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Acta Biochimica et Biophysica Sinica Advance Access originally published online on July 23, 2009
Acta Biochimica et Biophysica Sinica 2009 41(9):719-730; doi:10.1093/abbs/gmp060
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© The Author 2009. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Post-transcriptional regulation of NifA expression by Hfq and RNase E complex in Rhizobium leguminosarum bv. viciae

Yinghua Zhang1,2 and Guofan Hong1,*

1 State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
2 Graduate School of the Chinese Academy of Sciences, Shanghai 200031, China

* Correspondence address. Tel: +86-21-54921223; Fax: +86-21-54921011; E-mail: gfhong{at}sibs.ac.cn


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 References
 
NifA is the general transcriptional activator of nitrogen fixation genes in diazotrophic bacteria. In Rhizobium leguminosarum bv. viciae strain 8401/pRL1JI, the NifA gene is part of a gene cluster (fixABCXNifAB). In this study, results showed that in R. leguminosarum bv. viciae 8401/pRL1JI, host factor required (Hfq), and RNase E were involved in the post-transcriptional regulation of NifA expression. It was found that Hfq-dependent RNase E cleavage of NifA mRNA was essential for NifA translation. The cleavage site is located at 32 nucleotides upstream of the NifA translational start codon. A possible explanation based on predicted RNA secondary structure of the NifA 5'-untranslated region was that the cleavage made ribosome-binding sites accessible for translation.

Keywords    Hfq; RNase E; NifA; Rhizobium leguminosarum bv. viciae

Received: March 3, 2009; Accepted: April 21, 2009
   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Funding
 References
 
NifA belongs to the bacterial enhancer-binding protein family of transcriptional regulators that activate gene expression in concert with RNA polymerase containing the specialized {sigma}54 factor, which allows the polymerase core to recognize the –24/–12-type promoters [1–3]. In diazotrophic bacteria, NifA activates transcription of nitrogenase genes (Nif genes) and fix genes, particularly in the legume endosymbiotic bacteria. Although NifA genes are conserved in diazotrophic bacteria, their organization within Nif gene clusters differs [1]. In fast-growing rhizobia, such as Sinorhizobium meliloti, Rhizobium leguminosarum bv. trifolii, and R. leguminosarum bv. viciae, NifA is located in the symbiotic plasmid, between the fixABC operon and NifB gene.

There are different mechanisms for the regulation of NifA expression in root nodule bacteria (Sinorhizobium meliloti [4–7], Bradyrhizobium japonicum [8,9], and Azorhizobium caulinodans [10]). The regulation of NifA expression in R. leguminosarum bv. viciae has not been studied in detail. Nitrogenase is not expressed in free-living cells, probably because NifA is not functional under these conditions. The expression of the NifA gene is positively controlled by host factor required (Hfq) in Azorhizobium caulinodans [11,12] and Rhodobacter capsulatus [13]. However, whether and how Hfq regulates NifA gene expression in R. leguminosarum remains unknown.

The Hfq gene was initially identified as a gene encoding a bacterial Hfq for the replication of the bacteriophage Qβ RNA [14,15]. Inactivation of the Hfq gene in Escherichia coli causes a wide variety of phenotypes and alters the expression of many genes [16,17]. Hfq controls the translation [18,19] and decay [20] of some mRNAs in an sRNA-independent manner. Several antisense sRNAs, however, need Hfq to interact with target mRNA(s) that, in turn, modify mRNA translation and/or stability. Hfq also stabilizes the interacting sRNAs in vivo [21,22]. By facilitating the interaction between some sRNAs and their associated mRNA targets, the protein participates in the positive or negative regulation of translation initiation of these mRNAs [23,24]. The sRNA–mRNA interaction can result in the sequestration or exposure of the Shine–Dalgarno sequence from the mRNA targets or the initiation of mRNA degradation by RNase E [25] or RNase III [26]. In E. coli, 20% of the Hfq proteins co-purify with RNase E, but how the protein targets the mRNAs for degradation or stabilization is unknown [27].

Hfq can also induce mRNA stabilization with the help of an sRNA [28]. Several sRNAs that specifically bind the Hfq protein (e.g. DsrA, MicF, RyhB, SgrS, and RydC) control the translation of selected mRNAs in response to environmental stress [29].

RNase E is an essential endoribonuclease in bacteria. RNase E plays an important role in all aspects of RNA metabolism, including processing and/or decay of rRNAs, tRNAs, non-coding small RNAs, and mRNAs. Endoribonucleolytic cleavage by RNase E depends on 5'-terminal structures of RNAs [30,31] and occurs within single-stranded A- and/or U-rich segments [32–34].

In this study we found that in R. leguminosarum bv. viciae strain 8401 pRL1JI, NifA gene expression was regulated by Hfq and RNase E at the post-transcriptional level.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Funding
 References
 
Bacteria and plasmids
Escherichia coli DH5{alpha} was used as a host for the cloning experiments, and E. coli ER2566 for overproduction of the recombinant proteins. Biparental conjugation was performed to mobilize broad-host-range plasmids from E. coli to R. leguminosarum as described by Simon et al. [35]. Rhizobium leguminosarum strain 8401 is a derivative of a biovar phaseoli isolate (strain 8002); it is streptomycin resistant and has been cured of the Sym plasmid pRP2JI. Rhizobium leguminosarum strain 8401/pRL1JI carries the biovar viciae Sym plasmid. The plasmids used in this study are described in Table 1.


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  		Table 1 Plasmids used in this study

 
Media
Rhizobium leguminosarum strains were routinely grown in TY medium (5 g tryptone, 3 g yeast extract, and 0.66 g CaCl2 per liter) at 28°C. Starter cultures were prepared by inoculating a single colony from a TY plate stock into 3 ml of TY medium and incubating overnight at 28°C in a shaking incubator (300 rpm). A 0.3-ml portion of this culture was then subcultured into another 3 ml of TY medium and incubated to a suitable cell density (OD600 of 0.3–0.4). A 0.03-ml portion of this starter culture was then inoculated into 3 ml of nitrogen-rich medium [36] to give an initial optical density at 600 nm of 0.02. To investigate the nitrogen starvation effect, 3 ml of starter culture were centrifuged and 99% of the clarified medium was decanted; then the cell pellets were inoculated into 3 ml of nitrogen-free medium [36]. The nitrogen-free medium contained per liter: 5 g DL sodium lactate, 5 g disodium succinate hexahydrate, 1.67 g K2HPO4, 0.87 g KH2PO4, 0.1 g MgSO4·H2O, 0.05 g NaCl, 40 mg CaCl2·2H2O, 10 mg FeCl3·6H2O, 5 mg Na2MoO4·2H2O, 2.5 mg MnSO4·H2O, 0.7 mg ZnSO4·7H2O, 0.14 mg CoSO4·7H2O, 0.12 mg CuSO4·5H2O, 0.03 mg H3BO4, 0.02 mg biotin, 4 mg nicotinic acid, and 4 mg calcium pantothenate. The nitrogen-rich medium was the nitrogen-free medium supplemented with 1 g/L (NH4)2SO4 and 1 g/L yeast extract. The nitrogen-free medium contained no nitrogen source at all and the poor growth of R. leguminosarum strains relied on the limited nitrogen source coming from starter cultures. Escherichia coli strains were grown in Luria–Bertani medium at 37°C.

Construction of modified suicide plasmid pSUP202a
A 1.5-kb kanamycin resistance cassette was amplified by PCR from plasmid pSUP1011 [37] using primers P19 (5'-CGCGAAGCTTGCGGCCGCCACGCTGCCGCAA-GCACTC-3') containing HindIII site and NotI site (underlined) and P20 (5'-CGCGGGATCCCCAAAGC-GGCCATCGTGCC-3') containing a BamHI site (underlined). The fragment obtained was digested with BamHI/HindIII and designated fragment K. A 1.8-kb fragment containing the complete sacB gene was amplified by PCR from Bacillus subtilis genomic DNA using primers P21 (5'-CGCGGGATCCTCTAGACGCCATTC-TGTGCCGTTGCC-3'), which contains BamHI site followed by XbaI site (underlined), and P22 (5'-CGC-GGTCGACAATCCTTCAGTTAGAGATCC-3'), which contains an SalI site. The fragment obtained was digested with BamHI and SalI and designated fragment S. Fragment K and fragment S were ligated with HindIII/SalI digested pSUP202. The obtained plasmid was designated pSUP202a, which contained a kanamycin resistance gene (for positive screening with kanamycin) and the sacB gene (for negative screening with 10% sucrose [38]). The sacB gene encodes a levansucrase gene and levansucrase activity is lethal in the presence of sucrose for most gram-negative bacteria.

Construction of the R. leguminosarum bv. viciae Hfq mutant
The 2.5 kb Hfq region DNA was amplified by PCR from genomic DNA of R. leguminosarum bv. viciae strain 8401/pRL1JI using primers P27 (5'-GAGAATTGCCTA-TGTAAATGG-3') and P30 (5'-CGGCCCGTTGCCGA-TGCGGC-3'). The fragment was sequenced by Shanghai GeneCore BioTechnologies (Shanghai, China). The sequence data have been submitted to the GenBank databases under accession No. FJ648549.

The 1.4 kb fragment downstream of the Hfq stop codon was obtained by PCR amplification from genomic DNA of R. leguminosarum bv. viciae strain 8401 using primers P23 (5'-CGCGGGATCCCAGGATCAGCCGT-CATTTCG-3'), which contains a BamHI site, and P24 (5'-CGCGTCTAGACGGTCGCTTCGGTTTCGGACAGG-3') which contains an XbaI site. The 900 bp fragment upstream of the Hfq start codon was obtained by PCR amplification from genomic DNA of R. leguminosarum bv. viciae strain 8401 using primers P25 (5'-CGCG-GCGGCCGCCCGATGCCAGCGTGCATATCGAGG-3'), which contains a NotI site, and P26 (5'-CGCGGCGG-CCGCCGCGCCGCTTCTTTCTTTATTGCC-3'), which also contains a NotI site. The two fragments were then inserted into the BamHI/XbaI and NotI sites of the modified suicide plasmid pSUP202a. The resulting plasmid pSUP202a2, in which the Hfq coding region was replaced by a kanamycin resistance cassette, was transferred into R. leguminosarum bv. viciae strain 8401/pRL1JI by biparental conjugation. After screening with kanamycin and 10% sucrose, one true marker exchange mutant (resulting from double crossover) was confirmed by PCR using primers P27 (5'-GAGAATTGCCTATGT-AAATGG-3')/P28 (5'-GAGTGCTTGCGGCAGCGTG-3') and P29 (5'-GCATCCAGGAAACCAGCAGCG-3')/P30 (5'-CCGGCCCGTTGCCGATGCGGC-3').

DNA manipulations and sequence analysis
Rhizobium leguminosarum bv. viciae genomic DNA was extracted using a Promega genomic preparation kit (Madison, USA). DNA was sequenced by Shanghai GeneCore BioTechnologies. Nucleotide sequence data were analyzed with the DNA-star package. Homology searches were performed using the National Center for Biotechnology Information BLAST network server (http://www.ncbi.nlm.nih.gov/BLAST/).

Construction of Lac promoter-controlled fixABCXNifAB expression plasmid and strains
The 7.5 kb DNA fragment including fixA, fixB, fixC, fixX, NifA, and NifB genes was amplified by PCR from genomic DNA of R. leguminosarum bv. viciae strain 8401/pRL1JI using primers P35 (5'-TGTCGGCAACCC-TACAAAACCCC-3') and P6 (5'-TCAATTAGAGGGG-CCTAAAGCCG-3'). The fragment was sequenced by Shanghai GeneCore BioTechnologies. The sequence data have been submitted to the GenBank database under accession No. FJ648550.

The rrnB terminator was amplified with primers P3 (5'-CGCGGGTACCCTGTTTTGGCGGATGAGAG-3') containing a KpnI site/P4 (5'-CGCGGGTACCCAAAA-AGGCCATCCGTCAGG-3') containing a KpnI site from pKK223-3 plasmid DNA by PCR and inserted into the KpnI site of pRK415 to generate the expression plasmid pRK415a. The 7 kb DNA fragment of the fixABCXNifAB gene region without its potential endogenous promoter upstream of fixA was amplified with primers P5 (5'-GGGTCCCTAAAGCCCGAATCCG-3')/P6 (5'-TC-AATTAGAGGGGCCTAAAGCC-3') from the 7.5 kb DNA by PCR and introduced into the HindIII site of pRK415a by blunt end ligation so that the 7 kb fixABCXNifAB DNA was under control of the Lac promoter (Plac) and the rrnB terminator. The resulting plasmid was designated pRK415a-FN and transferred into R. leguminosarum bv. viciae strain 8401/pRL1JI wild type or Hfq mutant strain (M21) by biparental conjugation.

Northern hybridization
Total cellular RNA was isolated and analyzed by northern hybridization. Briefly, cells were grown at 28°C in nitrogen-limited or nitrogen-rich medium to reach an optical density at 600 nm of 0.3–0.4. After being harvested and washed with ice-cold TE (10 mM Tris–HCl, pH 8.0, and 1 mM EDTA), the cells were suspended in 100 µl of TE and extracted directly by vigorously mixing them with an equal volume of equilibrated phenol/chloroform. After centrifugation (10,000 rpm), part of the upper layer was used for analysis. RNA samples were fractionated by formaldehyde-agarose gel electrophoresis and transferred onto positively charged nylon membranes by the capillary method. DNA probes were prepared by PCR amplification using primers P7 (5'-ATGATTAAACCAGAGGCGC-3')/P8 (5'-TCACT-CCTTCTTCACATCGATACG-3') for NifA and P9 (5'- CAACATGAGAGTTTGATCCTGG-3')/P10 (5'-CGTC-TTACCAATTCCACAGC-3') for 16S rRNA. The probes were labeled with 32P by random primer labeling.

Purification of Rhizobium Hfq protein
Rhizobium Hfq was expressed and purified using the IMPACTTM-CN protein purification system of New England Biolab (NEB; Beijing, China).

The coding region of the Rhizobium Hfq gene was amplified with primers P1 (5'-GGTGGTTGCTCTT-CCAACGCGGAACGTTCTCAGAATC-3') containing a SapI site/P2 (5'-CGCGCTGCAGTCAGGACGCTGCTT-CTTCGCTCTCG-3') containing a PstI site from R. leguminosarum bv. viciae strain 8401/pRL1JI genomic DNA by PCR and inserted into the SapI /PstI sites of the expression vector pTYb11 to generate the expression plasmid pTYb11-RHfq. The expression plasmid was introduced into E. coli strain ER2566 and protein purification was carried out according to the manufacturer's manual.

Protein samples were analyzed by SDS–PAGE. Protein concentrations were determined by the Bradford method.

Rhizobium RNase E complex purification
RNase E complex was purified from R. leguminosarum bv. viciae 8401/pRLIJ1 wild type and Hfq mutant strain (M21) according to the method described previously [39,40]. Cells were cultured in TY medium. All purification steps were performed at 4°C. Buffers contained 2 µg/ml aprotinin, 0.8 µg/ml leupeptin, and 0.8 µg/ml pepstatin A. A suspension of 100 g Rhizobium cells in 100 ml of lysozyme-EDTA buffer containing 50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 5% glycerol, 3 mM EDTA, 1 mM dithiothreitol (DTT), 1.5 mg/ml lysozyme, and 1 mM phenylmethylsulfonyl fluoride (PMSF) at room temperature was prepared. After 40 min on ice, 50 ml of room temperature DNase-Triton buffer containing 50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 5% glycerol, 1 mM DTT, 3% Triton X-100, 30 mM magnesium acetate, 1 mM PMSF, and 20 µg/ml DNase I were added, followed by a 1-min low-speed blending. The lysate was kept on ice for 30 min and 37.5 ml of 5 M NH4Cl were slowly added. The lysate was stirred for an additional 30 min and clarified for 1 h at 12,000 rpm. Proteins of this supernatant were precipitated with 40% ammonium sulfate, dissolved in 112.5 ml of buffer A containing 10 mM Tris–HCl, pH 7.5, 5% glycerol, 0.5% genapol X-080, 1 mM EDTA, 0.1 mM DTT, 0.1 mM PMSF, and 50 mM NaCl and loaded on a sulphopropyl (SP)-Sepharose cation-exchanger column (Pharmacia), equilibrated with buffer containing 50 mM NaCl. After washing with buffer A containing first 50 and then 300 mM NaCl, RNase E was eluted with 1 M NaCl and 0.5% genapol X-080 in buffer A. Fractions were analyzed with SDS–PAGE. Peak fractions from the SP-column were diluted 2 folds with buffer A and layered on a 10–30% (w/v) glycerol gradient containing buffer A with 300 mM NaCl. Centrifugation was performed at 4°C for 15 h at 37,000 rpm. Fractions were collected from the top of the gradient tube. The endoribonuleolytic activity of each fraction was tested using the in vitro transcribed 82 nt RNA fragment of the NifA gene (see below) as substrates. The fractions of high activity were collected and analyzed with SDS–PAGE.

Protein identification by MS spectrometry
Protein identification by MS spectrometry was conducted by RCPA (Research Centre for Proteome Analysis, Key Lab of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China). Briefly, the procedure involved excision of the band from the gel, in-gel trypsin digestion, MALDI-TOF MS (4800 Plus MALDI TOF/TOF Analyzer) of the tryptic peptides, and database searching of the peptide mass fingerprinting data.

RNase E cleavage experiments
The 82 nt RNA fragment that was located in the (–47)–(+35) region of the NifA gene (NifA translational start was +1), was in vitro transcribed with T7 RNA polymerase (TaKaRa, Dalian, China) according to the manufacturer's manual. The template for in vitro transcription was constructed as follows. The PCR products of primers P11 (5'-CGCGGAATTCTTCGTGCAGCAGGG-TAAAAAGC-3') containing an EcoRI site/P12 (5'-CG-CGCTGCAGTAGAGTATATGGAGCCGCGCC-3') containing a PstI site were digested with EcoRI and PstI, then cloned into the corresponding sites of plasmid pGEM3zf(+). The recombinant plasmid was designated pGEM-82 and linearized by PstI to be used as a template. The in vitro transcribed RNA was labeled with 32P by incorporating 32P-CTP into it during the in vitro transcription reaction.

RNase E cleavage tests were carried as out described by Klein and Evguenieva-Hackenberg [40]. The reaction temperature was 28°C in this study.

Cleavage site determination
We determined the probable site of the 5'-untranslated region (UTR) of NifA mRNA cleavage by the RNase E complex. After the cleavage reaction, RNA fragments were size-fractionated, purified, and ligated to a 5'-DNA adapter (5'-TTTCTGCAGATGGCTAAGGGGCAATC-TTTACAAG-3') containing a PstI site and a 3'-adapter (5'-GCAGATCGTCAGAATTCCAG-3') containing an EcoRI site with T4 RNA ligase from NEB. The 3'-adapter was blocked by ddA with terminal transferase (NEB) at its 3'-terminus and phosphorylated by T4 polynucleotide kinase (NEB) at the 5'-terminus. The ligated RNA was reverse-transcribed into cDNA with the Access Quick reverse transcription-polymerase chain reaction (RT-PCR) system (Promega) with the 5'-adapter sequence and another primer complementary to the 3'-adapter. The RT-PCR product was amplified by PCR with the same primers and the DNA was cloned into the pGEM-T vector (Promega) for sequencing.

Total RNA was extracted from strain 8401/pRL1JI wild type cells carrying plasmid pRK415a-FN cultured in nitrogen-limited medium and the 3'-terminals of the predicted 4 kb mRNA were determined by 3'-RACE (rapid amplification of cDNA ends). RNA was polyadenylated using E. coli poly(A) polymerase, reverse transcribed using the primer 3'-RACE1: (5'-TCACGACTCACTATAGGA-TCCTTTTTTTTTTTTN-3'), amplified with the specific primer P36 (5'-TTTGCGCCGTCGATAACACC-3') and 3'-RACE2: (5'-TCACGACTCACTATAGGATCC-3'), and cloned into the TA cloning vector for sequencing.

β-Galactosidase activity assays
The translational fusion LacZ reporter plasmids were constructed as follows. A Tac promoter (Ptac) without its ribosome-binding site (RBS) was amplified with primers P17 (5'-GCGAAGCTTGGCTGTGCAGGTCGTAAATC-3') containing a HindIII site/P18 (5'-CGCGGGATCCGT-GTGAAATTGTTATCCG-3') containing a BamHI site from plasmid pKK223-3 by PCR and inserted into the HindIII and BamHI sites of the vector pMP220 to generate plasmid pMP220V. Fragment A was obtained by PCR amplification using primers P13 (5'-CGCGGGATCCG-CCTACAAACAGGGGAGG-3') containing a BamHI site/P14 (5'-CGCGGGATCCTTCGTGCAGCAGGGTA-AAAAGC-3') containing a BamHI site from pGEM-82 plasmid DNA. Fragment B was obtained by annealing two oligonucleotides P37 (5'-CGCGGGATCCAAAAAGCA-GTGCACCCTCCCCTGTTTGTAGGC-3') containing a BamHI site and P38 (5'-CGCGGGATCCGCCTACAAA-CAGGGGAGGGTGCACTGCTTTTT-3') containing a BamHI site. Then fragment A and B were inserted into the BamHI sites of plasmid pMP220V to generate translational fusion LacZ reporter plasmids pMP220A and pMP220B, respectively. Each reporter plasmid was transferred into R. leguminosarum bv. viciae strain 8401/pRL1JI wild type or Hfq mutant strain (M21) by biparental conjugation. β-Galactosidase activity assays were performed as described by Miller [41]. The locations of fragment A and B are indicated in Fig. 1.


Figure 1
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  		Figure 1 The effects of RNase E and Hfq on NifA translation  (A) Enlargement of the fixX-NifA intergenic region. The NifA translational start is numbered +1 and indicated by a line and horizontal arrow. (B) RNase E cleavage within the NifA 5'-UTR. 32P-labeled 82 nt in vitro transcribed NifA 5'-UTR RNA, which was located at (–47)–(+35), was incubated with RNase E complex purified from wild type cells (lane 2), or RNase E complex purified from Hfq mutant cells (lane 3), or RNase E complex purified from Hfq mutant cells plus Hfq (lane 4) at 28°C. After incubation for 1 h, samples were phenol extracted and analyzed on a 10% sequencing gel. (C) Schematic representation of the LacZ reporter translational fusions (C) and their activities (in Miller units) in wild type and Hfq mutant R. leguminosarum bv. viciae strain 8401/pRL1JI. In plasmid pMP220A, the 47 bp DNA corresponding to NifA 5'-UTR (designated fragment A) was inserted between the tac promoter (Ptac) and promoterless LacZ. In plasmid pMP220B, the 32 bp DNA corresponding to NifA 5'-UTR (designated fragment B) was inserted between the tac promoter (Ptac) and promoterless LacZ. Translation from pMP220A and pMP220B transcripts utilizes the NifA RBS. Plasmid pMP220V was a negative control with no RBS. The locations of fragment A and fragment B are indicated by lines and numbers. Cells were cultured in nitrogen-limited or nitrogen-rich medium to 0.3–0.4 (OD600). The numbers shown are averages obtained from at least three independent cultures. (D) Prediction of RNA secondary structure of the (–47)–(+3) region of NifA 5'-UTR by the MFOLD program (version 3.2) from Zuker [52]. The RNase E cleavage site, which is indicated by an arrow, is located between U(–33) and A(–32). The RBS is marked by a vertical line.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Funding
 References
 
Construction of R. leguminosarum bv. viciae Hfq mutant
The genetic organization of the sequenced 2.5 kb Hfq region from the genome of R. leguminosarum bv. viciae 8401/pRL1JI is shown in Fig. 2 upper panel. Upstream of the Hfq gene, there is a partial open reading frame (ORF) that was 971 bp long and encoded a D-alanine aminotransferase gene. Downstream of the Hfq gene there is also a partial ORF that is 1373 bp long and encoded a GTP-binding protein hflx.


Figure 2
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  		Figure 2 Physical map of the R. leguminosarum bv. viciae strain 8401/pRL1JI Hfq region  Upper panel: Hfq gene organization. In addition to the Hfq gene, two partial ORFs are located on the cloned 2.5 kb fragment of genomic DNA corresponding to the Hfq region. Hfq, host factor required for Qβ replication; hflx, putative GTP-binding protein of unknown function. Primers used in the construction of Hfq mutant are indicated by horizontal arrows. For the construction of the Hfq mutant, PCR products of primers P25/P26 were introduced into the NotI site of modified suicide plasmid pSUP202a (middle panel) and PCR products of primers P23/P24 were introduced into the BamHI and XbaI sites. The sacB encodes a levansucrase gene and levansucrase activity is lethal in the presence of sucrose. Lower panel: Hfq mutant strain M21 is shown. Two pairs of primers P27/P28 and P29/30 were used to test the double crossover of recombination.

 
The R. leguminosarum bv. viciae Hfq mutant was constructed by replacing the Hfq coding region with a kanamycin resistance cassette as described in "Materials and Methods". The colony morphology of the Hfq mutant strain M21 was normal.

Analysis of the NifA DNA region
In R. leguminosarum bv. viciae strain 8401/pRL1JI, the NifA gene locus is located in plasmid pRL1JI [42]. The upper panel of Fig. 3(A) shows the genetic organization of the sequenced 7.5 kb NifA region. Genes and ORFs in this cluster were designated on the basis of their similarity to genes and ORFs previously described in R. leguminosarum bv. viciae 3841 (AM236084.1). The whole sequence of this 7.5 kb DNA shares 90% identity with R. leguminosarum bv. viciae strain 3841 and strain UPM791. The fixA upstream region contains a potential {sigma}54-binding sequence preceded by consensus NifA-binding sequences, suggesting the existence of NifA-dependent promoters (Fig. 4).


Figure 3
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  		Figure 3 R. leguminosarum bv. viciae strain 8401/pRL1JI NifA mRNA expressed from broad-host-range plasmid pRK415a-FN  In this plasmid, fixABCXNifAB was under control of an upstream Lac promoter, which took the place of the potential endogenous NifA-dependent promoter upstream of fixA. (A) Structure of the NifA DNA region from R. leguminosarum bv. viciae strain 8401/pRL1JI. The grey arrows correspond to ORFs identified in a 7.5-kb DNA fragment containing the NifA gene from R. leguminosarum and adjacent DNA (EMBL accession No. FJ648550). (B) Schematic depiction of mRNAs expressed from plasmid pRK415a-FN. Upper panel: Lac promoter (Plac) controlled fixABCXNifAB expression in plasmid pRK415a-FN. The grey arrows correspond to ORFs; the Lac promoter (Plac) is indicated by a black square. Lower panel: predicted mRNAs expressed from plasmid pRK415a-FN. The 7 kb fixABCXNifAB mRNA was cut into a 4 kb fixABCX part and a 3 kb NifAB part by RNase E and Hfq. The existence of 7 kb and 3 kb mRNAs were proved by northern hybridization with a NifA probe. The 4 kb mRNA was not checked, so a question mark is placed on it. The position of the NifA probe used for northern hybridizations is indicated by a black square. The position of the specific primer used for 3'-RACE is indicated by a black square. (C) Northern blot probed with a probe for NifA (upper panel) or a control probe for 16S RNA (lower panel). Wild type (lanes 1 and 3) and Hfq mutant (lanes 4 and 5) R. leguminosarum bv. viciae strain 8401/pRL1JI harboring pRK415a-FN were grown in either nitrogen-limited medium (L) (lanes 1 and 4) or nitrogen-rich medium (R) (lanes 3 and 5). Wild type cells harboring pRK415a were grown in nitrogen-limited medium (L) (lane 2). Plasmid pRK415a was the control empty vector.

 


Figure 4
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  		Figure 4 The nucleotide sequences of the region upstream of fixA and the 5'-terminal of fixA  The numbers on the left indicate the positions of the first nucleotide according to the EMBL accession No. FJ648550 sequence. NifA- and {sigma}54-binding sites are enclosed in boxes.

 
Analysis of NifA mRNA expressed from plasmid pRK415a-FN
In R. leguminosarum bv. viciae, NifA is not expressed in free-living cells, even in a nitrogen-limited medium. We constructed plasmid pRK415a-FN as described in Materials and Methods. In this broad-host-range plasmid, fixABCXNifAB was expressed from the Lac promoter upstream of fixA [Fig. 3(B), upper panel]. This plasmid was transferred into R. leguminosarum bv. viciae strain 8401/pRL1JI wild type and Hfq mutant strain (M21) to investigate Hfq's effects. We detected NifA mRNA by northern hybridization using a NifA probe [Fig. 3(B), lower panel]. Two forms of NifA mRNAs were detected on northern blot: one transcript of approximately predicted size is 7 kb, the other is smaller (3 kb) [Fig. 3(C)]. The smaller form was only detectable in wild type cells carrying pRK415a-FN grown in nitrogen-limited medium. In wild type cells carrying control empty vector pRK415a, no NifA mRNA was detectable. In Hfq mutant cells, the smaller form of NifA mRNA was not detectable, suggesting that Hfq may play a role in NifA expression. According to its length, the 3 kb smaller transcript was likely to be NifAB. This transcript was possibly generated by post-transcriptional cleavage of the 7 kb larger mRNA since there was no other active promoter upstream of NifA in free living cells except the Lac promoter upstream of fixA. 3'-RACE was conducted to determine the conjectured cleavage site within the fixX-NifA intergenic region. The specific primer used in 3'-RACE was located 100 bp upstream of the NifA translational start. The obtained 3'-end was located 32 nt upstream of the NifA translational start. This supported the idea that a cleavage occurred within the 7 kb larger mRNA and this cleavage was dependent on Hfq and the nitrogen source.

Hfq can be associated with RNase E complex in R. leguminosarum bv. viciae
There were seven major bands on the SDS–PAGE analysis of the purified RNase E complex of strain 8401/pRL1JI wild type cells (Fig. 5). The protein bands of about 180, 65, and 15 kDa were proved to be RNase E, RNA helicase, and Hfq, respectively, by MALDI-TOF-MS analysis. The weak band of 15 kDa of the RNase E complex purified from Hfq mutant cells was obviously generated by bromophenol blue, which is commonly used as an indicator in electrophoresis.


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  		Figure 5 Hfq can be associated with RNase E complex measured by 10% Coomassie Brilliant Blue stained SDS–PAGE  Lanes 1 and 2: protein molecular weight standards; lane 3: RNase E complex purified from R. leguminosarum bv. viciae strain 8401/pRL1JI wild type cells; lane 4: RNase E complex purified from Hfq mutant cells. The 180 kDa RNase E, 65 kDa RNA helicase, and 15 kDa Hfq were identified by MS analysis.

 
RNase E complex can cut the in vitro transcribed NifA 5'-mRNA fragment and this cleavage is dependent on Hfq
We in vitro transcribed an 82 nt RNA fragment that was located in the (–47)–(+35) region of NifA mRNA [Fig. 1(B), right panel, NifA translational start is numbered +1] and conducted RNase E cleavage tests [Fig. 1(B), left panel]. RNase E complex purified from wild type cells cut the 82 nt substrate RNA into two smaller fragments. On the contrary, the RNase E complex lost this cleavage activity in the absence of Hfq. It seemed that Hfq was essential for the cleavage activity of the RNase E complex. To determine the cut site, after the cleavage reaction the two RNA product fragments were recovered from gel and cloned (see Materials and Methods) according to the method of Yao et al. [50]. The locations of the two fragments are indicated in Fig. 1(B). Thus we concluded that the cut site was located at 32 nt upstream of the NifA translational start. This was consistent with the 3'-RACE result. Thus we came to the conclusion that the 3 kb NifAB part was cleaved from the larger 7 kb fixABCXNifAB mRNA by the RNase E complex in the presence of Hfq.

Hfq-dependent RNase E cleavage is essential for NifA translation, probably by making RBSs accessible
The translational fusions of Ptac-A-lacZ and Ptac-B-lacZ were constructed and assayed for β-galactosidase activity in R. leguminosarum bv. viciae strains at the same cell density (see Materials and Methods).The reporter gene structures and the locations of fragments A and B are indicated in Fig. 1(C). β-Galactosidase activities were measured when cells were cultured in nitrogen-limited or nitrogen-rich medium. The translational activity of fragment A in wild type cells was 15 times higher than in Hfq mutant cells. However, the translational activity of fragment B did not depend on Hfq. The β-galactosidase activity of neither fragment A nor B was regulated by a nitrogen source. We predicted the secondary structure of NifA 5'-UTR [Fig. 1(D)] and found that the nucleotides of the RBS of the NifA gene were able to form a double-strand structure with adjacent nucleotides through base-pairing. This made the RBSs inaccessible to ribosomes, and so the translation of the NifA gene could not be initiated. The Hfq-dependent cleavage by RNase E at 32 nt upstream of the NifA translational start codon destroyed the double-strand structure and released the RBS; translation could then be initiated. This hypothesis could explain why fragment B had high translational activity in both wild type cells and Hfq mutant cells while fragment A only had high activity in wild type cells. Thus we came to a conclusion that Hfq-dependent RNase E cleavage was essential for NifA translation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Funding
 References
 
The NifA expression in symbiosis has not been studied in strain 8401/pRL1JI. In R. leguminosarum bv. viciae UPM791, NifA is expressed in symbiosis with peas from a {sigma}54-dependent NifA-autoregulated promoter located upstream of the orf71 orf79 fixW orf5 fixABCX NifA NifB operon, although basal levels of symbiotic NifA expression were obtained from a second promoter located upstream of the fixX-NifA intergenic region [51]. In strain 8401/pRL1JI, NifA- and {sigma}54-binding sites were identified upstream of fixA. It is reasonable to propose that 8401/pRL1JI NifA is expressed from this potential promoter upstream of fixA. We used the Lac promoter-controlled fixABCXNifAB expression system as the study target of NifA expression on the basis of this hypothesis.

The regulatory function of Hfq in gene expression is somehow mediated by RNase E activity, at least in some cases [27,43–46]. In general, RNase E forms a multi-protein complex with an exoribonuclease PNPase, an RNA helicase, enolase, and several other minor components [39,40,47–49]. In E. coli, the RNase E complex contains RNase E, PNPase, an RNA helicase, and enolase. Hfq has been reported to be co-purified with RNase E in E. coli [27]. However, enolase, Hfq, and PNPase were not found in the high molecular weight complex containing RNase E in R. capsulatus [39] and R. leguminosarum [40] but rather two RNA helicases (65 and 74 kDa) and a 50 kDa Rho factor. It is not clear whether Hfq is one of the components of RNase E complex in R. capsulatus since the proteins that migrated faster than 32.5 kDa on SDS–PAGE were not shown in [39]. We purified the RNase E complex of R. leguminosarum bv. viciae strain 8401/pRL1JI cells and analyzed the components of RNase E complex by SDS–PAGE followed by Coomassie Brilliant Blue staining. We observed seven major protein bands which migrated as 180, 110,100, 74, 65, 50, and 15 kDa on SDS–PAGE, respectively (Fig. 5). By MS analysis, the 180, 65, and 15 kDa bands were proved to be generated by RNase E, an RNA helicase, and Hfq, respectively. The 110 kDa band and the 100 kDa band might be generated by the degradation products of full length RNase E. The 74 kDa band might be generated by another RNA helicase. The 50 kDa band might be generated by Rho factor. Rhizobium leguminosarum Hfq is 80 amino acids long, but it migrated with a molecular mass of about 15 kDa, presumably following the behavior of the E. coli Hfq, which migrates as 14 kDa on SDS–PAGE in spite of its 11 kDa molecular weight.

Although the in vitro transcribed 82 nt RNA of NifA 5'-UTR could be cleaved by RNase E complex in the presence of Hfq in vitro, the in vivo cleavage was regulated by the nitrogen source [Fig. 3(C)]. Unfortunately, we cannot give a clear explanation for this difference now. In Azorhizobium caulinodans, Hfq gene expression is not regulated by nitrogen [11]. In strain 8401/pRL1JI, we purified an RNase E complex containing Hfq protein when cells were cultured in TY medium, which was nitrogen-rich. Consequently, Hfq might not be responsible for nitrogen regulation. Since RNase E cleavage is dependent on RNA structure, we guessed that other factor(s) and/or nucleotides upstream of fragment A might be responsible for nitrogen regulation. In E. coli, Hfq and RNase E often act together with sRNA to regulate gene expression. However, no sRNA has yet been reported in Rhizobia.

The mechanism revealed in this study is possibly common in R. leguminosarum because the NifA gene organization is the same and the sequence of the fix-NifA intergenic region shares 100% identity among strains UPM791, 8401/pRL1JI, and 3841. However, this mechanism is not common among other species of Rhizobia. Since the NifA gene organization of Sinorhizobium meliloti is the same as that of R. leguminosarum, it is most likely that this mechanism is suitable for Sinorhizobium meliloti. However, the RBS region of the NifA gene in Sinorhizobium meliloti is unable to form a double-strand structure like that in R. leguminosarum.

It has been reported that in E. coli, Hfq protects RNA against RNase E cleavage in vitro [20,55]. This is opposite to the conclusion of this paper. However, Brigid and Matthew have reported that the primary transcripts of MicX, a Vibrio cholerae sRNA, are processed in an RNase E- and Hfq-dependent fashion to a shorter, still active and much more stable form [53]. What cause the opposite role of Hfq in RNase E-dependent cleavage remains unclear. Given the role proposed above for RNase E in the processing of the 7 kb NifA transcripts, coupled with the absence of reports of Hfq-mediated RNA cleavage, it seems unlikely that Hfq is directly responsible for cleavage of this RNA. Instead, we hypothesize that Hfq, which has been shown to alter the structure of other RNA species, may promote folding of NifA 5'-UTR into a structure susceptible to RNase E cleavage. An alternative explanation for the absence of processed 3 kb NifA transcripts in an Hfq mutant is that processed transcripts may be extremely unstable in this genetic background. Analyses of sRNAs have revealed that Hfq can play a dramatic role in transcript stabilization [54] and it seems likely that Hfq stabilizes the 3 kb NifA transcripts as well. However, as we never observed the 3 kb NifA mRNA in the absence of Hfq and we could not observe the same 3' extremity by 3'-RACE experiment in an Hfq mutant, there is currently no evidence to suggest that it is formed. Consequently, if Hfq is not required for RNase E-mediated cleavage of the 7 kb NifA transcripts, as proposed above, then it is not clear why some low levels of the 3 kb NifA transcripts are not detected.


   Funding
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
References
 
This work was supported by Pan-Deng Project of China to GuoFan Hong.


   Acknowledgements
 
We sincerely appreciate the staff of RCPA (Research Centre for Proteome Analysis, Key Lab of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Science) for carrying out the protein mass spectrometry analysis.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 References
 

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