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Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 901-907 |
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doi:10.1111/j.1745-7270.2008.00467.x |
Symbiotic plasmid is required for NolR to
fully repress nodulation genes in Rhizobium� leguminosarum A34
Fengqing Li, Bihe Hou, and Guofan
Hong*
State Key Laboratory of Molecular Biology,
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, Shanghai 200031, China
Received: April 3, 2008�������
Accepted: May 16,
2008
This work was
supported by the grants from the Pan-Deng Plan and the Innovation Program of
China
*Corresponding
author: Tel, 86-21-54921223; Fax, 86-21-54921011; E-mail, [email protected]
NolR is a regulator of nodulation
genes present in Rhizobium� and Sinorhizobium. However, the
mechanism by which NolR participates in the inducible transcription of
nodulation genes remains unclear. To investigate whether there are other
factors� regulating the function of NolR, an insertion mutant of NolR in
Rhizobium leguminosarum strain 8401, which lacks the symbiotic plasmid,
was constructed by homologous recombination. We investigated the effects of
NolR in�activation on the expression of nodulation genes. Three inducible
nodulation� genes (nodA, nodF and nodM) were expressed
constitutively in NolR- mutant, MR114. Our results suggested� that
the symbiotic plasmid is required for NolR to fully repress nodulation genes in
Rhizobium leguminosarum A34. In addition, MR114 has provided a useful
tool for further� study of molecular interactions between NolR and other
factors.
Keywords �������NolR;
symbiotic plasmid; nod box
The development of nitrogen-fixing
plant root nodules by rhizobia requires an exchange of signals between the two
partners [1-6]. During
this plant-microbe interaction, bacterial� nodulation (nod) gene
expression is regulated both positively and negatively. The positive elements
include nodD, which is a regulatory protein common to all rhizobia� [7]; nodD1,
nodV, and nodW in Bradyrhizobium japonicum [8,9]; and syrM in Sinorhizobium
meliloti (S. meliloti) [10]. The negative elements include nodD2 and
NolA in Bradyrhizobium japonicum and NolR in Rhizobium and
Sinorhizobium [11-14].
NolR, encoded by the chromosomal NolR
gene, was first found in S. meliloti AK631 as a repressor controlling nod
gene expression; it was found to be necessary for optimal nodulation on alfalfa
[15]. NolR protein, in dimeric form, binds to the overlapping nodD1 and nodA
promoters� at the RNA polymerase binding site [16]. It has been proposed� that
NolR binds to the (A/T)TTAG-N(9)-A(T/A) target sequences [14]. Studies in S.
meliloti have shown that both the HTH motif and the C-terminal part of NolR
are essential for DNA binding [17,18]. NolR of S. meliloti
differentially down-regulates nod genes involved in the core nod factor
synthesis, which results in production of low quantities of fully decorated nod
factors [18]. The expression� of NolR is repressed by its own product as
well as by the nod gene inducer luteolin. Studies in Sinorhizobium
fredii HH103 have shown that inactivation or overex�pression of NolR
can affect the production of nod factors, signal responsive proteins and
exopolysaccharide and thus change the nodulation capacity� of Sinorhizobium
fredii on different hosts [19]. Proteomic studies on S. meliloti strains
have suggested that NolR might be a global regulator that influences the
expression of many proteins involved in various metabolic pathways and cellular
functions [20]. Subsequent studies have shown that NolR is a global regulatory
protein required to optimize� nodulation, bacterial growth and survival, and
conjugative transfer of a plasmid [21]. Although research has uncovered� an
increasing number of NolR functions, the mechanism by which NolR participates
in the inducible transcription of nod genes remains unclear.
In this study, we mutated NolR
in Rhizobium leguminosarum (R. leguminosarum) 8401,
a strain lacking� the symbiotic plasmid (pSym), by homologous recombination. We
then investigated the effects of NolR inactivation on the expression of nod
genes. Three inducible� nod genes (nodA, nodF and nodM)
were expressed constitutively� in 8401 NolR- mutant, MR114, suggesting that the
pSym is required for NolR to fully repress nod genes in R.
leguminosarum A34 (A34).
Materials and Methods
Bacterial strains, plasmids and
DNA fragments
All the bacterial strains and
plasmids used in this work and their relevant characteristics are listed in Table
1. Escherichia� coli�� strains were grown at 37 �C in Luria-Bertani
(LB) medium. Rhizobia were grown at 28 �C in TY medium [22]. If appropriate,
antibiotics were added at the following concentrations: for E. coli,
100 mg/ml of ampicillin, 50 mg/ml of kanamycin, and 20 mg/ml of chloramphenicol or
tetracycline; for rhizobia, 100 mg/ml of streptomycin, 25 mg/ml of kanamycin, 20 mg/ml of tetracycline� and 10 mg/ml of chloramphenicol. The sequences� of all the primers used are
listed in Table 2. The nodAD promoter fragment (AD13) was
obtained by EcoRI/PstI digestion of plasmid pUCWZ. The nodF,
nodM and nodO promoter fragments were named as F12, M12 and O12,
respectively; they were obtained from EcoRI/PstI fragments of
polymerase chain reation (PCR) products� amplified with total DNA of A34 as
template and primer pairs of F1E and F2P, M1E and M2P, and O1E and O2P,
respectively. The NolR promoter fragment (R12) was obtained from EcoRI/SphI
fragments of PCR products amplified with total DNA of A34 as template and
primer pair RP1E and RP2Sp. The sizes of AD13, F12, M12, O12 and R12 fragments
were 317 bp, 218 bp, 302 bp, 312 bp and 308 bp, respectively. All these
fragments were labeled with a-32P-dATP by Klenow fragment and used
in gel retardation assays.
For the construction of nodD-lacZ,
nodF-lacZ, nodM-lacZ and NolR-lacZ fusions, AD13, F12, M12 and R12 fragments were
cloned into pMP220 vector to obtain pMP220D, pMP220F, pMP220M and pMP220R,
respectively. To construct nodA-lacZ fusion, the AD13 fragment was cloned into pMP221 vector to obtain
pMP221A. These report plasmids were transferred into 8401 and its NolR- mutant by biparental conjugation
in order to investigate the effects of NolR inactivation on the expression of
the inducible nod genes and NolR itself [23].
Overexpression of NolR protein in
8401R
pU3R was constructed by inserting the NolR
gene into plasmid pKNDT to replace the nodD gene [24]. R1E and R2E
primers were used to amplify the NolR gene. Bpneo and R2E primers were
used to screen the positive colonies. 8401R was obtained by transferring pU3R into 8401.
Construction of 8401 NolR- mutant by homologous
recombination
The NolR::KmR disrupted gene fragment was
constructed by overlapping extension PCR as described by Ho et al [25].
Three fragments, RF1, Km and RF2, were obtained in the first-turn PCR using the
primer pairs of RKm1B and RKm2, RKm3 and RKm4, and RKm5 and RKm6B,
respectively, and total DNA of 8401, pUCKm, total DNA of 8401, respectively, as
templates. In the second-turn PCR, fragment RF1Km was obtained using annealing
products� of purified RF1 and Km as templates and primer pair RKm1B and RKm4.
In the third-turn PCR, we used the annealing products of purified RF1, Km and
RF2 as templates and primer pair RKm1B and RKm6B. The resulting� fragment
(R2K::Km) contains 8401 chromosome DNA fragment (2 kb), in which a 1.5 kb
kanamycin resistance� cassette was inserted at position 172 of NolR open
reading frame in 8401. Then, R2K::Km fragment was BamHI-digested and
inserted into the suicide plasmid pSUP202 to obtain p202R2K::Km [26]. This
plasmid was transferred into 8401 by biparental conjugation. Co-integrate�-containing
exconjugants (resulting from single crossover) were distinguished from true
marker exchange mutants (resulting from double crossover) by the vector�s
ampicillin resistance. One of the mutants obtained, MR114, was confirmed by
Southern blot analysis and PCR method.
DNA manipulation and sequence
analysis
Standard DNA work and Southern
blot were carried out as described by Sambrook et al [27]. Total DNA of
8401 was extracted using a Promega genomic preparation kit (Madison, USA). DNA
was sequenced by Shanghai GeneCore BioTechnologies (Shanghai, China).
Nucleotide sequence data were analyzed with the DNA-Star package. Homology
searches were carried out using NCBI BLAST
server (http://www.ncbi.nlm.nih.gov/blast/).
b-galactosidase activity assay
b-galactosidase activity assays
were carried out as described� by Miller [28], using 10 mM naringenin to induce� nodA,
nodF and nodM. Three independent experiments were carried out for
each strain at the same optical density� (OD600=0.4).
Gel retardation assay
Gel retardation assays were
carried out as described by Hu et al [23].
Results
Identification and sequencing of NolR
in 8401
To verify that NolR exists
in 8401 and A34, we used a labeled NolR fragment (PCR product of primer
pair TOMR1E and TOMR2E) from R. leguminosarum bv.
viciae strain TOM as a probe to hybridize three restriction
endonuclease-digested genomic DNA from 8401 and A34 (data not shown). The three
restriction endonuclease were BamHI, BglII and EcoRI. The
hybridization signals indicated that there was only one copy of NolR in
8401 and A34 and that NolR is located on the chromosome of 8401 and A34.
Two primers, 3841R1 and 3841R2,
were designed from the homologous region of a 2 kb fragment of R. leguminosarum
3841, which contained the NolR gene. These primers were used for PCR
amplification of a 2040 bp fragment of 8401. DNA sequencing of the 2040 bp
fragment (GenBank accession No. EU416274) showed that the NolR
gene extends between positions 903 and 1220, encoding 105 amino acids (aa), and
respectively shares 100%, 99% and 77% identity with the NolR proteins of R.
leguminosarum TOM, 3841 and S. meliloti AK631.
NolR in 8401 has different affinities for nodAD, nodF,
nodM, nodO and NolR promoters in vitro
When the protein extracts of 8401 and A34 were used in gel
retardation assays, the NolR-binding band was not detectable [Fig. 1(A),
lanes 2-5]. Using a Rhizobium-expressing system in which native Rhizobium
proteins could be overexpressed, we constructed a strain that overexpressed
NolR in order to investigate the binding activity of NolR in 8401 to nod
box. The gel shift assay showed that the protein extracts of 8401R could bind
to the nodAD promoter and that the major band was a NolR-DNA binding
band [Fig. 1(A), lane 7].
Using the protein extracts of 8401R, we performed the gel
retardation assay to observe the affinities of nodAD, nodF, nodM,
nodO and NolR promoters for NolR in vitro. Obviously, NolR
promoter had the greatest affinity for NolR [Fig. 1(B), lane 10]. This
result suggested that NolR might autoregulate its own expression by negative
feedback inhibition. NolR promoter had the greatest affinity for NolR
and was followed, from highest to lowest, by nodM,
nodA, nodO and nodF promoters [Fig. 1(B)]. This result
suggested that NolR might repress these latter genes to different extents.
Expression of NolR is negatively autoregulated and not
affected by the nod gene-inducer naringenin in 8401
Expression of NolR was studied by measuring the b-galactosidase
activity of a NolR-lacZ fusion plasmid introduced into 8401, A34 and
MR114 (Fig. 2). In 8401 and A34, the NolR-lacZ fusion
gene�s expression of NolR showed was very low, compared
with that of nodD in 8401 (Table 3). Since there were no visible
NolR-DNA complex bands in the gel shift assay in Fig. 1(A), it seems
likely that the amount of NolR protein in the 8401 and A34 strains is very low.
In 8401, NolR expression was only about 10 % of that in MR114 (Fig. 2),
suggesting a strongly negative autoregulation of NolR. It was also found
that inducer naringenin had almost no influence on NolR expression� in
8401, MR114 and A34.
The inducible nod genes expressed constitutively in MR114
Inactivation of NolR made three inducible nod genes, nodA,
nodF and nodM, express constitutively in mutant MR114 (Table
3).
We found that NolR repressed nod genes without inducer�
naringenin in 8401. In the absence of inducer naringenin, the expression of nodA-lacZ,
nodF-lacZ and nodM-lacZ fusions in 8401 were approximately 6.3%,
33.3% and 12.5%, respectively, of those in MR114. The repression extent of NolR
for these genes is basically in proportion to the affinity of NolR for their
promoters. So NolR repression� for inducible nod genes is likely
correlated with the affinities� of NolR for nod gene promoters.
In the presence of inducer naringenin, the expression of nodA-lacZ,
nodF-lacZ and nodM-lacZ fusions in 8401 was approximately 9.1%,
50% and 14.3%, respectively, of those in MR114. This showed that in 8401 the
presence� of inducer has little influence on NolR repression for inducible� nod
genes. Although the inducible nod genes express� constitutively in
MR114, the expression level of these genes is much lower than that in A34. In
summary, our results suggested that the pSym is required for NolR full
repression of nod genes in A34.
Discussion
In our previous study on the effect of nod box conformational
changes on the expression of nod genes [36], we proposed that there
might be a repressor involved in the regulation of that expression. NolR might
be this repressor. As research continues to examine NolR, more of its
functions� have been revealed; for example, Cren et al have proposed that
an inducer and other proteins control NolR�s negative autoregulation
[18]. It is difficult to investigate NolR function in such a complicated
system. Unlike most nod genes that are located on the symbiotic plasmid,
NolR is on the chromosome. To study its function against a much
simplified genetic background, we mutated NolR in 8401 by homologous
recombination. By comparing our results with previous reports, we were able to
demonstrate� the functions and the corresponding regulation mechanisms of NolR
in wild-type strains.
We found that NolR expression was not affected by inducer
naringenin in 8401 and A34. This result differed from previous reports that NolR
expression was affected by inducer luteolin in S. meliloti strain AK631
and ZB138, an AK631 derivative strain cured of pSym [18]. R. leguminosarum
and S. meliloti strains showed different phenotype of inducer�s
influence on NolR expression. One possible reason for this was that R.
leguminosarum�s genetic� background differs from S. meliloti. As we
know, there are three copies of nodD in S. meliloti, nodD1,
nodD2 and nodD3, but in A34, there is only one copy of nodD.
Therefore, there must be some other unknown differences in their genetic
backgrounds. As the 8401 phenotype differs� from that of S. meliloti
ZB138, the factors that cause this variance may be present on the chromosome of
R. leguminosarum or S. meliloti.
Inducible nod genes (nodA, nodF and nodM)
can be expressed strongly in the absence of nodD in MR114, which
indicates that NolR inactivation is sufficient for the transcription� of the
inducible nod genes. In MR114, the conformation of nod box, which
is not bound by NolR, is likely suitable for RNA polymerase to initiate the
transcription� of the inducible nod genes. So in a wild-type strain
harboring the pSym, such as A34, activated nodD might make the
conformation of nod box more suitable for RNA polymerase to initiate the
transcription.
Our results indicated that the presence of the pSym is required for
NolR to fully repress nod genes in A34, suggesting� that unidentified
factors present in the pSym could mediate the NolR repression capacity. As
shown in Fig. 1(A), protein extracts from 8401D resulted in more free
DNA fragments being bound than that from 8401R. It seemed probable that nodD
had greater affinity for the nodAD promoter than NolR in vitro,
assuming that the amount of nodD and NolR proteins expressed in the same system
were basically equal. At the same time, we found that the expression level of nodD-lacZ
is much higher than that of NolR-lacZ (>10 folds) in 8401 (Table 3
and Fig. 2), also suggesting that other factors might be required in
NolR repression for nod genes via binding to nod box in vivo.
It seems likely that nodD is the first candidate for mediating
inducer-NolR indirect interactions.
To further study which factors are involved in the inducer�-dependent
repression of NolR, we suggest introducing� some candidates into MR114.
Candidates able to rescue inducible nod genes� expression level in MR114
to a level comparable to wild-type strains� would be most suitable.
In conclusion, we mutated NolR in 8401 by homologous�
recombination and investigated the effects of NolR inactivation� on the
expression of nod genes and NolR gene in 8401 and MR114. We found
that three inducible nod genes (nodA, nodF and nodM)
express constitutively in MR114. Therefore, our results showed that the
symbiotic plasmid is required for NolR to fully repress nod genes in
A34. In addition, MR114 has provided a useful tool for further study of molecular
interactions between NolR and other factors.
Acknowledgements
We are grateful to Dr. Jie Feng and Dr. Qiang Li for their kind help and discussing the research with us.
References
1���� Long SR.
Rhizobium-legume nodulation: life together in the underground. Cell 1989, 56:
203-214
2���� D�nari� J, Debell� F,
Prom� JC. Rhizobium lipo-chitooligosaccharide nodulation factors: signaling
molecules mediating recognition and morphogenesis. Annu Rev Biochem 1996, 65:
503-535
3���� van Rhijn P,
Vanderleyden J. The rhizobium-plant symbiosis. Microbiol Rev 1995, 59: 124-142
4���� Downie JA. Signalling
strategies for nodulation of legumes by rhizobia. Trends Microbiol 1994, 2: 318-324
5���� G�ttfert M. Regulation
and function of rhizobial nodulation genes. FEMS Microbiol Rev 1993, 10: 39-63
6���� Schlaman HR, Okker RJ,
Lugtenberg BJ. Regulation of nodulation gene expression by nodD in rhizobia. J
Bacteriol 1992, 174: 5177-5182
7��� G�ttfert M, Holzh�user
D, B�ni D, Hennecke H. Structural and functional analysis of two different nodD
genes in Bradyrhizobium japonicum USDA110. Mol Plant Microbe Interact
1992, 5: 257-265
8���� G�ttfert M, Hitz S,
Hennecke H. Identification of nodS and nodU, two inducible genes
inserted between the Bradyrhizobium japonicum nodYABC and nodIJ
genes. Mol Plant Microbe Interact 1990, 3: 308-316
9���� Loh J, Garcia M, Stacey G. NodV, nodW, a second flavonoid
recognition system regulating nod gene expression in Bradyrhizobium
japonicum. J Bacteriol 1997, 179: 3013-3020
10��� Mulligan JT, Long SR. A
family of activator genes regulates expression� of Rhizobium meliloti
nodulation genes. Genetics 1989, 122: 7-18
11��� Sadowsky MJ, Cregan PB,
Gottfert M, Sharma A, Gerhold D, Rodriguez-Quinones F, Keyser HH et al.
The Bradyrhizobium japonicum NolA gene and its involvement in the
genotype-specific� nodulation of soybeans. Proc Natl Acad Sci USA 1991, 88: 637-641
12��� Garcia M, Dunlap J, Loh J,
Stacey G. Phenotypic characterization and regulation of the NolA gene of
Bradyrhizobium japonicum. Mol Plant Microbe Interact 1996, 9: 625-636
13��� Loh J, Stacey MG, Sadowsky
MJ, Stacey G. The Bradyrhizobium japonicum NolA gene encodes three
functionally distinct proteins. J Bacteriol 1999, 181: 1544-1554
14��� Kiss E, Mergaert P, Olah B,
Kereszt A, Staehelin C, Davies AE, Downie JA. Conservation of NolR in
the Sinorhizobium and Rhizobium� genera of the Rhizobiaceae family.
Mol Plant Microbe� Interact 1998, 11: 1186-1195
15��� Kondorosi E, Gyuris J,
Schmidt J, John M, Duda E, Hoffmann B, Schell J et al. Positive and
negative control of nod gene expression in Rhizobium meliloti is
required for optimal nodulation. EMBO J 1989, 8: 1331-1340
16��� Kondorosi E, Pierre M,
Haumann U, Buire M, Hoffmann B, Schell J, Kondorosi A. Identification of NolR,
a negative transacting factor controlling the nod regulon in Rhizobium
meliloti. J Mol Biol 1991, 222: 885-896
17��� Cren M, Kondorosi A,
Kondorosi E. An insertional point mutation inactivates NolR repressor in Rhizobium
meliloti 1021. J Bacteriol 1994, 176: 518-519
18��� Cren M, Kondorosi A,
Kondorosi E. NolR controls expression of the Rhizobium meliloti nodulation
genes involved in the core Nod factor synthesis. Mol Microbiol 1995, 15: 733-747
19��� Vinardell JM, Ollero FJ,
Hidalgo �, L�pez-Baena FJ, Medina C, Ivanov-Vangelov K, Parada M et al.
NolR regulates diverse symbiotic signals of Sinorhizobium fredii HH103.
Mol Plant Microbe Interact 2004, 17: 676-685
20��� Chen H, Higgins J, Kondorosi
E, Kondorosi A, Djordjevic MA, Weinman JJ, Rolfe BG. Identification of nolR-regulated
proteins in Sinorhizobium meliloti using proteome analysis.
Electrophoresis 2000, 21: 3823-3832
21��� Chen H, Gao K, Kondorosi E,
Kondorosi A, Rolfe BG. Functional genomic analysis of global regulator NolR in Sinorhizobium
meliloti. Mol Plant Microbe Interact 2005, 18: 1340-1352
22��� Li Q, Feng J, Hu HL, Chen XC,
Li FQ, Hong GF. A HU-like gene mutation in Rhizobium leguminosarum bv.
viciae affects the expression of nodulation genes. Mol Microbiol 2004, 51: 861-871
23��� Hu HL, Liu ST, Yang Y, Chang
WZ, Hong GF. In Rhizobium leguminosarum, nodD represses its own
transcription by competing with RNA polymerase for binding sites. Nucleic Acids
Res 2000, 28: 2784-2793
24��� Feng J, Li FQ, Hong GF.
Expression and purification of Rhizobium leguminosarum nodD. Protein
Expr Purif 2002, 26: 321-328
25��� Ho SN, Hunt HD, Horton RM,
Pullen JK, Pease LR. Site-directed mutagenesis by overlap extension using the
polymerase chain reaction. Gene 1989, 77: 55-59
26��� Simon R, Priefer U, P�hler A.
Vector plasmids for in vivo and in vitro manipulations of
Gram-negative bacteria. In: P�hler A, ed. Molecular Genetics of the Bacteria
Plant Interaction. Heidelberg: Spring-Verlag 1983
27��� Sambrook J, Fritsch EF,
Maniatis T. Molecular Cloning: A Laboratory Manual. New York: Cold Spring
Harbor 1989
28��� Miller J. Experiment in
Molecular Genetics. New York: Cold Spring Harbor 1972
29��� Johnston AW, Beringer JE.
Identification of the Rhizobium strains in pea root nodules using
genetic markers. J Gen Microbiol 1975, 87: 343-350
30��� Downie JA, Ma QS, Knight CD,
Hombrecher G, Johnston AWB. Cloning of the symbiotic region of Rhizobium
leguminosarum: the nodulation genes are between the nitrogenase genes and
the nifA-like gene. EMBO J 1983, 2: 947-952
31��� Downie JA, Knight CD,
Johnston AWB, Rossen L. Identification of genes and gene products involved in
the nodulation of peas by Rhizobium leguminosarum. Mol Gen Genet 1985,
198: 225-262
32��� Simon R, Priefer U, P�hler A.
A broad range host mobilization system for in vivo genetic engineering:
transposon mutagenesis in gram-negative bacteria. Biotechnology 1983, 1: 784-791
33��� Bagdasarian M, Lurz R, R�ckert
B, Franklin FC, Bagdasarian MM, Frey J, Timmis KN. Specific purpose plasmid
cloning vectors. II. Broad host range, high copy number RSF1010-derived vectors
and a host-vector system for gene cloning in Pseudomonas. Gene 1981, 16:
237-247
34��� Spaink HP, Okker RJ,
Wijffelman CA, Tak T, Goosen-de Roo L, Pees E, van Brussel AA et al.
Symbiotic properties of rhizobia containing a flavonoid independent hybrid nodD
product. J Bacteriol 1989, 171: 4045-4053
35��� Chang WZ, Hong GF. Two
functional regions were discovered within nodA promoter. Chin J
Biotechnol 1997, 13: 83-87
36��� Chen XC, Feng J, Hou BH, Li
FQ, Li Q, Hong GF. Modulating DNA bending affects nodD-mediated transcriptional
control in Rhizobium leguminosarum. Nucleic Acids Res 2005, 33: 2540-2548