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Interaction between NifL and NifA in the nitrogen-
fixing Pseudomonas stutzeri A1501
Zhihong Xie,
1
3 Yuetang Dou,
1
Shuzheng Ping,
1
Ming Chen,
1
Guoying Wang,
2
Claudine Elmerich
3
4 and Min Lin
1
Correspondence
Min Lin
linmin57@vip.163.com
1
Biotechnology Research Institute, CAAS, Beijing, PR China
2
Biology College, China Agricultural University, Beijing, PR China
3
Institut des Sciences du Ve
´
ge
´
tal, CNRS UPR-2355, Gif-sur-Yvette, France
Received 29 May 2006
Revised 30 August 2006
Accepted 1 September 2006
Pseudomonas stutzeri strain A1501 isolated from rice fixes nitrogen under microaerobic conditions
in the free-living state. This paper describes the properties of nifL and nifA mutants as well as the
physical interaction between NifL and NifA proteins. A nifL mutant strain that carried a mutation non-
polar on nifA expression retained nitrogenase activity. Complementation with a plasmid containing
only nifL led to a decrease in nitrogenase activity in both the wild-type and the nifL mutant,
suggesting that NifL acts as an antiactivator of NifA activity. Using the yeast two-hybrid system and
purified protein domains of NifA and NifL, an interaction was shown between the C-terminal domain
of NifL and the central domain of NifA, suggesting that NifL antiactivator activity is mediated by
direct protein interaction with NifA.
INTRODUCTION
Regulation of nitrogen fixation (nif) gene expression, in
response to the environmental signals ammonium and O
2
,
depends on the transcriptional activator, NifA, a member of
the
s
54
-dependent family of bacterial activators (Morett &
Segovia, 1993). The mode of activation of nif gene
transcription by NifA appears to be common to a large
number of diazotrophs, whereas the regulation of NifA
synthesis and activity differs from one organism to another
(Merrick, 2004). NifA activity in the gamma subgroup of
proteobacteria is controlled by the antiactivator NifL.
Indeed, NifA and NifL form an atypical two-component
sensor–regulator system, and NifL mod ulates the activity of
NifA by direct protein–protein interaction (Martı
´
nez-
Argudo et al., 2004). NifA proteins are structurally similar
to each other. This enabled the description of NifA as a
multidomain protein (Studholme & Dixon, 2003 and
references therein). The catalytic domain of NifA, which
interacts with the RNA polymerase, shares a high degree of
identity with an AAA+-type ATPase. The C-terminal part is
the DNA-binding domain and contains an HTH motif. The
N-terminal domain carries a GAF domain. The NifL protein
resembles histidine kinase protein (HPK), but NifL is not
subject to autophos phorylation. The amino-terminal region
contains a PAS domain, and it has been proposed that this
region may be involved in O
2
sensing (Zhulin et al., 1997).
The C-terminal region shows similarity to the GHKL
superfamily of ATPases.
The nitrogen fixation ability within the genus Pseudomonas
has been questioned for a long time (for reviews see Chan
et al., 1994; Lalucat et al., 2006). It is now established that
several strains of Pseudomonas stutzeri can fix nitrogen
(Vermeiren et al., 1999; Rediers et al., 2004). P. stutzeri
A1501, isolated from rice, fixes nitrogen in the free-living
state, under microaerobic conditions in media devoid of
ammonia (You et al., 1991; Lin et al., 2000; Desnoues et al.,
2003). A 30 kb DNA region containing the nitrogen fixation
(nif and rnf) genes has been previously characterized and the
regulatory nifLA region mapped within the main nif cluster.
By using different lacZ fusions, it was observed that nifA
controlled the expression of other nif (and rnf) operons and
that chromosomal nifLA–lacZ fusion expression was
strongly reduced in the presence of oxygen and ammonia
(Desnoues et al., 2003). As NtrC and RpoN were also found
to control nifLA expression (Desnoues et al., 2003), this
suggested that the regulation circuitry in P. stutzeri
resembled more the situation in Klebsiella pneumoniae
than in Azotobacter species, where nifLA exp ression is not
impaired in the presence of ammonia (Blanco et al., 1993),
although P. stutzeri is phylogenetically closer to Azotobacter
(Rediers et al., 2004) than to Klebsiella. The objective of this
work was to further document the role of NifLA in P. stutzeri
A1501. In particular, the properties of a nifL mutant strain
non-polar on nifA expression as well as the phys ical
interaction between NifL and NifA were investigated.
3Present address: Department of Biochemistry, Cellular and Molecular
Biology, the University of Tennessee, Knoxville, TN 37996, USA.
4Present address: De
´
partement de Microbiologie, Biologie Mole
´
culaire
du Ge
`
ne chez les Extre
ˆ
mophiles, Institut Pasteur, 75724 Paris Cedex
15, France.
Abbreviation: GST, glutathione S-transferase.
0002-9171
G
2006 SGM Printed in Great Britain 3535
Microbiology (2006), 152, 3535–3542 DOI 10.1099/mic.0.29171-0
METHODS
Strains and plasmids. Plasmids and bacterial strains are listed in
Table 1. P. stutzeri was grown in minimal lactate medium or in LB
medium at 30 uC as described by Desnoues et al. (2003). The yeast
strain used for the two-hybird analysis was Saccharomyces cerevisiae
PJ69-4A harbouring three reporter genes, lacZ, ADE2 and HIS3,
under the control of the GAL promoter (James et al., 1996). Yeast
cells were grown in YPD rich medium or in SD minimal medium
supplemented with appropriate amino acids, at 30 uC, as described
in the manual supplied with the Matchmaker Two-Hybrid System
(Clontech). Antibiotics were used at the following concentrations
(
mgml
21
): ampicillin (Amp), 100; kanamycin (Km), 50; tetracycline
(Tc), 10.
Nitrogenase activity. Nitrogenase activity was determined with
bacterial suspensions incubated at an OD
600
of 0?1, in N-free mini-
mal lactate medium, at 30 uC, under an argon atmosphere contain-
ing 1 % oxygen and 10 % acetylene, according to the derepression
protocol described by Desnoues et al. (2003). Nitrogenase specific
activity is expressed as nmol ethylene min
21
(mg protein)
21
.
Protein concentrations were determined by the Bio-Rad protein
assay (BSA protein standard). Each experiment was repeated at least
three times.
Molecular techniques. Plasmid isolation, genomic DNA extrac-
tion, gel electrophoresis, restriction mapping, transformation and
molecular cloning, Western blotting and amplification by PCR
(Amersham kit) were performed by standard methods (Sambrook &
Russell, 2001) or as recommended by the manufacturers of the pro-
ducts used. Restriction enzymes were purchased from Promega and
oligonucleotides from Shanghai Biotech Company. Nucleotide
sequencing was performed by the Takara Company.
RT-PCR. Total RNA was isolated by acid-phenol extraction and the
single-stranded cDNA synthesis was performed using the
ProtoScript First Strand cDNA Synthesis Kit (New England Biolabs).
RNA (1
mg) isolated from bacteria grown under nitrogen fixation
conditions was used for RT-PCR to amplify a 415 bp fragment
with primers specific for nifA, RT-PCRnifAF and RT-PCRnifAR
(Table 2). After 35 cycles of PCR amplification (94 uC for 1 min;
58 uC for 1 min; 72 uC for 1 min), PCR products were separated on
0?8 % agarose gels and visualized by ethidium bromide staining.
Construction of nifL mutants. Construction of strain 1507B, a
nifL deletion non-polar mutant, was similar to strain 1507 pre-
viously described (Desnoues et al., 2003), except that it carries the
KIXX cassette in the same orientation as the nifA gene. A nifL polar
mutant, designated 1507A, was constructed for this work, as follows.
Oligonucleotide primers pL-F and pL-R (see Table 2) were designed
to amplify a 1647 bp BamHI–HindIII fragment that was cloned at
the unique BamHI–HindIII sites of the pSUP202 vector. Then, the
KIXX cassette was inserted, as a SmaI fragment, into the unique
ApaI site within the nifL coding sequence, previously treated with
the Klenow fragment of DNA polymerase I to fill in ends. The orien-
tation of the Km resistance gene was the opposite of that of the nifA
gene. Plasmids were transferred into P. stutzeri recipients by conju-
gation, using Escherichia coli S17-1 as the donor, as previously
Table 1. Strains and plasmids
Strain/plasmid Relevant characteristics Source/reference
P. stutzeri
A1501 Wild-type, Chinese Culture Collection: CGMCC 0351 Lin et al . (2000);
Desnoues et al. (2003)
1506 nifA-km deletion mutant, Nif
2
Km
r
Desnoues et al. (2003)
1507B nifL-km deletion mutant, non-polar on nifA expression, Nif
+
Km
r
Nicole Desnoues
1507A nifL-km mutant, polar on nifA expression, Nif
2
Km
r
This study
S. cerevisiae
PJ69-4A MATa trp1-109 leu2-3,112 ura3-52 his3-200 gal4
D gal80 LYS2 ::GAL1-HIS3
GAL2-ADE2 met2 ::GAL7-lacZ
James et al. (1996)
Plasmids
pET-28a(+) Cloning vector carrying a His
6
linker for expression and purification of
proteins, Km
r
Novagen
pGAD-C
1
* Expression vector for yeast two-hybrid system, GAL4(768–881), LEU2, Amp
r
James et al. (1996)
pGBD-C
1
* Expression vector for yeast two-hybrid system, GAL4(1–147), TRP2, Amp
r
James et al. (1996)
pGAD-SIP and pBDU-SNF I Positive control for yeast two-hybrid system, Amp
r
James et al. (1996)
pGEX-2T Cloning vector which expresses GST protein, Amp
r
Pharmacia
pGST-NifLc pGEX-2T derivative carrying a 1145 bp fragment encoding the C-terminal
portion of nifL, Amp
r
This study
pHis
6
-NifA pET28a(+) derivative carrying 1587 bp PCR product of nifA,Km
r
This study
pSUP202 Suicide vehicle, Ap
r
Tc
r
Cm
r
Simon et al. (1983)
pUC4-KIXX Source of km cassette Pharmacia
pVA1 pVK100 derivative carrying a nifA promoterless gene in opposite orientation
of the km gene, Tc
r
This study
pVA3 As pVA1, but with nifA under the control of the km promoter, Tc
r
This study
pVK100 RK2 replicon, Tra
2
Km
r
Tc
r
Allen & Hanson (1985)
pVL pVK100 derivative carrying nifL This study
*For pGAD and pGBD derivatives containing entire or portions of nifA and nifL, constructed in this work, see Fig. 2, Table 2 and Table 3.
3536 Microbiology 152
Z. Xie and others
described (Desnoues et al., 2003). Recombination in the host
genome at the correct location was checked for by PCR amplifica-
tion with appropriate oligonucleotide primers.
Construction of nifL- and nifA-containing plasmids.
Oligonucleotides flanking the coding region of nifA and nifL were
designed (see Table 2) to amplify the corresponding genes by PCR.
For nifA, a 1634 bp promoterless DNA fragment was amplified and
cloned as a HindIII fragment into the Km resistance gene of
pVK100 under the control of the km promoter for pVA3 and in the
opposite orientation for pVA1. For nifL, the amplified 1899 bp frag-
ment contained 239 bp of the non-coding sequence; it was cloned as
a HindIII–XhoI fragment into the Km resistance gene of pVK100 in
the same orientation as the Km resistance gene, to yield pVL.
Yeast two-hybrid analysis. The oligonucleotides designed to
amplify the fragments encoding the desired domains of NifA and
NifL are listed in Table 2. PCR amplification of strain A1501 geno-
mic DNA was performed with the Proof Start DNA polymerase
(Qiagen) and the resulting amplicons were cloned in-frame into
pGBD-C
1
or pGAD-C
1
vectors (Table 1). The fusion junctions were
verified in all the constructed plasmids by nucleotide sequence ana-
lyses. S. cerevisiae competent cells were prepared using the method
described in the Matchmaker II protocol and pGAD and pGBD
derivatives were co-transformed into the yeast recipient. The interac-
tion between NifA and NifL domains was screened for by growth on
SD medium lacking Leu, His, Trp and Ade (Table 3). The filter and
quantitative liquid
b-galactosidase assay were done according to the
protocol described by the Matchmaker II system (Clontech).
Activity is expressed as Miller units (Miller, 1972).
Expression and purification of NifL and NifA fusion proteins.
A1501 nifA was amplified by PCR using oligonucleotides pnifA F
and R (Table 2) and the amplicon was cloned into pET28a(+)
vector (Novagen) to generate a His
6
-tagged NifA fusion protein.
Similarly, oligonucleotides pnifL-C, F and R were used to amplify
the C-terminal portion of nifL, which was constructed to fuse in-
frame with GST (glutathione S-transferase) in the pGEX-2T vector
(Pharmacia). Cultures containing the recombinant plasmids were
grown aerobically in LB broth and expression from the T7 promoter
was induced by addition of 1 mM IPTG. His
6
-NifA fusion protein
was purified by nickel affinity chromatography on Hi-trap chelating
columns (Pharmacia) and GST-NifLc fusion protein was purified by
batch elution from affinity glutathione-Sepharose 4B beads
(Pharmacia) as recommended by the manufacturer. SDS-PAGE of
protein samples was performed by standard methods (Sambrook &
Russell, 2001).
Analysis of protein complexes. The pull-down assay was per-
formed according to the method of Hasan et al. (2004). Purified
GST-NifLc (10 ng) was immobilized on glutathione-Sepharose 4B
beads and washed thoroughly with wash buffer (20 mM Tris/HCl
pH 7?5, 2 mM EDTA, 100 mM NaCl, 12 mM
b-mercaptoethanol
and 0?1 % Triton X-100). Immobilized GST-NifLc was then incu-
bated with the purified His
6
-NifA for 1 h at 4 uC. After washing,
bound proteins were eluted in SDS-PAGE sample buffer and sepa-
rated by 8 % SDS-PAGE. Western blotting with anti-histidine or
anti-GST antibodies (Invitrogen) was performed by standard meth-
ods (Sambrook & Russell, 2001).
Table 2. Oligonucleotides primers
Primer Sequence* Amplicon
size (bp)
UseD
RT-PCRnifA F 59-ACCGGAAACCCTGCTCGAATCGGA-39 415 nifA transcription
RT-PCRnifA R 59-GCTTGAGTTTGCGACCCTGCT-39
pL F 59-CG
GGATCCCAACAGCCCCCTAACCGTATG-39 1647 Construction of 1507A
pL R 59-CT
AAGCTTATCAGCTGGCCGAGAAGGGCA-39
pVA F 59-CCG
AAGCTTTCAGATCTTGCGCATATGA-39 1634 Construction of pVA1 and pVA3
pVA R 59-CCG
AAGCTTACGGTGCATATCGATAGC-39
pVL F 59-GAAT
CTCGAGATAGCGCAAC-39 1899 Construction of pVL
pVL R 59-ATGC
AAGCTTCCCCTGTCA-39
pnifA F 59-CG
GGATCCGAAGCTCGCATGAACG-39 1587 Entire nifA, Y2H and pHis
6
-NifA
pnifA R 59-CC
ATCGATGATCTTGCGCATATGAATG-39
pnifAn F 59-ATC
GAATTCATGAACGCCACATTCGCCG-39 617 N-terminal portion of nifA, Y2H
pnifAn R 59-GAT
AGATCTTTCGCGGCGTAGCTCGTC-39
pnifAm F 59-GAA
GGATCCATAGAGGACGGCCAGGAAG-39 829 Central portion of nifA, Y2H
pnifAm R 59-CAT
ATCGATACCGGTGAGGGAGACCAC-39
pnifAc F 59-GAA
GGATCCCAGCAGGGTCGCAAACTC-39 374 C-terminal portion of nifA, Y2H
pnifAc R 59-CC
ATCGATGATCTTGCGCATATGAATG-39
pnifL F 59-CG
GAATTCCAACAGCCCCCTAACCGT-39 1644 Entire nifL, Y2H
pnifL R 59-CG
GGATCCGCTGGCCGAGAAGGGCA-39
pnifLn F 59-ATC
GAATTCGCTTTGCAACGGATACGG-39 545 N-terminal portion of nifL, Y2H
pnifLn R 59-GAT
AGATCTGCTGTCGACCACCGCCT-39
pnifLc F 59-ATC
GAATTCCAGCGCGTCAGCAACCAG-39 1145 C-terminal portion of nifL, Y2H and pGST-NifLc
pnifLc R 59-GAT
AGATCTGCTGGCCGAGAAGGGCAGTTC-39
*Restriction sites are underlined.
DY2H: amplicons were cloned in pGAD and pGBD vectors for the yeast two-hybrid experiments; see Table 3 and Fig. 2.
http://mic.sgmjournals.org 3537
NifL/NifA interaction in Pseudomonas stutzeri
RESULTS
Properties of a nifL mutant strain non-polar on
nifA expression and effect of overexpression of
NifA and NifL
A nifA mutant, as well as a nifL mutant polar on nifA
expression, was previously shown to be devoid of
nitrogenase activity (Desnoues et al., 2003). To further
investigate the role of NifL in the regulation of nif gene
expression in P. stutzeri, we used strain 1507B, in which
most of the nifL coding sequence (1506 bp ClaI fragment)
was substituted by a KIXX cassette cloned in the same
orientation as the nifA gene. The non-polar effect of the nifL
mutation on nifA expression was first checked by RT-PCR.
A nifA transcript was indeed detected, both in the wild-type
and in the 1507B nifL mutant, while no nifA transcript was
produced in the nifL polar mutant constructed for this work,
1507A (Fig. 1a). The lack of nifA transcript in the nifL
mutant 1507A strongly suggested that, as in other nitrogen
fixers containing a nifL gene, nifA and nifL were transcribed
from the same promoter in strain A1501. In addition, in
strain 1507B the presence of nifA transcript was consistent
with nifA transcription from the km promoter of the
cassette.
The nifL non-polar mutant strain 1507B displayed residual
nitrogenase activity, up to 30 % of that of the wild-type
strain (Fig. 1b). It was then possible to investigate the effect
of the increased production of NifA and NifL on nitrogenase
activity by complementation analysis with plasmids expres-
sing nifA or nifL. Expression of nifA in pVA3 was monitored
from the km promoter of the vector. Indeed, pVA3 restored
30 % nitrogenase activity to the nifA-km mutant (1506) and
nifL-km polar (1507A) strains (Fig. 1b), but the comple-
mentation reached up to 60 % when kanamycin was added
to the culture medium (not shown). As expected, pVA1, in
which nifA was cloned in the opposite orientation, did not
affect nitrogen fixation (Fig. 1b). Plasmid pVA3 also
increased nitrogenase activity of the wild-type and of the
nifL non-polar mutant 1507B (Fig. 1b). In contrast, plasmid
pVL, which carries the nifL gene, decreased nitrogenase
activity of the wild-type and of the nifL non-polar mutant
(Fig. 1b). Thus, NifL negatively modulates NifA activity.
This is consistent with a role for NifL as NifA antiactivator.
Physical interaction between NifL and NifA
in vivo
Deduced translation products of A1501 NifA and
Azotobacter NifA share 82 % identity, suggesting that the
domain structure of the P. stutzeri protein is very similar to
that defined in Azotobacter NifA, and hence in other NifAs
(Studholme & Dixon, 2003). This enabled us to design
appropriate oligonucletides that could amplify portions
encoding the N-terminal (NifAn), central (NifAm) and C-
terminal (NifAc) domains, respectively, as schematized in
Fig. 2. The same was applied to NifL, which shared 72 %
identity with Azotobacter NifL. In that case, the nifL gene was
divided into only two regions, encompassing the N-terminal
PAS (NifLn) and the C-terminal (NifLc) domains, respec-
tively (Fig. 2). Each of the domains was cloned into the yeast
pGAD vector, which carries the activating domain of the
yeast GAL4 transcriptional activator and into the pGBD
Table 3. Interaction between NifL and NifA detected by the yeast two-hybrid system
Plasmid combination Growth on
selective media*
b-Galactosidase activityD
(Miller units)
Interacting peptides
pGAD-SIP and pGBDU-SNF I ++++ 57?6±1?5 Positive control
pGAD-C
1
and pGBD-C
1
2 2?7±0?3 Negative control
pGAD-nifA and pGBD-nifL ++ 23?6±1?1 NifL and NifA
pGAD-nifL and pGBD-nifA ++ 25?3±2?3 NifL and NifA
pGAD-nifA and pGBD-nifLn (N-terminal domain) 2 4?1±0?3 None
pGAD-nifA and pGBD-nifLc (C-terminal domain) ++ 22?9±1?3 NifL C-terminal domain and NifA
pGAD-nifL and pGBD-nifAn (N-terminal domain) 2 5?3±0?5 None
pGAD-nifL and pGBD-nifAm (central domain) ++ 18±2?7 NifL and NifA central domain
pGAD-nifL and pGBD-nifAc (C-terminal domain) 2 3?15±0?7 None
pGAD-nifLc (C-terminal domain) and
pGBD-nifAm (central domain)
++ 12?0±2?4 NifL C-terminal domain and
NifA central domain
pGAD-nifAm (central domain) and
pGBD-nifLc (C-terminal domain)
++ 13?1±1?6 NifA central domain and NifL
C-terminal domain
pGAD-nifA and pGBDC
1
2 4?4±0?4 None
pGAD-nifL and pGBDC
1
2 3±0?2 None
*Growth was assayed on three different media: SD lacking Leu, Trp and His; SD lacking Leu, Trp and Ade; and SD lacking Leu, Trp, His and Ade
and containing X-Gal. + indicates that growth was observed on all three media and that colonies were blue on the X-Gal plates; 2 indicates that
growth was not observed on any of the three media tested.
D
b-Galactosidase activities are the means±SD of three determinations.
3538 Microbiology 152
Z. Xie and others
vector, which carries the DNA-binding domain of GAL4.
After co-transformation into yeast strain PJ69-4A, an
interaction between the peptides fused to the pGAD and
pGBD vectors is required so that the transcription from the
promoters under the control of GAL4 can proceed
(Table 3). As one of the reporter genes is lacZ, an enzymic
assay for
b-galactosidase gives an estimation of the relative
strength of the interaction (James et al., 1996). Fig. 3 shows
an example of the growth on selective media and Table 3
summarizes the data obtained for growth and
b-galactosi-
dase activity. From these data it is co ncluded that an
interaction is detected between the entire NifA and NifL,
and that the binding is limited to the C-terminal part of NifL
and the central domain of NifA.
Physical interaction between NifLc and NifA
in vitro
To further confirm the interaction between NifLc and NifA,
the corresponding DNA fragments were cloned into
415 bp
L 1 2 3 A1501 1506 1507B 1507A
No plasmid
pVA3
pVA1
pVL
40
Nitrogenase activity
U (mg protein)
_
1
32
24
16
8
(a) (b)
Fig. 1. Expression of nifA and nitrogenase activity in A1501 wild-type and mutant strains. (a) Amplification of nifA transcript
by RT-PCR. Lanes: L, DNA size marker; 1, wild-type; 2, 1507B non-polar nifL mutant; 3, 1507A nifL polar mutant. The arrow
indicates the 415 bp amplification product of nifA. (b) Nitrogenase activity in wild-type and mutant strains complemented with
plasmids carrying nifA and nifL and incubated under nitrogen-fixation conditions: A1501, wild-type; 1507B, nifL-km non-polar
mutant; 1507A, nifL-km polar mutant; 1506, nifA-km mutant. pVA3 contains nifA under the control of the km promoter; pVA1
contains nifA in the opposite orientation; pVL contains nifL.
Fig. 2. Schematic representation of the domains of NifL (top)
and NifA (bottom). See text for explanations. The oligonucleo-
tides designed to amplify the gene portions encoding the differ-
ent domains are listed in Table 3.
Fig. 3. Specificity of interaction between NifL and NifA. S. cer-
evisiae PJ69-4A strains carrying different plasmid combinations
were grown on SD medium lacking Leu, Trp, His and Ade and
containing X-Gal, and the colony filter-lift assay in the presence
of X-Gal was performed according to the protocol of the
Matchmaker manual: 1, positive control; 2, pGAD-nifAc (C-
terminal)+pGBD-nifL (entire nifL coding region); 3, pGAD-nifA
(entire nifA coding region)+pGBD-nifL (entire nifL coding
region); 4, pGBD-nifAn (N-terminal)+pGBD-nifL (entire nifL
coding region); 5, pGBD-nifAm (central domain)+pGBD-nifLc
(C-terminal); 6, negative control (pGAD-C1, pGBD-C1).
http://mic.sgmjournals.org 3539
NifL/NifA interaction in Pseudomonas stutzeri
expression vectors to obtain a GST-NifLc and a His
6
-NifA
fusion protein as described in Methods. After purification
and SDS-PAGE analysis, the GST-NifLc product had an
apparent molecular mass in the range 67 kDa, consistent
with the size of the NifL C-terminal part, 40 ?7 kDa (from
residues 162–537; Fig. 2), and the GST moiety of 27 kDa.
The migration of the His
6
-NifA product, estimated at
64?2 kDa, was similar to that of the GST-NifLc fusion
(Fig. 4). However, the two fusion proteins could be easily
differentiated using anti-histidine or anti-GST antibodies
after Western blotting, as shown below.
The result of NifLA complex formation and elution using
the pull-down assay is shown in Fig. 4. Lanes 1 and 3
correspond to control experiments performed only with
NifA (lane 1) or with NifLc (lane 3), while lane 2 contained
the elution product after the complex formation between
NifLc and NifA. Fig. 4(a) shows protein staining. Anti-GST
antibody, shown in Fig. 4(b), revealed material containing
the GST-NifLc in lane 3 but not in lane 1, whereas anti-His
antibody, in panel (c), revealed material containing His
6
-
NifA in lane 1 but not in lane 3. Both antibodies revealed a
protein product in lane 2 (Fig. 4b, c), suggesting that it
corresponded to a mixture of NifA and NifLc. This is
strongly in favour of complex formation between NifL and
NifA, suggesting a specific binding between NifA and NifLc.
DISCUSSION
It was previously established that a nifL mutant polar on nifA
expression led to a Nif
2
phenotype in P. stutzeri A1501
(Desnoues et al., 2003). In this report, assay for nitrogenase
activity of the nifL non-polar mutant 1507B showed it
displayed a significant residual activity, suggesting that NifL
is not an essential product for nitrogen fixation in strain
A1501. This result is similar to that reported first in the case
of K. pneumoniae (see Filser et al., 1983) and subsequently
found in other strains con taining a nifL gene, such as
Azotobacter vinelandii (Blanco et al., 1993) and Azoarcus sp.
(Egener et al., 2002). In addition, the decrease of nitrogenase
activity observed both in the wild-type and in the nifL non-
polar mutant in the presence of NifL excess produced by
plasmid pVL (Fig. 1) is consistent with previous observa-
tions in K. pneumoniae (Buchanan-Wo llaston et al., 1981).
Thus, NifL most likely acts in modulating NifA activity in
A1501, and this suggests that NifL is an antiactivator of NifA
activity.
The yeast two-hybrid system is a useful technique to detect
protein–protein interaction (James et al., 1996). It has been
successfully used to explore binding between several
components of the ntr and nif regulatory systems (Lei
et al., 1999; Martı
´
nez-Argudo et al., 2002; Rudnick et al.,
2002; Pawlowski et al., 2003; Chen et al., 2005). Thus, a
direct protein–protein binding was detected between NifA
and NifL in K. pneumoniae, Azotobacter vinelandii and
Enterobacter cloacae (Lei et al., 1999; Martı
´
nez-Argudo et al.,
2002; Liao et al., 2002). Results reported in P. stutzeri reveal
also a direct protein–protein interaction between NifA and
NifL and more precisely between the NifL GHKL (NifLc)
and NifA AAA+ (N ifAm) domains. Indeed, N ifL PAS
domain (NifLn) does not display binding activity to NifA
(Table 3).
A prerequisite to further study of the mechanisms of
interaction between NifL and NifA is to obtain the protein
products in a soluble form (Lee et al., 1993). In this work we
used convenient expression vectors to overproduce His-
tagged NifA and GST fusion to NifLc. This enabled a rapid
purification of both proteins and allowed us to demonstrate
in vitro complex formation between NifLc and NifA (Fig. 4).
This is consistent with the in vivo binding observed. It also
suggests that under conditions not compatible with nitrogen
fixation, inactivation of NifA is probably due to a protein
complex between N ifL and NifA so that nif genes cannot be
transcribed and hence nitrogenase cannot be synthesiz ed.
Mechanisms by which NifL and NifA modulate nif gene
expression have been mainly studied in K. pneumoniae and
A. vinelandii. The activity of NifLA complexes is modulated
by GlnK (a PII parologue protein), ATP/ADP ratio and 2-
oxoglutarate, but it is clear that mechanisms of signal
communication between NifA and NifL are different in
these two species (Martı
´
nez-Argudo et al., 2004). In both
systems, GlnK, which senses the nitrogen status of the cell,
was shown to interact with the NifLA via direct protein–
protein interaction (Stips et al., 2004; Martı
´
nez-Argudo
et al., 2004). In the case of Azotobacter, when nitrogen is
limiting, GlnK, in its uridylylated form, does not interact
with NifL, and thus NifL does not antagonize NifA activity
Fig. 4. Analysis of protein complexes after elution from the Sepharose column. (a) SDS-PAGE. (b) Western blot with GST-
Tag antibody. (c) Western blot with His-Tag antibody, Lanes: 1, control His
6
-NifA; 2, His
6
-NifA+GST-NifLc; 3, control
GST-NifLc.
3540 Microbiology 152
Z. Xie and others
(Martı
´
nez-Argudo et al., 2004). In contrast, GlnK is required
for the relief of NifL inhibit ion in K. pneumoniae (He et al.,
1998; Stips et al., 2004). In the present report, we have
limited our investigation to the demonstration of an
interaction between NifL and NifA, using both in vivo
and in vitro techniques. It is not known whether a PII
protein is involved in the modulation of NifLA activity. P.
stutzeri A1501 carries a single copy of a glnB-like gene (this
laboratory, unpublished observation), as in Azotobacter. The
inactivation of the glnB-like gene and the purification of its
protein product is in progress. Althoug h P. stutzeri nifL is
not an essential gene for nitrogen fixation, the NifL pro tein
plays a regulatory role as antiactivator of NifA activity.
The purification of both NifA and NifL proteins opens
interesting perspectives to further study the mechanisms of
interaction of NifL and NifA in this species.
ACKNOWLEDGEMENTS
The authors wish to thank Ms Nicole Desnoues for the construction of
strain 1507B, Dr Sanfeng Chen for providing the plasmid vectors for
the Y2H experiments and Ms Patricia Shapiro for reading the
typescript. This work was supported by funds from National Programs
of China ‘973’ (grant no. 2001CB108904) and ‘863’ (grant no.
2001AA214021).
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