1 School of Biological Science, University of East Anglia, Norwich NR4 7TJ, UK
2 Department of Molecular Microbiology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK
Correspondence
R. Gary Sawers
gary.sawers{at}bbsrc.ac.uk
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ABSTRACT |
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INTRODUCTION |
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However, a very different protein, called RirA, appears to be the major Fe-responsive transcriptional repressor in at least two rhizobial species, namely Rhizobium leguminosarum (Todd et al., 2002, 2005
) and Sinorhizobium meliloti (Viguier et al., 2005
). These two species both have a protein with amino acid sequence similarity to bona fide Fur, and in R. leguminosarum this protein can complement an E. coli fur mutant and it can bind to Fur boxes (Wexler et al., 2003
). Nonetheless, in Rhizobium and Sinorhizobium this homologue appears to be relegated to a minor role involved in the uptake of manganese, not iron (Chao et al., 2004
; Díaz-Mireles et al., 2004
; Platero et al., 2004
). We have renamed this Fur-like protein Mur, because it regulates the sitABCD operon, which encodes an ABC-type transporter for Mn2+, repressing its transcription under conditions of Mn2+ sufficiency. A similar role for Mur of S. meliloti in the regulation of its sitABCD operon was also demonstrated. Furthermore, as noted previously (Wexler et al., 2003
), Mesorhizobium loti has no Fur-like protein in its deduced proteome, nor, interestingly, does it have a sitABCD operon.
It had been shown by gel shifts (Díaz-Mireles et al., 2004) that R. leguminosarum Mur can bind to the cis-acting regulatory region of sitABCD, most probably to two versions of a conserved sequence, termed the MRS motif, which differs significantly in sequence from canonical Fur boxes (Díaz-Mireles et al., 2004
; Escolar et al., 1999
; Baichoo & Helmann, 2002
).
The Fur-like protein (FurBj) of Bradyrhizobium japonicum, which is more distantly related to Sinorhizobium and to Rhizobium, also has some unusual characteristics. Although FurBj regulates another regulatory gene, irr, in response to Fe availability (Hamza et al., 2000), the sequence to which it binds has little or no similarity to that of a conventional Fur box (Friedman & O'Brian, 2003
).
Here, we identify the R. leguminosarum Mur-binding site, confirming that Mur requires Mn2+ to bind in vitro and that its cognate DNA-binding sequences do not resemble those described for conventional Fur.
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METHODS |
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Plasmid construction.
The sitlacZ transcriptional fusion plasmid pBIO1552 was made by PCR amplification of the 504 bp DNA region that extended from the end of the putative deaminase gene adm directly 5' of the sitABCD operon to within the sitA gene using primers Sit-EcoRI (5'-CGCGAATTCGTGGAAAGCGCGGCTCCGACAGAAG-3') and Sit-SphI (5'-CGCGCATGCTTGGTGATCGATTCCACGATCGCGGC-3') and genomic DNA isolated from R. leguminosarum 3841 as template in the reactions (the relevant EcoRI and SphI sites are underlined). The amplified fragment was digested with EcoRI and SphI and cloned into the wide-host-range lacZ promoter-probe plasmid pMP220 (Spaink et al., 1987) to form pBIO1552. The DNA regions spanning either TS1 or TS2 were individually cloned into pMP220 as 304 bp and 222 bp DNA fragments, respectively, after PCR amplification using oligonucleotides Sit-EcoRI and the SphI-containing MRS2R (5'-ACATGCATGCTTGACGACCCTTGTTTCGACTATT-3') to make pBIO1480 (TS1) and Sit-SphI and the EcoRI-containing MRS1F (5'-CCGGAATTCGAAACAAGGGTCGTCAAGCATTTTTTGC-3') to make pBIO1479 (TS2). Starting with pBIO1552 as template DNA, the putative 10 RNA polymerase recognition sequence of transcript start 1 (TS1) was mutated from AATAGT to AAGCTT (generating a HindIII restriction site) using the Stratagene Ex-Site PCR-based site-directed mutagenesis kit and oligonucleotides TS1-HindF (5'-GCTTCGAAACAAGGGTCGTCAAAGCAT-3') and TS1-HindR (5'-TTGTGCAATTAAGAATGAATTGCAAC-3'). The nucleotide sequences that constitute the restriction site are underlined. The resulting plasmid was named pBIO1528. Similarly, the predicted 10 RNA polymerase recognition sequence of the putative TS2 promoter was mutated from TTCGACT to ATCGATT (generating a ClaI site) using oligonucleotides TS2-ClaF (5'-ATCGATTCTCCGCGGCTTTGGCAATTCGGTC-3') and TS2-ClaR (5'-CAAGTTGCGATTCAGAATGATTTGC-3'). The nucleotide sequences that constitute the restriction site are underlined. The resulting plasmid was named pBIO1526.
The inserts of all the plasmids formed by cloning PCR products were ratified by DNA sequencing.
In vivo and in vitro genetic manipulations.
Plasmid conjugation, isolation and transformation were as described by Wexler et al. (2001). Primer extension analyses of the mur and the sitABCD transcripts were done as described by Sawers & Böck (1989)
using 25 µg total RNA isolated from R. leguminosarum J251 or appropriate derivatives using a Qiagen RNeasy kit. In some cases the strain from which the RNA was isolated contained the corresponding cloned gene, in order to amplify the signal. The primers used were 0·2 pmol 32P-labelled oligonucleotide Mur-1 (5'-CATGCCGCGTTCGGTGCAAAGCTCC-3') or sit-PE (Díaz-Mireles et al., 2004
) (5'-GGTTGTCTGTTGGGCAGCGGCGGG-3') as appropriate. The 5' end of the mur transcript was precisely located by running in parallel a DNA sequence ladder generated using plasmid pBIO939 (de Luca et al., 1998
) as template and labelled Mur-1 oligonucleotide as the sequencing primer. The DNA sequence ladder used with the sit operon transcript was generated using plasmid pBIO1457 (Díaz-Mireles et al., 2004
). DNA sequencing employed a T7 DNA sequencing kit as described by the manufacturer (USB).
DNase I footprinting.
A 318 bp DNA fragment spanning the transcription start site and regulatory sequences of sitABCD was amplified using oligonucleotides sit-PE (see above) and sit-ds (5'-GGTGCGTTGTTCGATGAGGTTGACCG-3'), with pBIO1552 DNA as template. To demonstrate binding of Mur to the coding and anti-coding strands, 50 pmol of either sit-ds or sit-PE, respectively, was labelled with 50 µCi (1850 kBq) [-32P]ATP prior to inclusion in the PCR reaction. The reaction conditions were essentially as described by Tucker et al. (2004)
. A 0·5 µg aliquot of labelled DNA fragment was mixed with 2·5 µl of 10x binding buffer (100 mM Tris/HCl, pH 8·0, 300 mM KCl), 10 µg bovine serum albumin, 0·01 unit poly(dI-dC) (Roche), purified Mur protein freshly diluted in 1x binding buffer (Díaz-Mireles et al., 2004
) to a final concentration of between 0 and10 µM, 1 µl 2·5 mM MnCl2 (where specified), in a final volume of 25 µl. After incubating the mixture for 30 min at 25 °C, 1 µl (0·2 units freshly diluted in 1x binding buffer and 50 % w/v glycerol) DNase I (Amersham Biosciences) was added and after 15 s the reaction was stopped by adding 100 µl stop solution (50 µg proteinase K ml1, 20 mM EDTA, 0·5 %, w/v, SDS, 0·2 mg glycogen ml1) and 100 µl 2-propanol. The DNA was precipitated at 20 °C for 30 min and after centrifugation and washing with cold 80 % (v/v) ethanol, the air-dried pellet was resuspended in 8 µl formamide dye. Reaction products were separated on a denaturing sequencing gel of 6 % (w/v) acrylamide and 7 M urea. The locations of the Mur-binding sites were determined using radioactively labelled
X174 DNA digested with HinfI.
Other methods.
-Galactosidase enzyme assays on R. leguminosarum cells, grown in minimal Y medium, were done essentially as described by Rossen et al. (1985)
. Experiments were repeated three times using independent cultures.
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RESULTS AND DISCUSSION |
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To locate the mur transcriptional initiation site, total RNA was isolated from strain J251 containing pBIO939 after growth in TY medium. Primer extension analysis revealed one major 5' end at an adenosine, 74 bp 5' of the mur translation initiation codon (Fig. 1). The 5' end of a further, very weak transcript was observed at a cytosine 15 bp 5' of the major initiation site.
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When the region 10 bps 5' of the TS2 putative transcript start was mutated (in plasmid pBIO1526, see Table 1) expression of the sitlacZ fusion was reduced by little more than twofold, with both Mur- and Mn2+-dependent regulation being maintained. The overall twofold reduction in expression of the mutated plasmid relative to the wild-type construct pBIO1552 is possibly attributable to increased instability of the transcript due to the mutation. In contrast, when the 10 sequence of TS1 was mutated (pBIO1528), no expression was detected. Thus, expression of sitABCD depends upon the presence of a functional TS1 promoter. However, sequences downstream of the TS1 promoter, which were mutated in pBIO1526, may also augment sitABCD expression, perhaps by affecting mRNA stability.
Primer extensions verified that inactivation of the TS1 promoter in pBIO1528 abolished transcription in the fusion derivative, whereas mutating the 10 region of TS2 had only minor effects on the transcription patterns (Fig. 2b).
Taken together, these observations indicate that the smaller TS2 transcript results from processing of the longer TS1 transcript or from reverse transcriptase stalling at the GC-rich sequence in the neighbourhood of TS2 in the primer extension experiments.
The Mn2+-Mur complex interacts specifically with two DNA sites in the sitABCD regulatory sequence
In a previous study (Díaz-Mireles et al., 2004), Mur was shown to bind in gel-shift assays to the 1222 bp and to the 200504 bp DNA fragments depicted in Fig. 2(a)
. The DNA sequences around TS1 and TS2 have two putative heptameric inverted repeats, termed MRS1 and MRS2, which were proposed to be recognized by Mur (Díaz-Mireles et al., 2004
). We set out to locate the Mur-binding sites precisely by DNase I footprinting. The target was a 318 bp PCR-amplified DNA fragment that extended from position 108 relative to the TS1 transcription initiation and included the first 99 bp of the sitA structural gene (see Fig. 2
). Two distinct sites of interaction were seen both on the anti-coding (Fig. 3
a) and the coding strand (Fig. 3b
), and these sites overlap the previously identified MRS1 and MRS2 motifs. Binding was saturated with 5 µM Mur protein, since increasing Mur to 10 µM did not affect the protection pattern observed (data not shown). Both the MRS1 and MRS2 sites appeared to have similar affinity for Mur, suggesting that the binding to both sites is unlikely to be cooperative.
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The extent of DNA sequence protected from DNase I digestion by Mn2+-Mur was 34 bp for MRS1 and 31 bp for MRS2, suggesting that the 14 kDa Mur protein might bind as a tetramer, which is generally observed for other Fur family members (Pohl et al., 2003; Friedman & O'Brian, 2003
, 2004
). However, this must be verified biochemically. MRS1 and MRS2 are separated by 16 bp and the almost central location of the transcription initiation site means that when the sitABCD promoter is bound by Mn2+-Mur, binding of RNA polymerase would be prevented (Fig. 4
a).
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The Fe2+-dependent Fur protein from B. japonicum (FurBj) binds, in vitro, to a 19 bp recognition sequence in the promoter region of the irr gene of this species (Friedman & O'Brian, 2003). This sequence not only differs from the MRS motifs reported here but also is very different from typical Fur recognition sequences (Baichoo & Helmann, 2002
; Escolar et al., 1999
).
The DNA recognition sequence of conventional Fur, for example from E. coli, P. aeruginosa and Bacillus subtilis, is 19 bp, to which two Fur dimers are thought to bind (Baichoo & Helmann, 2002; Fuangthong & Helmann, 2003
). The two Fur dimers are proposed to recognize heptameric inverted repeats with a 7-1-7 organization, two of which, offset by 6 bp, occur in the conventional Fur box (see Fig. 4b
). The Zur and PerR regulators are also members of the Fur superfamily and are proposed to bind similar 7-1-7 sequences (Fuangthong & Helmann, 2003
). The DNA recognition sequences for Fur, PerR and Zur differ from each other in two key base pairs, allowing selective and exclusive binding by the cognate regulator, suggesting that the DNA-recognition helices of at least some members of the Fur superfamily must be highly discriminatory (Fuangthong & Helmann, 2003
).
The Mur-binding sequence has some similarity to the FurBj-binding site. This is confined to two direct repeat sequences of 6 bp TTGCAA/C for the R. leguminosarum Mur recognition sequence and TTGCA/GT/G for FurBj (Fig. 4b). FurBj and MurRl can both bind to canonical E. coli or P. aeruginosa Fur boxes (Friedman & O'Brian, 2003
; Wexler et al., 2003
) and the cloned Mur of R. leguminosarum can partially correct the regulatory defect of an E. coli Fur mutant in response to Fe2+ but not to Mn2+ availability (Wexler et al., 2003
; E. D.-M., unpublished observations). However, their normal functions in R. leguminosarum and B. japonicum are substantially different from that of the conventional Fur Fe-responsive transcriptional regulator. In R. leguminosarum, Mur responds to Mn2+ and not to Fe2+ in its ability to repress the sitABCD Mn2+-uptake operon, the role of the global Fe-responsive regulator having been taken over by the very different RirA protein (Todd et al., 2002
, 2005
). Furthermore, as shown here, the only known recognition sequence of Mur in R. leguminosarum itself has essentially no similarity to a canonical Fur box. The sequences of Mur in R. leguminosarum, S. meliloti and its homologues in other closely related genera (Agrobacterium, Bradyrhizobium, Brucella, Bartonella and Caulobacter) are closely related to each other and form a clade that is more closely related in sequence to bona fide Fur of E. coli than to the other members of the Fur superfamily, Irr, PerR and Zur. It appears that the progenitor of the fur genes of R. leguminosarum and S. meliloti evolved to respond to a different metal (manganese) and to recognize a different DNA sequence, and is no longer needed for iron homeostasis. In the case of B. japonicum, its Fur still responds to iron but its role may be more limited in terms of the range of genes that it regulates and, certainly, its recognition site, too, is very different from the conventional Fur box, although it has some similarity to the Mur-binding sites of R. leguminosarum. We cannot exclude the possibility that Mur regulates other promoters in R. leguminosarum, perhaps in response to other divalent cations. However, growth of R. leguminosarum in the presence of 10 µM Fe3+, Zn2+, Co2+, Cu2+ or Ni2+ did not affect the levels of expression of the sitlacZ reporter (E. D.-M., unpublished).
To date there have been few molecular genetic studies on the Fur-like proteins of other -proteobacteria (Park et al., 2001
). It will be of interest to see the extent to which they resemble those of the rhizobia or other lineages of bacteria in their functions. It will also be intriguing to determine, from a structural point of view (Pohl et al., 2003
), how these rhizobial regulatory proteins discriminate such different cis-acting sequences compared to those of the much-studied E. coli Fur and, at least in the case of Mur described here, how they recognize different DNA motifs in response to the availability of different transition metals.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 14 July 2005;
revised 31 August 2005;
accepted 9 September 2005.
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