results Isolation of the Azospirillum brasilense rpoN gene Comparison of the RpoN protein sequences of several bacteria revealed significant overall conservation, with stretches of high homology in the NHzterminal and COOH-terminal regions. In particular, a sequence of nine perfectly conserved amino acids (RpoN-box) was identified, which has been found in all species examined thus far (Merrick 1993). Based on these conserved sequences, DNA primers were derived (see Materials and methods) and used in a PCR with genomic DNA of Azospirillum brasilense Sp7 as template. Using the RpoN-box derived primer (primer I), in combination with either one of the other two primers (primers 2 and 3; see Fig. 2) PCR yielded two amplification products of the expected sizes (0.2 and 1.2 kb, respectively). Both PCR products were isolated and the amplified DNA was purified. The 1.2-kb PCR product was used for a reamplificationreaction, using primers 1 and 2, which yielded the expected 0.2-kb fragment. The 0.2-kb PCR product was cloned into the Blue Script vector and subjected to DNA sequence analysis. The deduced amino acid sequence of the open reading frame carried by this cloned DNA fragment showed a significant similarity with several bacterial RpoN proteins. The highest similarity was found with Bradyrhizobiumjaponicum RpoNl (Kullik et al. 1991) (74% identity, 83% similarity) and Rhizobium meliloti RpoN (Ronson et al. 1987) (65% identity, 81% similarity; data not shown). To examine if more than one copy of rpoN was present in Azospirillum brasilense, the 0.2-kb PCR product was used as a probe for Southern blots carrying Azospirillum brasilense genomic DNA digested with SalI, PstI, HindIII, EcoRI, or BamHI. In each digest, a single hybridizing band was observed, suggesting that there is a single copy of rpoN in Azospirillum brasilense Sp7. The 0.2-kb PCR product was subsequently used to screen a Azospirillum brasilense Sp7 genomic library,constructed in the vector pLAFR1 (see Materials and methods). Three positive clones were obtained. Their restriction patterns were found to be identical and one was chosen for further analysis (pFAJ29).

Fig. 1. Physical map of the Azospirillum brasilense Sp7 rpoN region. The ORFs flanking the Azospirillum brasilense Sp7 rpoN gene show significant similarity to the ORFs in the Sfupstream and 3' downstream regions of the B. japonicum and Rhizobium meliloti rpoN genes.

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Genetic complementation with the Azospirillum brasilense rpoN gene Plasmid pFAJ29 was transferred by CaClz transformation into an rpoN mutant of E. coli (strain TH1; Hunt and Magasanik 1985). The rpoN mutation in this strain causes a deficiency in the utilization of arginine as nitrogen source (Aut- phenotype). Plasmid pFAJ29 was not able to restore growth of TH1 on minimal medium with arginine as sole nitrogen source. In the case of at least two bacteria (B. japonicum and Rhodobacter capsulatus), rpoN expression has been reported to be environmentally regulated (by oxygen, and ammonium and oxygen, respectively) (Kullik et al. 1991; Jones and Haselkorn 1989). Therefore strain THl harboring pFAJ29 was grown under microaerobic conditions, but growth on arginine could still not be restored. Subsequently,rpoN mutants of Rhizobium meliloti and Azorhizobium caulinodans were used for complementation experiments. These mutant strains fail to grow on minimal medium with nitrate as the sole nitrogen source (Ronson et al. 1987; Stigter et al. 1992). When plasmid pFAJ29 was introduced, by conjugation, into these mutant strains, growth was found to be fully restored, suggesting that plasmid pFAJ29 carries a functional Azospirillum brasilense rpoN locus. Sequence analysis of the Azospirillum brasilense Sp7 rpoN gene and flanking regions A restriction map of the insert of pFAJ29 was constructed (see Fig. I). Southern hybridization experiments using the 0.2-kb PCR product (see above) as probe revealed that the Azospirillum brasilense Sp7 rpoN gene was located on a 7.5-kb SalI fragment and a 2.3-kb PstI fragment. Both fragments were subcloned into pUC18 and their DNA sequence was partially determined. DNA sequence analysis of a 2.6-kbp rpoN region revealed the presence of three open reading frames (ORFs), transcribed in the same orientation. The nucleotide sequence and deduced amino acid sequence of the Azospirillum brasilense Sp7 rpoN gene and ORFl are shown in Fig. 2. The sequence alignment of the Azospirillum brasilense Sp7 rpoN gene product and other RpoN proteins is shown in Fig. 3. The Azospirillum brasilense Sp7 rpoN gene is 1569 bp long and encodes a protein of 523 amino acids (aa). At position 122 in Fig. 2, the ATG was designated as the putative start codon because of sequence comparisons with other rpoN sequences.

Milcamps et al.

47 1

Fig. 2. Nucleotide sequence of the Azospirillurn brasilense Sp7 rpoN gene and flanking regions. The deduced amino acid of the Sp7 rpoN gene and a segment of the Supstream ORF (ORFI) are shown. Stop codons are indicated by asterisks. The three PCR primers used to isolate the Azospirillurn brasilense Sp7 rpoN gene are underlined. TACATCTTGCACGATGGTGTGGTACTAATGG Y I L H D G V V L M

31

AGGGAGAACCGGCCGAGATCGTGGCGCACGAGGACGTGCGCCGTCTACCTCGGGGACCGGTTCAGCCTCTAGTGCGGTTTCCCTGCCC E G E P A E I V A H Q D V R R V Y L G D R F S L *

121

ATGGCGCTCAGCCAACGCCTCGATCTTCGTCAGTCCCAGTCCCTGGTGATGACTCCGCAGCTGCAGCAGGCCATCAAGCTGCTGCAGCTG M A L S Q R L D L R Q S Q S L V M T P Q L Q Q A I K L L Q L

211

TCGAACATCGAACTGTCGGATTTCGTGGATCGGGAGATCGAGCAGAACCCTTTGCTGGAGCGCGACGGCGGTCCCGGCGAGGGCGGCGGC

301


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GAATCCGGGGGCGACGGTGCCGGTGACGGCGGGGGCGAGCCCGGAGCGGTGGACCTTGCCGCGCCGGAGGAGCGCCAGCCGCCGATGACC E S G G D G A G D G G G E P G A V D L A A P E E R Q P P M T

391

GACGGGCGCACCCGCGACACGGTGGAGATGACGTCGTCGGAGACGATGGTCGGCCTCCGACGCGCCGCTCCACCGATTTCGAGAAT D G R T R D T V E M T S S E T M G S A S D A P L D T D F E N

481

GTCTATTCCGACGACCGTTTCTCCGACGGGACGGACGGCTCCAGCGACGTCTACGGCTCCTGGCAGGAGCGCGGCGGCCGCGGCGGCTTC V Y S D D R F S D G T D G S S D V Y G S W Q E R G G R G G F

571

GAGGACGACGAGTCCAACCTGGAAGCCACGCTGACCGGCCAGAAGAGCCTGCGCGACCATCTGACCGAGCAGTTGAAGATCGACCTGCCG

661

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GACCTCGGCGACCAGCTCATCGGTCTGGCGCTGATCGACATGCTGGACGGCCGGCTGGATCACCGGGCTGGAGGTCGAGAGCCTCGCG D L G D Q L I G L A L I D M L D E A G W I T G L E V E S L A

751

GGGCAGCTCGGCTGCGCGCCGGAGCGGGTGGAACGGGTGCTCGCCGCCTGCCAGCGCTTCGACCCGCCGGGCATCTTCGCGCGGTCGTTG G Q L G C A P E R V E R V L A A C Q R F D P P G I F A R S L

841

AAGGAATGTCTGGCGATCCAGCTGCGCGAGAAGAACCGCTTCGACCCGGCGATGGAGGCCCTGCTCGACCATCTGGAGCTGCTGGCGGCG

931

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CGCAACCTGCCGGCGCTGATGAAGGTCTGCGGCGTGGACGCCGAGGACGTCGCCGACATGGTGGCGGAGATCCGCAAGCTGAACCCCAAG R N L P A L M K V C G V D A E D V A D M V A E I R K L N P K

1021

CCGGCGCTCAGCTTCGACCACACGCCGGCCCAGCTCGTCACCCCCGACATCCTGATGCGCGCCAACCCGGGCGGCGGCTGGCTGATCGAC

1111

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A

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CTGAACCCGGACACGTTGCCGCGGGTGCTGGTCAACCACCGTTATTTCGCGCGCATCTCCGGCACCGCCCGCAACAAGGCGGACAAGGAA 1201 L N P D T L P R V L V N H R Y F A R I S G T A R N K A D K E TACATCACCGAGCGCTTCCAATCGGCCAACTGGCTGGTCAAGTCGCTGCACCAGCGCGCCACCACGATTTTGAAGGTGGCCAGCGAGATC Y I T E R F Q S A N W L V K S L H Q R A T T I L K V A S E I

1291

ATCCGGCAGCAGGACGCCTTTTTCATCCACGGCGTCTCGCATTTGAAGCCGCTGATCCTGCGCGACATCGCCGAGGCGATCGGCATGCAC

1381

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CAGGGGGCCGACGGGCAGGCCGCCCACTCGGCGGAGGCCGTGCGCTACCGCATCAAGGCGATGATCGACGCCGAGAAGCCGGACGACGTG Q G A D G Q A A H S A E A V R Y R I K A M I D A E K P D D V

1561

TTGTCGGACGATAAAATCGTCGAAATCCTTCGTGGTGAAGGGATCGACATTGCGCGCCGGACGGTCGCGAAGTATCGTATCGTGCGC

1641

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ATTCCGTCATCGGTGCAGCGACGCCGTGCCAAGATGTCACGCATGTAACCGGGCCGGGAGGGGGTCGTCCTTGATTGACAGCCCATACCG

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CCGTCACTATGGTCCCCGTCCGCACGTGGCGGCCTTCCGTCGTCCTATG

However, no obvious Shine-Delgarno-like sequence was detected in front of the start codon. The Azospirillum brasilense RpoN protein shows the highest similarity with the RpoN proteins of the rhizobial group. Typical domains of the sigma 54 factors could be distinguished in the 1348-1405 region (helix-turn-helix motif), and the

1601-1631 region (RpoN box). The GC content of the rpoN gene was 89.6%, within the range characteristic for Azospirillurn brasilense (Milcamps et al. 1993). Downstream of the rpoN gene, an ORF of about 199 aa (ORF2) was detected and its deduced polypeptide was found to share significant similarity with the proteins encoded by

Can. J. Microbiol. Vol. 42, 1996

472 GGPGEGGG NDEASGGE SDDMGAE VQPADEPTIS VPIADEA.VS SEPESPEL EGDHEPAV QTDLHDEIDT

ESGGDGAGDG APAEAGQFSD APTEVDQVSG DREDAG..PH VRIGPSVMRH DPPNPQ.... EVEAERDASL Q....QPQDN PDMLOBHQDQ

SSDVYG AQDMP ARDGAM PELLGQ PELLGQ GAGSGS EHAGGQ IDDELp

SWQERGG TTYTEWG TTYTEWGG WKSMPGAG. WXSMPGA S GSSIEWG IDWSRAG WQGETTQS.


B

BEDAEDYDS Ab Bj2 Bjl

Rm NGR Ac CC St Av

APEERQPPMT DGRTRDTVEM TSSETMGSAS DA GGPGEAFEPG QEEWUSKDLG .TRAEI.... EQ

................................

TGGETDEAAG Q S D L Y D S M S EAGGAEESVD HGDLYDSATT PTPPDSGAPV SGDWMESDMG ADRE...... .ADALE QEGYESGAAS ED. ..GGTLE

RSGERL.... SE SPGERL.... RS SSREAI.... ET

...................... .............. QKE....MPE EL EGDWHERIPS EL

GGEPGAVDLA SDGGHNDEPG

80 80 70

P.......AE V.......TA

71 72 69

..........

.......

EEZL

TQVDAVADTT B O DPLDT..... 69 STLDTTPGBY 7 7

8

FE SG SG D ND DR SF

..

% ..

.......... 1 4 2 236 230 182

Rm FVGGR NGR SSPVO

219 221 218 207 192 a15

Ab

SRM..... SALGNVLS NMWS LPRPRDS LPRQPAS

B j2 Bj1

......

.............. TAMSDRSRNP EPA. TMNSRASGGT GLDK ERCRQAASA. DCGFFAAAN. DCGFPAAAN.

.... .... .... ....... .............. MKEAV..... .............. V.........

F

Rm NGR AC CC St AV

M......... RpoN bcx

..............

525 537 484

513 525

513 497 476 50 1

Milcamps et al.

473


Fig. 3. Alignment of the deduced amino acid sequences of the rpoN genes of Azospirillum brasilense (Ab), Bradyrhizobiumjaponicum (Bjl) and (Bj2), Rhizobium meliloti (Rm), Rhizobium sp. NGR (NGR), Azorhizobium caulinodans (Ac), Klebsiellapneumoniae (Kp), Caulobacter crescentus (Cc), and Azotobacter vinelandii (Av). Identical amino acids (at least five out of nine) are highlighted in black. The DNA binding motif (HTH) and the conserved RpoN box are underlined.

Table 2. Phenotype of wild-type and rpoN mutant strains of Azospirillum brasilense Sp7.

Growth on rich medium Growth on minimal medium Growth on dicaroxylic acids (malate, succinate, fumarate) Growth on nitrate Growth on glutarnine, histidine, arginine, and proline [14C]CH3NH3+uptake Nitrogen fixation EPS synthesis IAA production Motility

Wild type Sp7

Mutant FAJ301/FAJ302

+ + + +

-

+ + -

Note: For a description of the appropriate growth media and assay conditions, see Materials and methods.

ORF104 and ORF203 of Rhizobium meliloti and B. japonicum, respectively. The region 5' upstream of the Azospirillum brasilense Sp7 rpoN coding region was sequenced in search of a promoter consensus sequence. Instead, the 3' end of an additional ORF (ORFI) was detected and its deduced polypeptide shared similarity with proteins encoded by corresponding ORFs upstream of the rpoN gene in several other bacteria (Rhizobium meliloti, Ronson et al. 1987; Azorhizobium caulinodans, Stigter et al. 1993; K. pneumoniae, Merrick and Coppard 1989; Rhizobium sp. NGR234, van Sloten and Stanley 1991; P. putida, Inouye et al. 1989; Thiobacillus ferooxidans, Berger et al. 1990; Salmonella typhimurium, Popham et al. 1991). The absence of sequences characteristic for promoters or transcriptional terminators, led us to suggest that the Azospirillum brasilense Sp7 rpoN gene might be cotranscribed with ORF1. The proposed genetic organization of the Azospirillum brasilense Sp7 rpoN region is shown in Fig. 1.

Construction of an Azospirillum brasilense rpoN mutant The 2.3-kb PstI fragment, containing a SmaI site located in the middle of the rpoN gene, was subcloned into pSUP202, generating the plasmid pFAJ30. A 1.3-kb kanamycin cassette, derived from pUC4K, was inserted into the SmaI site, resulting in plasmids pFAJ30-1 and pFAJ30-2. pFAJ30-1 and pFAJ30-2 contain the kanamycin cassette inserted into the rpoN gene in either orientation, relative to the transcriptional direction of the rpoN gene. Both constructs were introduced into wild-type Azospirillum brasilense strain Sp7. Kanamycin-resistant mutants (AbFAJ301 and AbFAJ302) were isolated and verified

by Southern blot analysis for double homologous cross-over events.

Analysis of the Azospirillum brasilense Sp7 rpoN mutant for growth, nitrogen metabolism, EPS, and IAA synthesis Several phenotypic traits described for other rpoN-deficient bacteria were examined in case of the Azospirillum brasilense rpoN mutant strains (Table 2). As expected, the nitrogen fixation ability of the rpoN mutant strains was abolished. Also the assimilation of nitrate was disturbed in the mutant strain. Unlike observed with the Rhizobium and Bradyrhizobium rpoN mutant strains, the Azospirillum brasilense rpoN mutants were not found to be affected in the utilization of dicarboxylic acids. EPS synthesis, brown pigment production, and IAA synthesis were not observed to be affected in the rpoN mutant strains. Analysis of Azospirillum brasilense Sp7 rpoN mutant for motility Since in the case of some bacteria rpoN has been described to play a role in motility, this phenotypic trait was also analyzed for the Azospirillum brasilense rpoN mutant strain. Light microscopy of rpoN Azospirillum brasilense cells in liquid medium, revealed a total lack of motility. Electron microscopic studies of negatively stained Azospirillum brasilense cells, from semisolid medium in which the lateral flagella are normally induced, revealed that the rpoN mutant lacked both type of flagella (see Fig. 4). Therefore, we conclude that the Azospirillum brasilense Sp7 rpoN gene also influences motility via both the polar and lateral flagella.

474



Fig. 4. Electron microscopic graphs of negatively stained Azospirillum brasilense Sp7 wild type and the Sp7 rpoN mutant strain. Sp7 wild type has a polar flagellum and several lateral flagella (A). Both flagella types are absent in the mutant strain (B). Bar, 0.5 pm.

Analysis of Azospirillum brasilense Sp7 rpoN, ntrB, and ntrC mutants for ammonium uptake To study the role of nitrogen regulatory genes in the expression of the ammonium carrier of Azospirillum brasilense (Hartmann and Kleiner 1982), ammonium uptake was measured in the rpoN mutant strain constructed here and the ntrB and ntrC mutant strains isolated by Liang et al. (1993). The ntrC gene product in Azospirillum hrasilense Sp7 has been shown to be involved in the regulation of nitrate utilization, the switchoff of nitrogenase by ammonia, and to a lesser extent, nifA expression. Ammonium uptake was measured in wild type and ntr strains by using [14C]methylammoniumas a radioactive ammonium analogue. This method has been successfully applied to measure ammonium uptake in lower eukaryotes and in prokaryotes (Kleiner 1985 and references therein). The results of this methylammonium uptake assay are presented in Fig. 5. These results suggest that the rpoN, ntrB and ntrC genes are all necessary for active ammonium uptake.

Discussion Previous structural and functional analysis of the promoter region of the nif and fix genes of Azospirillum brasilense

revealed a rpoN-dependent consensus motif (at position -241-12 with respect to the transcriptional start), the presence of upstream activator sequences (UASs) and the requirement of NifA as activator (Milcamps and Vanderleyden 1993). The nifA gene of Azospirillum brasilense strain Sp7 has been isolated and the DNA sequence was determined by Liang et al. (1991). In this study, we have isolated and described a second regulatory factor for nitrogen fixation: the rpoN (ntrA) gene of Azospirillum brasilense Sp7. The nucleotide sequence of the Azospirillum brasilense Sp7 rpoN and flanking regions was determined. Analysis of the deduced amino acid sequence of the rpoN gene revealed significant similarity with RpoN proteins of other bacteria. The highest degree of similarity was found with the B. japonicum rpoNl gene product (Kullik et al. 1991). The genetic organization of the Azospirillum brasilense Sp7 rpoN region was found to be similar to that found in B. japonicum and other bacteria. In most bacteria analyzed, one to five ORFs have been identified downstream of the rpoN gene and the nucleotide sequences of these ORFs tend to be highly conserved. Upstream of the Azospirillum brasilense rpoN, we identified an ORF, the deduced polypeptide of which shows significant similarity to a family of ATP-binding proteins. A similar situation is described for several rhizobial strains

475

Milcam~set al.


Fig. 5. Uptake of [14C]CH3NH3+ in wild-type Azospirillum brasilense Sp7 and rpoN, ntrB, and ntrC mutant strains. o,Sp7 wild type; m, Sp7 as negative control (grown in carrier repressing +, 7 148 (ntrC::TnS).The mean conditions); *, FAJ301 (rpoN::Krncassette); x, 7194 (ntrB::TnS); and standard deviations are derived from four independent experiments. The mean standard deviation for strains 7148,7194, FAJ301, and Sp7 as negative control was 0.11 nmol [14C]CH3NH3+/mg protein. Note that the symbols o,*, x, and + overlap.

Time (min)

(Fisher 1994). The function of this ORF is not known and has been studied only in the case of Rhizobium meliloti and Rhizobium sp. NGR234 (Albright et al. 1989; van Sloten and Stanley 1991). Since mutagenesis of this ORF was found to be impossible, it was suggested that mutations in this ORF are lethal. In the case of B. japonicum, two copies of rpoN (rpoN1 and rpoN2) have been described, both of which are involved in nitrogen fixation (Kullik et al. 1991).Also in the case of Azorhizobium caulinodans, two rpoN copies have been postulated to be present (Stigter et al. 1992). In the case of Rhodobacter sphaeroides, two rpoN copies have also been isolated, although only one of these copies appears to be functional (Meyer and Tabita 1992). Using the rpoN gene of Azospirillum brasilense Sp7 as a probe for Southern hybridization experiments revealed that only one copy of rpoN is likely to be present in Azospirillum brasilense. Therefore, we postulate that Azospirillum has only one copy of the rpoN gene. We were able to show that the Azospirillum brasilense Sp7 rpoN gene could complement rpoN mutants of Rhizobium meliloti and Azorhizobium caulinodans, but not the rpoN mutant of E. coli. The absence of complementation in the latter mutant background may be due to the lack of Azospirillum brasilense Sp7 promoter recognition in E. coli. In several bacterial species, RpoN is involved in the regulation of nitrogen assimilation processes. For example, Azotobacter vinelandii, K. pneumoniae, Azorhizobium caulinodans, Rhizobium meliloti, and B. japonicum rpoN mutants are deficient in growth on nitrate as sole nitrogen source (Kustu et al. 1989). In addition, nitrate assimilation of Azospirillum brasilense is regulated by rpoN. This is not surprising since ntrB and ntrC mutants of Azospirillum brasilense are reported to be deficient in growth on nitrate (Liang et al. 1993).

Measurements of CH3NH3+uptake indicate that the Azospirillum brasilense rpoN gene, as well as the ntrB and ntrC genes, are involved in ammonium transport. Studies in E. coli and K. pneumoniae (Jajakumar et al. 1986; Kleiner 1985) indicate that in these microorganisms the ntr system also regulates the activity of an ammonium carrier. The absence of ammonium uptake in the Azospirillum brasilense rpoN, ntrB, and ntrC mutants seems, however, to be in conflict with the observation that the rpoN, ntrB, and ntrC mutants grow normally on medium with a low ammonium concentration (Liang et al. 1993; this study). In addition, it was shown by Zhang et al. (1994) that single ntrC mutants of Azospirillum brasilense, grown in the presence of ammonium, show only a partial loss of nitrogenase activity in comparison with the wild-type strain. Studies with ntrC-draG double mutants of Azospirillum brasilense (Zhang et al. 1994), however, revealed a normal switchoff of the nitrogenase in response to low ammonium concentrations (0.2 mM). This suggests that absence of total nitrogenase switch-off in ntrC mutants is rather due to an altered regulation of the dinitrogenase reductase activating glycohydrolase (draG gene product), than to an inability of ntrC mutants to sense low ammonium concentrations (Zhang et al. 1994). These contrasting observations can be explained by assuming the existence of (at least) two types of ammonium carriers in Azospirillum brasilense. Only one of them, which is regulated by the ntr system, can transport [l4C]methylammonium. The simultaneous existence of an inducible methylammonium uptake system and a constitutive ammonium uptake system has also been suggested for Rhodobacter sphaeroides (Cordts and Gibson 1987), Rhodobacter capsulata (Cordts and Gibson 1987), and Anacystis nidulans (Boussiba et al. 1984). The constitutive ammonium uptake



system in all three organisms has a negligible affinity for methylammonium. Under conditions of nitrogen fixation, reduced levels of glutamine synthetase activity have been measured in the Azospirillum brasilense ntrC and ntrB mutant strains, ranging from 40 to 80% of wild-type activity (de Zamaroczy et al. 1993; Zhang et al. 1994; this study). Glutamine synthetase can convert methylammonium to y-glutamylmethylamide. Since this latter metabolite is much less efficiently excreted from the cells than methylammonium, the reaction catalyzed by the glutamine synthetase represents a metabolic trap that leads to accumulation of radiolabel (Jajakumar et al. 1986). Although the reduction of this metabolic trapping may, on the long term, lead to a reduced accumulation of radioactivity, it is not likely that this reduction is responsible for the complete loss of [14C]methylammoniumtransport in the Azospirillum brasilense ntrC and the ntrB mutant strains. It was shown in E. coli that mutants deficient in glutamine synthetase with 2% of the wild-type activity have a [14C]methylammoniumtransport activity of about 70% of the wild type (Jajakumar et al. 1986). In the assay conditions used by these authors, as well as in our assays, cells were not washed before radioactivity incorporation was measured. Since no ammonium excretion in the ntr mutants has been measured (this study), competition of excreted NH4+,which has a much higher affinity for the carrier, can be ruled out. Since rpoN-dependent promoters have been described for several Azospirillum brasilense nif andfix genes, the nitrogen fixation dependent phenotype of the rpoN mutant was expected. More surprising, however, was the nonmotile phenotype. Involvement of rpoN in flagella production is not a novel phenomenon in bacteria, since it has been observed for P. aeruginosa and P. putida rpoN mutants (Totten et al. 1990; Inouye et al. 1989). The requirement of RpoN for biosynthesis of flagella has also been reported for Caulobacter and Campylobacter (Newton 1989; Alm et al. 1993). In the case of Azospirillum, two types of flagella have been described: one polar flagellum and several lateral flagella. The polar flagellum seems to be expressed constitutively, while the lateral flagella are induced on solid medium and apparently used for swarming (Hall and Kriegg 1983). Electron microscopy of the Azospirillum brasilense Sp7 rpoN mutant showed absence of both flagella types, indicating that the sigma 54 factor is most likely required for expression of the structural genes, encoding both polar and lateral flagella. Thus, it appears as if the Azospirillum brasilense Sp7 rpoN gene is involved in controlling diverse cellular functions. However, the rationale behind coordinate regulation of nitrogen metabolism related functions and flagellar biosynthesis, as well as the exact molecular mechanism of the rpoN-mediated regulatory circuit in Azospirillum brasilense, remains to be elucidated.

Acknowledgments We thank Dr. C. Elmerich for kindly providing us with the Azospirillum brasilense ntrB and ntrC mutants and we are grateful to Dr. M. de Zamaroczy for his helpful discussion and' comments regarding the studies on ammonium transport. We also thank V. Keijers and U. Rossbach for their help with the DNA sequence analysis, and Dr. B. Vanderscheuren for her

expertise with the electron microscope. A.M. is a recipient of the Collen Research Foundation; A.V.D. is a recipient of the Belgian National Fonds voor Wetenschappelijk onderzoek. Part of this work was supported by funds of the Nationaal Fonds voor Wetenschappelijk onderzoek (FGWO nr 3009593) and the Flemish Government (GOA 1993) to J.V., and the National Science Foundation Center for Microbial Ecology (BIR 9120006) and the Department of Energy (DEFG 0290ER20021) to F. J.dB.

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I

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(

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I

I

i I

1

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