1 School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK
2 John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK
Correspondence
A. W. B. Johnston
a.johnston{at}uea.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Since its discovery (Hantke, 1981), one Fe-dependent regulator has attracted most attention, at least in Gram-negative bacteria. This protein, Fur (ferric uptake regulator), is a transcriptional repressor of more than 90 different genes in for example Escherichia coli and Pseudomonas aeruginosa (Hantke, 2001a
). Many of these genes, such as those involved in synthesis or uptake of siderophores, are directly involved in Fe nutrition. Other genes, including those for production of exotoxin by pathogens, encode adaptive responses to Fe-deficient conditions. In its repressive mode, Fur protein attached to Fe2+ binds to motifs (fur boxes) that abut target promoters, preventing transcription. Fur also appears to positively regulate some genes (Hantke, 2001a
). This, too, may be an indirect repressive effect in which E. coli Fur represses transcription of ryhB, which encodes a small RNA molecule that in turn inhibits genes whose expression is enhanced in Fe-rich conditions (Masse & Gottesman, 2002
). A molecular structure for Fur of the N2-fixing bacterium Rhizobium leguminosarum biovar viciae, the microsymbiont of peas and vetches, and the subject of the present study, has recently been presented (Kolade et al., 2002
).
In some Gram-positive bacteria, Fe-responsive gene regulation is mediated by members of the DtxR family, identified in Corynebacterium diphtheriae as a regulator of Fe-dependent diphtheria toxin (Boyd et al., 1990). Although Fur and DtxR have no sequence homology, their structures may have some similarities (de Peredo et al., 2001
). In addition to Fur itself, there are at least three other subgroups in the Fur superfamily. PerR is an oxidative-stress-response regulator (Bsat et al., 1998
) and Zur regulates genes involved in Zn uptake (Hantke, 2001b
).
The fourth member of the Fur family is Irr, which occurs in bacteria known collectively as the rhizobia. These induce N2-fixing nodules on the roots of legumes and in this symbiotic state, have a very high demand for Fe, since several polypeptides made specifically in the nodule (e.g. nitrogenase and leghaemoglobin) are Fe-proteins (see Johnston et al., 2001). Irr was identified in Bradyrhizobium japonicum (which nodulates soybeans) as a transcriptional repressor of hemB, which specifies
-aminolaevulinic acid dehydratase, in the haem biosynthetic pathway (Hamza et al., 1998
). Irr is subject to complex post-translational modification such that it is degraded when bound to haem, which is delivered by the enzyme ferrochelatase (Qi et al., 1999
; Qi & O'Brian, 2002
). Irr is restricted to a few
-proteobacteria including rhizobia, Agrobacterium, the animal pathogen Brucella and Rhodopseudomonas palustris, a photosynthetic bacterium very closely related to B. japonicum. Transcription of irr is moderately repressed in Fe-replete conditions, this being dependent on a protein (FurBj) that is homologous to Fur (Hamza et al., 1999
), even though the irr promoter region has no sequence similarity to canonical fur boxes (Hamza et al., 2000
). Interestingly, FurBj also positively regulates hemA, which specifies
-aminolaevulinic acid synthase, the first step in haem biosynthesis (Hamza et al., 2000
).
These observations suggested that rhizobial Fur differs from that of other bacteria, including E. coli, in which Fur has been studied in detail. A further difference occurs in the regulation of the rhizobial hmu genes, which specify haem transporters in B. japonicum and R. leguminosarum. As in other bacteria that use haem as an Fe source, rhizobial hmu genes are repressed in Fe-replete conditions (Nienaber et al., 2001; Wexler et al., 2001
). In many other bacteria, this is mediated by Fur (e.g. Ochsner et al., 2000
), but fur mutants of B. japonicum and R. leguminosarum express their hmu genes normally. Also, transcription of R. leguminosarum tonB, which is required for haem utilization, was Fe-regulated but again this was unaffected by a fur mutation (Wexler et al., 2001
).
The ability to use haem is unusual, being normally a property of pathogens. Therefore, the failure of rhizobial hmu genes to be Fur-regulated may not be typical of Fe uptake systems. Most strains of R. leguminosarum make vicibactin (VB), a trihydroxamate siderophore (Dilworth et al., 1998). Genes for VB synthesis and uptake were identified, as follows: fhuAF and fhuCDB specify the receptor and ABC uptake transporter for VB; the vbsC, vbsGSO and vbsADL operons specify enzymes for VB synthesis; and rpoI encodes an RNA polymerase
factor that initiates transcription of vbsGSO and vbsADL. All these operons are expressed at high levels only in Fe-depleted conditions (Carter et al., 2002
; Stevens et al., 1999
; Yeoman et al., 1999
, 2000
).
Interestingly, each of the above operons is deregulated in R. leguminosarum with mutations in rirA, a newly found gene whose product has no sequence similarity to Fur but which has close homologues in other rhizobia, Agrobacterium and Brucella (Todd et al., 2002). This further suggests novelty in Fe-responsive regulation in rhizobia. Here, we establish if Fur of R. leguminosarum (FurRl) has any properties of classical Fur proteins and test if there is functional redundancy between Fur and Irr.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
In vivo and in vitro genetic manipulations.
Plasmids were conjugated from E. coli to R. leguminosarum by triparental mating. Tnlac insertions in irr were obtained and introduced into the genome of R. leguminosarum as in Wexler et al. (2001). Sequencing used the dideoxy chain-termination method. Primers to amplify the hemA promoter region from R. leguminosarum genomic DNA and to determine the irr mRNA starts by primer extensions (Sawers & Böck, 1989
) are listed in Table 2
.
|
Mobility shift assays.
DNA mobility shift assays, using three different DNA fragments, were performed as described by Ochsner et al. (1995), with minor modifications. One of these fragments, synthesized by MWG Biotech, was a 25 bp synthetic DNA fragment containing an E. coli fur box, labelled with fluorescein at the 5' terminus of each single-stranded oligonucleotide. The oligonucleotides were based on a 19 bp fur box sequence (Escolar et al., 1999
) extended by three randomly chosen nucleotides at the 5' and 3' termini (5'-att gat aat gat aat cat tat cga c-3' and 5'-GTC GAT AAT GAT TAT CAT TAT CAA T-3'). Double-stranded DNA was prepared by mixing equal concentrations of the single-stranded oligonucleotides and heating at 90 °C for 10 min followed by cooling to room temperature over 1 h. The other two DNA fragments were (i) a 352 bp EcoRIHindIII fragment from pVDS, which contains the Fur-regulated pvdS promoter of P. aeruginosa (Ochsner et al., 1995
) and (ii) a 344 bp EcoRIHindIII fragment containing the R. leguminosarum irr promoter, end-labelled with [35S]dATP
S (Table 2
). Non-denaturing gels contained 0·2 mM Mn2+. Various concentrations (050 µM) of purified Fur protein were added to the fluorescein-labelled DNA (2 µM), in the presence of unlabelled, non-competitor DNA [200 µM poly(deoxyinosinic-deoxycytidylic) acid, sodium salt]. For radiolabelled fragments purified Fur (0200 nM) was added to approximately 100 ng DNA. Following PAGE, dried gels were exposed to a Molecular Dynamics phosphor-imager screen, digitally quantified with a Storm 840 laser scanner (Molecular Dynamics) and visualized with ImageQuant software.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
We had also identified an operon, sfuABC (Todd et al., 2002), which is also likely involved in Fe uptake. Its products closely resemble SfuA, SfuB and SfuC of Serratia and YfuABC of Yersinia, two similar ABC iron importers of a type normally found only in pathogens and whose expression is Fur-regulated (Angerer et al., 1992
). The promoter of R. leguminosarum sfuABC was cloned in the gfp reporter plasmid pOT2, forming pBIO1378. When transferred to wild-type J251 and to J325, pBIO1378 conferred high-level expression (green UV fluorescence) in both backgrounds on -Fe but not +Fe agar (Fig. 1
). Thus, sfuABC is Fe-regulated, but not via Fur.
|
Effects of R. leguminosarum Fur on hemA expression
In B. japonicum, transcription of the hemA gene is higher in Fe-replete than in Fe-depleted media, this regulation depending on Fur of this species (Hamza et al., 2000). We investigated the effects of FurRl on the expression of R. leguminosarum hemA, which we identified from its genome sequence (http://www.sanger.ac.uk/Projects/R_leguminosarum/). A 405 bp fragment 5' of hemA, which spanned its promoter(s), was amplified from this sequenced strain's genomic DNA using hemRlS and hemRlE primers (Table 2
). The PCR product was cloned into the reporter plasmid pMP220 to form pBIO1437, which was mobilized into J251 and into J325. For both of the resultant transconjugants, hemAlacZ expression was enhanced, about threefold, in high-Fe conditions (Table 3
). Thus, expression of hemA responds to Fe, but this does not involve Fur.
R. leguminosarum Fur functions heterologously in E. coli as a ferric uptake regulator
Given its lack of effect on Fe-responsive genes, we tested if FurRl has at least some properties of Fur from -proteobacteria. This was done both genetically and biochemically. First, its ability to correct a fur mutant of E. coli was examined. R. leguminosarum fur was cloned in pUC18, oriented to be under the control of the lac promoter (pBIO1153) or lacking a functional promoter (pBIO1154). Both plasmids were transferred into two E. coli strains containing a bfdlacZ fusion (bfd specifies bacterioferritin-associated ferredoxin and is Fur-regulated). One strain (JRG2652) is Fur+; the other (JRG2653) has a fur deletion.
-Galactosidase was assayed for each strain, grown in high- and low-Fe media with IPTG. As expected,
-galactosidase expression was Fe-regulated in JRG2652, but in the fur mutant, JRG2653, Fe regulation of bfdlacZ was absent (Table 4
). However, when pBIO1153 (but not pBIO1154) was present in JRG2653, Fe-dependent regulation was partially restored. The expressed FurRl antagonized E. coli Fur in wild-type JGR2652, perhaps by forming a heterodimer with E. coli Fur, the hybrid being less effective than native E. coli Fur.
|
|
Analysis of R. leguminosarum irr and of irr fur mutants
The lack of effect of FurRl on the expression of Fe-responsive genes in R. leguminosarum, coupled to the fact that Irr is confined to the rhizobia, raised the possibility that there may be functional redundancy between Irr and Fur, even though these proteins have non-identical functions in B. japonicum. We therefore identified irr of R. leguminosarum, with a view to obtaining a double mutant strain, defective in both Irr and Fur.
The R. leguminosarum genome sequence has one gene whose product is very similar (73 % identical) to B. japonicum Irr. A probe internal to R. leguminosarum irr was used to probe E. coli colonies harbouring a pLAFR1-based cosmid gene bank of R. leguminosarum genomic DNA (de Luca et al., 1998). A 4·2 kb fragment that included irr was subcloned from one hybridizing cosmid into pUC18 to form pBIO1340. The insert was sequenced to confirm the presence of intact irr. Interestingly, R. leguminosarum Irr has no cysteines and lacks the haem-binding pocket that is needed for degradation of Irr of B. japonicum (Qi et al., 1999
). We noted that Irr of Agrobacterium, Brucella, Sinorhizobium, Mesorhizobium and one of the two Irr proteins of Rps. palustris also lacked this motif, suggesting that B. japonicum Irr may be unusual in its haem-dependent instability.
To mutate R. leguminosarum irr, the 4·2 kb EcoRI fragment from pBIO1340 was cloned into pLAFR1, to form pBIO1374, which was mutagenized with Tnlac. A mutant plasmid (pBIO1375) with an insertion 36 bp 3' of the irr translational start was isolated, with irr and lacZ of Tnlac in the same orientation. This mutation was marker-exchanged into the genome of wild-type J251 and into the fur mutant J325 to form strains J386 and J387, respectively. Having ratified (by Southern blots) the two homogenotes, we examined several of their phenotypes.
As in B. japonicum (Hamza et al., 2000), the R. leguminosarum irr mutants, J386 and J387, showed strong pink UV fluorescence (Fig. 1
), consistent with accumulation of PPIX and the loss of repression of hemB. Fluorescence spectra (excited at 405 nm and emission measured at 633 nm) of J386 extracts identified the pink fluorescent compound as PPIX (not shown). Although growth of these mutants in liquid minimal media with ferric citrate, haemin or low Fe as sole Fe sources was slightly reduced compared to those of J251, on CAS agar the haloes of both mutants were indistinguishable from that of the wild-type. Strains J386 and J387 were also inoculated onto peas; both mutants nodulated and fixed N2 in a similar manner to wild-type strain J251.
Since J386 and J387 both contain an irrlacZ fusion, to assay the effects of Irr on expression of Fe-responsive genes, we used a reporter gene other than lacZ for these fusions. Accordingly, several plasmids were made, based on the wide-host-range vector pOT2, in which different Fe-responsive promoters control the gfp reporter (Table 1). These were pBIO1378 (sfugfp), pBIO1364 (vbsAgfp), pBIO1369 (rpoIgfp), pBIO1371 (tonBgfp), pBIO1370 (hmugfp), pBIO1363 (vbsSgfp) and pBIO1365 (fhuAgfp). They were transferred into J251 (wild-type), J325 (fur) and into derivatives of strains J386 (irr) and J387 (fur irr), cured of pPH1JI (used for the marker-exchange and which confers resistance to gentamicin, as does pOT2). Transconjugants were plated on high- and low-Fe agar medium and examined under UV light. The results are illustrated in Fig. 1
with the sfugfp fusion plasmid pBIO1378 (note the yellow colour, due to combined fluorescence from GFP and PPIX). Fluorescence was seen only in Fe-deficient agar, confirming the Fe-mediated repression of sfu. The differential expression in high and low Fe was unaffected by mutations in fur (J325) or in irr (J386 and J387). Similar results occurred with all the other gfp fusion plasmids (not shown).
Transcription of R. leguminosarum irr
Expression of a B. japonicum irrlacZ transcriptional fusion is less (by about 3050 %) in cells grown in high-Fe medium (Hamza et al., 1998, 2000
). To examine R. leguminosarum irr expression, we transferred pBIO1373, which contains irrlacZ, into wild-type J251 and into J325 and assayed
-galactosidase activity after growth in different Fe conditions (Table 3
). In both recipients, expression of irrlacZ was enhanced, by about 20 %, under low-Fe conditions. Essentially the same results were obtained when the irrlacZ fusion was present in the fur mutant, J325. Thus, in R. leguminosarum, unlike B. japonicum, the modest, Fe-dependent effect on irr expression is not mediated via Fur. Consistent with this, purified FurRl did not bind in vitro to a 344 bp PCR fragment spanning the irr promoter region, which had been mapped by primer extension (not shown). Also, there is no sequence near the R. leguminosarum irr promoter region that resembles the B. japonicum Fur-binding site (Hamza et al., 2000
).
Genomic analyses of the Fur superfamily in relation to rhizobial homologues
Given the unusual nature of the rhizobial Fur regulator, we determined the sequence relatedness (using CLUSTAL W) among members of Fur superfamily (Fur, Irr, Zur and PerR) in the rhizobia and their very close relatives Brucella and Agrobacterium. These sequences were compared with a selection of members of the Fur superfamily in other bacterial taxa. The deduced proteomes of Agrobacterium, Bradyrhizobium, Brucella, Rhizobium and Sinorhizobium all contained one protein whose sequence clearly placed them in the Fur (in sensu stricto) clade of the Fur superfamily. The Fur-like proteins of these genera were closely related to each other and were all within the branch that included the ratified Fur proteins of (e.g.) E. coli and Pseudomonas. However, the deduced proteome of Mesorhizobium loti has no protein whose sequence places it within this Fur clade, indicating that it can survive with no Fur-like protein whatsoever.
The analysis confirmed that proteins most closely related to the canonical Irr of B. japonicum were confined to the rhizobia and their very close relatives, though it was noted that both Brucella and Rps. palustris (a very near neighbour of Bradyrhizobium) both have two Irr-like proteins in their proteome. All the rhizobia also contain a single protein that is closely related to Zur, the Zn-responsive regulator of E. coli, but none has a PerR-like protein.
Conclusions
The results presented here and in studies on Fur of B. japonicum (Nienaber et al., 2001) show that the regulation of many Fe-responsive genes in the rhizobia is not mediated by Fur. Further, none of the transcripts examined here contained sequences that resembled fur boxes, nor did we find such cis-acting sequences in the expected regions (i.e. 5' of putative operons) anywhere in the genome of R. leguminosarum. It may be that FurRl recognizes different regulatory DNA motifs, as appears to be the case in B. japonicum (Hamza et al., 1999
).
Fur proteins of rhizobia have some properties attributed to classical Fur proteins. FurBj (Hamza et al., 1999) and FurRl (this work) both bind to canonical Fur boxes in a metal-dependent way and, as shown here, a fur mutant of E. coli was partially corrected by the cloned fur of R. leguminosarum. The lack of an expected phenotype in fur mutants is not due to functional redundancy between Fur and Irr, judged from the phenotype of irr fur double mutants.
It seems that, instead of Fur, Rhizobium uses RirA as a regulator of Fe-responsive operons. Excepting hemA, all the genes described here are deregulated in rirA strains of R. leguminosarum but the mechanisms involved are unknown (Todd et al., 2002). RirA has not been studied in bacteria other than R. leguminosarum, but close homologues occur in the proteomes only of its very close relatives (Sinorhizobium, Mesorhizobium, Agrobacterium, Brucella), suggesting that its role as a regulator may be confined to a very small group of bacteria.
Future studies, perhaps using transcriptomics or proteomics, may be needed to determine if R. leguminosarum Fur regulates any genes in this species and if so, whether iron, or some other molecule, is the co-repressor molecule.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Angerer, A., Klupp, B. & Braun, V. (1992). Iron transport systems of Serratia marcescens. J Bacteriol 174, 13781387.[Abstract]
Beringer, J. E. (1974). R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84, 188198.[Medline]
Beynon, J. L., Beringer, J. E. & Johnston, A. W. B. (1980). Plasmids and host-range in Rhizobium leguminosarum and Rhizobium phaseoli. J Gen Microbiol 120, 421429.
Boyd, J., Oza, M. N. & Murphy, J. R. (1990). Molecular cloning and DNA-sequence analysis of a diphtheria tox iron-dependent regulatory element (dtxR) from Corynebacterium diphtheriae. Proc Natl Acad Sci U S A 87, 59685972.[Abstract]
Bsat, N., Herbig, A., Casillas-Martinez, L., Setlow, P. & Helmann, J. D. (1998). Bacillus subtilis contains multiple Fur homologues: identification of the iron uptake (Fur) and peroxide regulon (PerR) repressors. Mol Microbiol 29, 189198.[CrossRef][Medline]
Carter, R. A., Worsley, P. S., Sawers, G. & 7 other authors (2002). The vbs genes that direct synthesis of the siderophore vicibactin in Rhizobium leguminosarum: their expression in other genera requires ECF factor RpoI. Mol Microbiol 44, 11531166.[CrossRef][Medline]
Crosa, J. H. (1997). Signal transduction and transcriptional and post-transcriptional control of iron-regulated genes in bacteria. Microbiol Mol Biol Rev 61, 319336.[Abstract]
de Luca, N. G., Wexler, M., Pereira, M. J., Yeoman, K. H. & Johnston, A. W. B. (1998). Is the fur gene of Rhizobium leguminosarum essential? FEMS Microbiol Lett 168, 289295.[CrossRef][Medline]
de Peredo, A. G., Saint-Pierre, C., Latour, J. M., Michaud-Soret, I. & Forest, E. (2001). Conformational changes of the ferric uptake regulation protein upon metal activation and DNA binding; first evidence of structural homologies with the diphtheria toxin repressor. J Mol Biol 310, 8391.[CrossRef][Medline]
Dilworth, M. J., Carson, K. C., Giles, R. G. F., Byrne, L. T. & Glenn, A. R. (1998). Rhizobium leguminosarum bv. viciae produces a novel cyclic trihydroxamate siderophore, vicibactin. Microbiology 144, 781791.
Escolar, L., Perez-Martin, J. & de Lorenzo, V. (1999). Opening the iron box: transcriptional metalloregulation by the Fur protein. J Bacteriol 181, 62236229.
Friedman, A. M., Long, S. R., Brown, S. E., Buikema, W. J. & Ausubel, F. M. (1982). Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene 18, 289296.[CrossRef][Medline]
Hamza, I., Chauhan, S., Hassett, R. & O'Brian, M. R. (1998). The bacterial Irr protein is required for coordination of heme biosynthesis with iron availability. J Biol Chem 273, 2166921674.
Hamza, I., Hassett, R. & O'Brian, M. R. (1999). Identification of a functional fur gene in Bradyrhizobium japonicum. J Bacteriol 181, 58435846.
Hamza, I., Qi, Z., King, N. D. & O'Brian, M. R. (2000). Fur-independent regulation of iron metabolism by Irr in Bradyrhizobium japonicum. Microbiology 146, 669676.
Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557580.[Medline]
Hantke, K. (1981). Regulation of ferric iron transport in Escherichia coli K12: isolation of a constitutive mutant. Mol Gen Genet 182, 288292.[Medline]
Hantke, K. (1987). Selection procedure for deregulated iron transport mutants (fur) in Escherichia coli K12: fur not only affects iron metabolism. Mol Gen Genet 210, 135139.[Medline]
Hantke, K. (2001a). Iron and metal regulation in bacteria. Curr Opin Microbiol 4, 172177.[CrossRef][Medline]
Hantke, K. (2001b). Bacterial zinc transporters and regulators. Biometals 14, 239249.[CrossRef][Medline]
Hirsch, P. R. & Beringer, J. E. (1984). A physical map of pPH1JI and pJB4JI. Plasmid 12, 139141.[Medline]
Johnston, A. W. B., Yeoman, K. H. & Wexler, M. (2001). Metals and the rhizobial-legume symbiosis uptake, utilization and signalling. Adv Microb Physiol 45, 113156.[Medline]
Kolade, O. O., Bellini, P., Wexler, M., Johnston, A. W. B., Grossmann, J. G. & Hemmings, A. M. (2002). Structural studies of the Fur protein from Rhizobium leguminosarum. Biochem Soc Trans 30, 771774.[Medline]
Masse, E. & Gottesman, S. (2002). A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli. Proc Natl Acad Sci U S A 99, 45204625.
Messing, J., Crea, R. & Seeburg, P. H. (1983). A system for shotgun DNA sequencing. Nucleic Acids Res 9, 309314.
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Nienaber, A., Hennecke, H. & Fischer, H.-M. (2001). Discovery of a haem uptake system in the soil bacterium Bradyrhizobium japonicum. Mol Microbiol 41, 787800.[CrossRef][Medline]
Ochsner, U. A., Vasil, A. I. & Vasil, M. L. (1995). Role of the ferric uptake regulator of Pseudomonas aeruginosa in the regulation of siderophores and exotoxin A expression: purification and activity on iron-regulated promoters. J Bacteriol 177, 71947201.[Abstract]
Ochsner, U. A., Johnson, Z. & Vasil, M. L. (2000). Genetics and regulation of two distinct haem-uptake systems, phu and has, in Pseudomonas aeruginosa. Microbiology 146, 185198.
Qi, Z. & O'Brian, M. R. (2002). Interaction between the bacterial iron response regulator and ferrochelatase mediates genetic control of heme biosynthesis. Mol Cell 9, 155162.[Medline]
Qi, Z., Hamza, I. & O'Brian, M. R. (1999). Heme is an effector molecule for iron-dependent degradation of the bacterial iron response regulator (Irr) protein. Proc Natl Acad Sci U S A 96, 1305613061.
Sawers, G. & Böck, A. (1989). Novel transcriptional control of the pyruvate formate-lyase gene: upstream regulatory sequences and multiple promoters regulate anaerobic expression. J Bacteriol 171, 24852498.[Medline]
Spaink, H. P., Okker, R. J. H., Wijffelman, C. A., Pees, E. & Lugtenberg, B. J. J. (1987). Promoters in the nodulation region of the Rhizobium leguminosarum symbiotic plasmid pRL1JI. Plant Mol Biol 9, 2739.
Stevens, J. B., Carter, R. A., Hussain, H., Carson, K. C., Dilworth, M. J. & Johnston, A. W. B. (1999). The fhu genes of Rhizobium leguminosarum, specifying siderophore uptake proteins: fhuDCB are adjacent to a pseudogene version of fhuA. Microbiology 145, 593601.[Abstract]
Todd, J. D., Wexler, M., Sawers, G., Yeoman, K. H., Poole, P. S. & Johnston, A. W. B. (2002). RirA, an iron-responsive regulator in the symbiotic bacterium Rhizobium leguminosarum. Microbiology 148, 40594071.
Wexler, M., Yeoman, K. H., Stevens, J. B., de Luca, N. G., Sawers, G. & Johnston, A. W. B. (2001). The Rhizobium leguminosarum tonB gene is required for the uptake of siderophore and haem as sources of iron. Mol Microbiol 41, 801816.[CrossRef][Medline]
Wood, W. B. (1966). Host specificity of DNA produced by Escherichia coli; bacterial mutations affecting the restriction and modification of DNA. Mol Biol 16, 118133.
Yeoman, K. H., Delgado, M.-J., Wexler, M., Downie, J. A. & Johnston, A. W. B. (1997). High affinity iron acquisition in Rhizobium leguminosarum requires the cycHJKL operon and the feuPQ gene products, which belong to the family of two-component transcriptional regulators. Microbiology 143, 127134.[Abstract]
Yeoman, K. H., May, A. G., de Luca, N. G., Stuckey, D. B. & Johnston, A. W. B. (1999). A putative ECF sigma factor gene, rpoI, regulates siderophore production in Rhizobium leguminosarum. Mol PlantMicrobe Interact 12, 994999.[Medline]
Yeoman, K. H., Wisniewski-Dye, F., Timony, C., Stevens, J. B., de Luca, N. G., Downie, J. A. & Johnston, A. W. B. (2000). Analysis of the Rhizobium leguminosarum siderophore-uptake gene fhuA: differential expression in free-living bacteria and nitrogen-fixing bacteroids and distribution of an fhuA pseudogene in different strains. Microbiology 146, 829837.
Received 18 November 2002;
revised 21 January 2003;
accepted 28 January 2003.