FEMS Microbiology Letters 100 (1992) 113-120 © 1992 Federation of European Microbiological Societies 0378-1097/92/$05.00 Published by Elsevier
113
FEMSLE 80029
Regulation of nitrogen fixation in Azospirillum brasilense Sp7: Involvement of nifA, glnA and glnB gene products Yao Yun Liang, MiNos de Zamaroczy, Florence Ars~ne, Annick Paquelin and Claudine Elmerich Unitd de Physiologie Cellulaire and URA 1300 CNRS, Ddpartement des Biotechnologies, lnstitut Pasteur, Paris, France
Received 10 June 1992 Accepted 18 June 1992
Key words: Azospirillum brasilense; Nitrogen fixation regulation; Glutamine synthetase; Pn protein; NifA protein 1. SUMMARY The expression of nifA-, niH- and nifB-lacZ fusions was examined in different mutants of Azospirillum brasilense. Mutations in nifA, glnA and glnB severely impaired the expression of nifH- and nifB-lacZ fusions. By contrast, a nifA-lacZ fusion was not affected in a nifA or a glnB background and was only partially impaired in glnA mutants. It is proposed that in A. brasilense, the PII protein and glutamine synthetase are involved in a post-translational modification of NifA.
2. INTRODUCTION
AzospirUlurn can fix nitrogen under flee-living conditions or in association with grasses [1]. The
Correspondence to: C. Elmerich, Unit~ de Physiologie Cellulaire and URA 1300 CNRS, D~partement des Biotechnologies, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.
genetics of nitrogen fixation is best documented in A. brasilense Sp7. This led to the characterization of a 30-kb region of DNA containing nifHDK, nifE, nifUS and fixABC homologues [2]. The nucleotide sequence of nifH was established. Under conditions of nitrogen fixation, transcription proceeds from a o-54-dependent promoter [3], that is preceded by two upstream elements typical of genes controlled by nifA [3,4]. The nifA gene from strain Sp7 was recently cloned and sequenced and it was shown that NifA positively controlled nifH expression [5]. Construction of a nifA-lacZ fusion and mapping of the mRNA start site showed that nifA was expressed from an unidentified promoter, both under conditions of nitrogen fixation and in the presence of oxygen and ammonia [5]. The latter conditions were not compatible with nitrogen fixation nor with the expression of a nifH-lacZ fusion [5]. This suggested that the nifA product was synthesized in an inactive form in the presence of oxygen and ammonia [5]. A. brasilense Sp7 glutamine auxotrophs, impaired both in glutamine synthetase (GS) activity and in nitrogen fixation, have been isolated [6]. In
114
particular, strain 7029 displayed a Nif- phenotype, whereas strain 7028 was Nil c (i.e. fixing nitrogen in the presence of ammonia). A plasmid containing the GS structural gene, glnA, from A. brasilense and K. pneumoniae, restored a wild-type phenotype to these mutants [7], suggesting that a functional GS was essential for nitrogen fixation. An open reading frame, upstream of glnA, was found to have a high similarity with glnB [8], the structural gene for the P~I protein. In enteric bacteria the latter is involved both in adenylylation of GS and in the regulation of its synthesis ([9] and references therein). In order to determine precise involvement of NifA, GS and Pn in the regulation of nil gene expression in A. brasilense, lacZ fusions to nifA, nifH and nifB have been used to monitor the expression of their promoters under conditions of nitrogen fixation and ammonia assimilation.
3. M A T E R I A L S AND M E T H O D S
3.1. Bacterial strains, plasmids, media, growth conditions, nitrogenase and/3-galactosidase assays Bacterial strains and plasmids are listed in Table 1. Growth media, assays for nitrogen fixation by the acetylene reduction test, and for /3galactosidase activity were as described [2,5]. De-
termination of /3-galactosidase activity, including kinetics experiments, was performed on two cultures in parallel, in three independent experiments.
3.2. Construction of a nifB-lacZ fusion The nucleotide sequence of a 440-bp fragment containing the beginning of nifB and the upstream sequence is shown in Fig. 1A. A HindIII-BamHI fragment containing the promoter region of nifB and the seven first codons of the coding sequence was constructed and cloned in an M13 vector. This fragment was obtained by PCR as described [13], using the oligonucleotide primers shown in Fig. lB. After verification of the nucleotide sequence, the HindIII-BamHI fragment was subcloned into pGD926, to yield pAB135 (Fig. 1B).
3.3. Construction of a glnB insertion mutant Details of the construction of the glnB 7606 mutant will be reported elsewhere. A pSUP202 derivative containing a kanamycin cartridge derived from pUC4K at the unique BglII site of the glnB coding sequence was first constructed. The mutation was introduced into A. brasilense Sp7 genome by homologous recombination according to [2]. It was checked by hybridization that the
Table l
Azospirillum brasilense strains and plasmids Strain or plasmid Strain Sp7 7067 7028 7029 7606 Plasmid pAB576 pAB358 pAB53 pGD926 pAB 135 SUP202 pUC4K
Relevant properties
Source or reference
wild-type
[10] [5] [6,7] [6,7] this work
nifA-Tn5 mutant, NilglnA mutant, Gin- Nif c glnA mutant, Gin Nif glnB-kan mutant, Nil pVK100 derivative containing nifA- lacZ pRK290 derivative containing nifH-lacZ pVK100 derivative containing nifA expressed from the kan promoter pRK290 derivative Tc r, lacZY pGD926 derivative containing nifB- lacZ AmprTcrCm r containing oriT from RP4 source of a kan cartridge
[5] [5] [5] [11] Fig. 1, this work
[121 Pharmacia
115 cartridge had recombined at the correct location (data not shown)
4. RESULTS
4.1. Inuoh.,ement of NifA in the expression of the nifB-lacZ fusion It was previously shown that in A. brasilense Sp7 nifB and nifA are located on the same 5.6-kb EcoRI fragment [5]. Figure 1A shows the partial nucleotide sequence of the 5' terminal end of
nifB, the encoded polypeptide of which shares a high similarity with B. japonicum NifB [14]. The DNA region upstream of the putative start codon displayed a T G G - N 9 - T G C T sequence reminiscent of a ~S4-dependent promoter, as well as a TGT-N10-ACA sequence resembling a NifA DNA binding site. This was confirmed by monitoring the expression of the nifB-lacZ fusion carried by plasmid pAB135 into the wild-type and a nifA background (strains Sp7 and 7067, respectively). Figure 2A shows the kinetics of /3-galactosidase activity of the nifB-lacZ (plasmid pAB135) and the nifH-lacZ (plasmid pAB358) fusions in the wild-type background, under conditions of nitrogen fixation (0.5% oxygen/N-free medium). Kinetics were similar although the maximal activity was lower in the case of the nifBlacZ fusion. In media containing ammonia, under an atmosphere containing 0.5% oxygen or under air, the activity was less than 50 U min i (rag protein)- 1, i.e. of the same order of magnitude as the background level. Thus, the nifB-lacZ fusion behaves like the previously constructed nifH-lacZ fusion [5]. Moreover, no expression was found in a nifA background (data not shown), suggesting that NifA positively controlled nifB as it does
nifH. 4.2. Kinetics of expression of a nifA-lacZ fusion In order to determine the precise role of oxygen and ammonia in the regulation of nifA transcription, kinetics of/3-galactosidase activity were performed with a nifA-lacZ fusion (plasmid pAB576) carried by the wild-type strain Sp7 incubated under several physiological conditions (Fig. 2B). As reported before [5], the nifA-lacZ fusion
displayed a significant background of activity in the presence of ammonia and air, that corresponded to about 20% of the specific activity observed after 4 h derepression under conditions of nitrogen fixation. The kinetics performed also confirmed that under low oxygen tension, addition of 20 mM ammonia limited derepression of nifA transcription to roughly 60%. Thus, both ammonia and air play a role in regulation of nifA expression. In addition, the expression of the nifA-lacZ fusion was not impaired on a nifA background [5].
4.3. Expression of the nif-lacZ fusions in glnA mutants Phenotypic characterization of the two glnA mutants, strain 7029 ( G i n - N i l - ) and 7028 ( G l n Nil c) has been previously documented [6,7]. It was verified that introduction of a plasmid containing a functional glnB gene, but devoid of glnA, into the two mutants did not restore a nitrogen fixation wild-type phenotype. Moreover, the mutants were not complemented by introduction of plasmid pAB53, which carried nifA expressed from the kanamycin resistance gene promoter of the vector. Thus, the Nif- phenotype of strain 7029 cannot be attributed to P , or NifA deficiency and the Nif c phenotype of strain 7028 is not modified by an excess of PH or NifA. Table 2 reports the expression of the nifA-lacZ fusion in the two mutants of A. brasilense. As expected, in strain 7028, which reduces acetylene to a higher extent at low oxygen tension in the presence of 20 mM ammonia than in the absence of ammonia, expression of the nifA, nifH and nifB-lacZ fusions behaved similarly. By contrast, however, with nifH and nifB the nifA-lacZ fusion was also expressed in the presence of air and ammonia. This suggested that NifA activity in this mutant was independent of the presence of ammonia in the medium, but that the NifA product was still sensitive to oxygen. Strain 7029, which cannot reduce acetylene, retained 25-30% nifA-lacZ activity under the three physiological conditions tested. In agreement with the Nif- phenotype observed, the nifH and nifB fusions are not expressed under any circumstance tested.
116
A AGAGGGACTTCAAACACAGTTGCC f
GCCCC~-~'~CGGGTCCCC6~-~GGCGGCGTGTTTGTCGGCCATTGTTGGGCTTTGTCCGC CTTTGTCGGATTGCCGACATGGAGCACAGGGCCGACACTGCCGTCTAACCCTCTGAAACT CCACACTTCACCCGGT~CCCCGAACT~TTTAACGACCCGACCAAGGGCGCCGACG M
A
N
V
I
S
L
D
S
I
L
G
C
A
S S S C G S S D G P A D M A P E V W E TCCTCCTCCTGCGGGTCGTCGGACGGCCCCGCCGACATGGCGCCGGAGGTGTGGGAGAAG
K
GCGCGGCTGGGAAGGGAGTGGACGATGGCCAACGTTATTTCGCTCGACAGCATCCTGGGC V
G
E
L
K
A
A
A
E
A
P
P
T
A
A
A
S
A
GTGGGTGAGCTGAAAGCCGCCGCCGAGGCGCCGCCCACCGCCGCCGCCTCCGCGTGCGCG
V
K
N
H
P
C
Y
S
E
E
A
H
H
Y
F
A
R
M
GTGAAGAACCATCCCTGCTACTCCGAGGAGGCGCATCACTACTTCGCCCGCATGC
B
pAB135
trp 'ED' nifB'
lac'Z
lacY
[ R
]....
l H
L
S
R
Bm
A
N
V
I
S
L
D
TACCGGTTGCAATAAAGCGACCTAGGTAA BamHI
CCCAAGCTTAGAGGGACTTCAAACACAGTTGCC HindlII Fig. 1. Construction of a nifB-lacZ fusion. (A) Nucleotide sequence of the promoter region and of the beginning of the nifB coding sequence from A. brasilense Sp7. Open and shadowed boxes show sequences resembling NifA and o.54 D N A binding sites, respectively. A putative ribosome binding site is underlined. Arrows indicate the limits of the PCR amplified fragment. Homology with B. japonicum NifB [14] is indicated by stars which correspond to identical amino acids or with similar chemical properties. (B) Map of the pAB135 region that contains the nifB-lacZ fusion. Relevant features of the cloning vector pGD926 are described elsewhere [11]. The oligonucleotides used as primers are indicated below the map. Bm, BamHI; H, HindllI; R, EcoRl; S, Sinai.
117
4. 4. Phenotypic characterization of a glnB insertion mutant and involvement of Pit in nif-lacZ fusion expression
Table 2
A mutant of strain Sp7 carrying a kanamycin cartridge inserted in the glnB coding sequence was constructed. The resulting 7606 mutant strain did not require glutamine and displayed growth
Fusion
• 5000 A
/3-Galactosidase activity of n i f - l a c Z brasilense strains Sp7 and mutants
gene fusions in A.
%/3-Galactosidase activity 0.5% 0 2 -NH 4
0.5% 0 2 + NH 4
Air + NH 4
Sp7 nifA - lacZ nifH-lacZ nifB-laZ
100 100 100
57 < 2 < 2
21 _< 2 < 2
7028 n i f A - l a c Z n i f H - lacZ
32 42
79 85
69 _< 2
•r.
= 4000"
nifB-lacZ
15
101
_< 2
~
3000-
7029 nifA - lacZ nifH-lacZ n i f B - lacZ
25
113
FEMSLE 80029
Regulation of nitrogen fixation in Azospirillum brasilense Sp7: Involvement of nifA, glnA and glnB gene products Yao Yun Liang, MiNos de Zamaroczy, Florence Ars~ne, Annick Paquelin and Claudine Elmerich Unitd de Physiologie Cellulaire and URA 1300 CNRS, Ddpartement des Biotechnologies, lnstitut Pasteur, Paris, France
Received 10 June 1992 Accepted 18 June 1992
Key words: Azospirillum brasilense; Nitrogen fixation regulation; Glutamine synthetase; Pn protein; NifA protein 1. SUMMARY The expression of nifA-, niH- and nifB-lacZ fusions was examined in different mutants of Azospirillum brasilense. Mutations in nifA, glnA and glnB severely impaired the expression of nifH- and nifB-lacZ fusions. By contrast, a nifA-lacZ fusion was not affected in a nifA or a glnB background and was only partially impaired in glnA mutants. It is proposed that in A. brasilense, the PII protein and glutamine synthetase are involved in a post-translational modification of NifA.
2. INTRODUCTION
AzospirUlurn can fix nitrogen under flee-living conditions or in association with grasses [1]. The
Correspondence to: C. Elmerich, Unit~ de Physiologie Cellulaire and URA 1300 CNRS, D~partement des Biotechnologies, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.
genetics of nitrogen fixation is best documented in A. brasilense Sp7. This led to the characterization of a 30-kb region of DNA containing nifHDK, nifE, nifUS and fixABC homologues [2]. The nucleotide sequence of nifH was established. Under conditions of nitrogen fixation, transcription proceeds from a o-54-dependent promoter [3], that is preceded by two upstream elements typical of genes controlled by nifA [3,4]. The nifA gene from strain Sp7 was recently cloned and sequenced and it was shown that NifA positively controlled nifH expression [5]. Construction of a nifA-lacZ fusion and mapping of the mRNA start site showed that nifA was expressed from an unidentified promoter, both under conditions of nitrogen fixation and in the presence of oxygen and ammonia [5]. The latter conditions were not compatible with nitrogen fixation nor with the expression of a nifH-lacZ fusion [5]. This suggested that the nifA product was synthesized in an inactive form in the presence of oxygen and ammonia [5]. A. brasilense Sp7 glutamine auxotrophs, impaired both in glutamine synthetase (GS) activity and in nitrogen fixation, have been isolated [6]. In
114
particular, strain 7029 displayed a Nif- phenotype, whereas strain 7028 was Nil c (i.e. fixing nitrogen in the presence of ammonia). A plasmid containing the GS structural gene, glnA, from A. brasilense and K. pneumoniae, restored a wild-type phenotype to these mutants [7], suggesting that a functional GS was essential for nitrogen fixation. An open reading frame, upstream of glnA, was found to have a high similarity with glnB [8], the structural gene for the P~I protein. In enteric bacteria the latter is involved both in adenylylation of GS and in the regulation of its synthesis ([9] and references therein). In order to determine precise involvement of NifA, GS and Pn in the regulation of nil gene expression in A. brasilense, lacZ fusions to nifA, nifH and nifB have been used to monitor the expression of their promoters under conditions of nitrogen fixation and ammonia assimilation.
3. M A T E R I A L S AND M E T H O D S
3.1. Bacterial strains, plasmids, media, growth conditions, nitrogenase and/3-galactosidase assays Bacterial strains and plasmids are listed in Table 1. Growth media, assays for nitrogen fixation by the acetylene reduction test, and for /3galactosidase activity were as described [2,5]. De-
termination of /3-galactosidase activity, including kinetics experiments, was performed on two cultures in parallel, in three independent experiments.
3.2. Construction of a nifB-lacZ fusion The nucleotide sequence of a 440-bp fragment containing the beginning of nifB and the upstream sequence is shown in Fig. 1A. A HindIII-BamHI fragment containing the promoter region of nifB and the seven first codons of the coding sequence was constructed and cloned in an M13 vector. This fragment was obtained by PCR as described [13], using the oligonucleotide primers shown in Fig. lB. After verification of the nucleotide sequence, the HindIII-BamHI fragment was subcloned into pGD926, to yield pAB135 (Fig. 1B).
3.3. Construction of a glnB insertion mutant Details of the construction of the glnB 7606 mutant will be reported elsewhere. A pSUP202 derivative containing a kanamycin cartridge derived from pUC4K at the unique BglII site of the glnB coding sequence was first constructed. The mutation was introduced into A. brasilense Sp7 genome by homologous recombination according to [2]. It was checked by hybridization that the
Table l
Azospirillum brasilense strains and plasmids Strain or plasmid Strain Sp7 7067 7028 7029 7606 Plasmid pAB576 pAB358 pAB53 pGD926 pAB 135 SUP202 pUC4K
Relevant properties
Source or reference
wild-type
[10] [5] [6,7] [6,7] this work
nifA-Tn5 mutant, NilglnA mutant, Gin- Nif c glnA mutant, Gin Nif glnB-kan mutant, Nil pVK100 derivative containing nifA- lacZ pRK290 derivative containing nifH-lacZ pVK100 derivative containing nifA expressed from the kan promoter pRK290 derivative Tc r, lacZY pGD926 derivative containing nifB- lacZ AmprTcrCm r containing oriT from RP4 source of a kan cartridge
[5] [5] [5] [11] Fig. 1, this work
[121 Pharmacia
115 cartridge had recombined at the correct location (data not shown)
4. RESULTS
4.1. Inuoh.,ement of NifA in the expression of the nifB-lacZ fusion It was previously shown that in A. brasilense Sp7 nifB and nifA are located on the same 5.6-kb EcoRI fragment [5]. Figure 1A shows the partial nucleotide sequence of the 5' terminal end of
nifB, the encoded polypeptide of which shares a high similarity with B. japonicum NifB [14]. The DNA region upstream of the putative start codon displayed a T G G - N 9 - T G C T sequence reminiscent of a ~S4-dependent promoter, as well as a TGT-N10-ACA sequence resembling a NifA DNA binding site. This was confirmed by monitoring the expression of the nifB-lacZ fusion carried by plasmid pAB135 into the wild-type and a nifA background (strains Sp7 and 7067, respectively). Figure 2A shows the kinetics of /3-galactosidase activity of the nifB-lacZ (plasmid pAB135) and the nifH-lacZ (plasmid pAB358) fusions in the wild-type background, under conditions of nitrogen fixation (0.5% oxygen/N-free medium). Kinetics were similar although the maximal activity was lower in the case of the nifBlacZ fusion. In media containing ammonia, under an atmosphere containing 0.5% oxygen or under air, the activity was less than 50 U min i (rag protein)- 1, i.e. of the same order of magnitude as the background level. Thus, the nifB-lacZ fusion behaves like the previously constructed nifH-lacZ fusion [5]. Moreover, no expression was found in a nifA background (data not shown), suggesting that NifA positively controlled nifB as it does
nifH. 4.2. Kinetics of expression of a nifA-lacZ fusion In order to determine the precise role of oxygen and ammonia in the regulation of nifA transcription, kinetics of/3-galactosidase activity were performed with a nifA-lacZ fusion (plasmid pAB576) carried by the wild-type strain Sp7 incubated under several physiological conditions (Fig. 2B). As reported before [5], the nifA-lacZ fusion
displayed a significant background of activity in the presence of ammonia and air, that corresponded to about 20% of the specific activity observed after 4 h derepression under conditions of nitrogen fixation. The kinetics performed also confirmed that under low oxygen tension, addition of 20 mM ammonia limited derepression of nifA transcription to roughly 60%. Thus, both ammonia and air play a role in regulation of nifA expression. In addition, the expression of the nifA-lacZ fusion was not impaired on a nifA background [5].
4.3. Expression of the nif-lacZ fusions in glnA mutants Phenotypic characterization of the two glnA mutants, strain 7029 ( G i n - N i l - ) and 7028 ( G l n Nil c) has been previously documented [6,7]. It was verified that introduction of a plasmid containing a functional glnB gene, but devoid of glnA, into the two mutants did not restore a nitrogen fixation wild-type phenotype. Moreover, the mutants were not complemented by introduction of plasmid pAB53, which carried nifA expressed from the kanamycin resistance gene promoter of the vector. Thus, the Nif- phenotype of strain 7029 cannot be attributed to P , or NifA deficiency and the Nif c phenotype of strain 7028 is not modified by an excess of PH or NifA. Table 2 reports the expression of the nifA-lacZ fusion in the two mutants of A. brasilense. As expected, in strain 7028, which reduces acetylene to a higher extent at low oxygen tension in the presence of 20 mM ammonia than in the absence of ammonia, expression of the nifA, nifH and nifB-lacZ fusions behaved similarly. By contrast, however, with nifH and nifB the nifA-lacZ fusion was also expressed in the presence of air and ammonia. This suggested that NifA activity in this mutant was independent of the presence of ammonia in the medium, but that the NifA product was still sensitive to oxygen. Strain 7029, which cannot reduce acetylene, retained 25-30% nifA-lacZ activity under the three physiological conditions tested. In agreement with the Nif- phenotype observed, the nifH and nifB fusions are not expressed under any circumstance tested.
116
A AGAGGGACTTCAAACACAGTTGCC f
GCCCC~-~'~CGGGTCCCC6~-~GGCGGCGTGTTTGTCGGCCATTGTTGGGCTTTGTCCGC CTTTGTCGGATTGCCGACATGGAGCACAGGGCCGACACTGCCGTCTAACCCTCTGAAACT CCACACTTCACCCGGT~CCCCGAACT~TTTAACGACCCGACCAAGGGCGCCGACG M
A
N
V
I
S
L
D
S
I
L
G
C
A
S S S C G S S D G P A D M A P E V W E TCCTCCTCCTGCGGGTCGTCGGACGGCCCCGCCGACATGGCGCCGGAGGTGTGGGAGAAG
K
GCGCGGCTGGGAAGGGAGTGGACGATGGCCAACGTTATTTCGCTCGACAGCATCCTGGGC V
G
E
L
K
A
A
A
E
A
P
P
T
A
A
A
S
A
GTGGGTGAGCTGAAAGCCGCCGCCGAGGCGCCGCCCACCGCCGCCGCCTCCGCGTGCGCG
V
K
N
H
P
C
Y
S
E
E
A
H
H
Y
F
A
R
M
GTGAAGAACCATCCCTGCTACTCCGAGGAGGCGCATCACTACTTCGCCCGCATGC
B
pAB135
trp 'ED' nifB'
lac'Z
lacY
[ R
]....
l H
L
S
R
Bm
A
N
V
I
S
L
D
TACCGGTTGCAATAAAGCGACCTAGGTAA BamHI
CCCAAGCTTAGAGGGACTTCAAACACAGTTGCC HindlII Fig. 1. Construction of a nifB-lacZ fusion. (A) Nucleotide sequence of the promoter region and of the beginning of the nifB coding sequence from A. brasilense Sp7. Open and shadowed boxes show sequences resembling NifA and o.54 D N A binding sites, respectively. A putative ribosome binding site is underlined. Arrows indicate the limits of the PCR amplified fragment. Homology with B. japonicum NifB [14] is indicated by stars which correspond to identical amino acids or with similar chemical properties. (B) Map of the pAB135 region that contains the nifB-lacZ fusion. Relevant features of the cloning vector pGD926 are described elsewhere [11]. The oligonucleotides used as primers are indicated below the map. Bm, BamHI; H, HindllI; R, EcoRl; S, Sinai.
117
4. 4. Phenotypic characterization of a glnB insertion mutant and involvement of Pit in nif-lacZ fusion expression
Table 2
A mutant of strain Sp7 carrying a kanamycin cartridge inserted in the glnB coding sequence was constructed. The resulting 7606 mutant strain did not require glutamine and displayed growth
Fusion
• 5000 A
/3-Galactosidase activity of n i f - l a c Z brasilense strains Sp7 and mutants
gene fusions in A.
%/3-Galactosidase activity 0.5% 0 2 -NH 4
0.5% 0 2 + NH 4
Air + NH 4
Sp7 nifA - lacZ nifH-lacZ nifB-laZ
100 100 100
57 < 2 < 2
21 _< 2 < 2
7028 n i f A - l a c Z n i f H - lacZ
32 42
79 85
69 _< 2
•r.
= 4000"
nifB-lacZ
15
101
_< 2
~
3000-
7029 nifA - lacZ nifH-lacZ n i f B - lacZ
25
