School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK1
John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK2
Author for correspondence: Andrew W. B. Johnston. Tel: +44 1603 592264. Fax: +44 1603 592250. e-mail: a.johnston{at}uea.ac.uk
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ABSTRACT |
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Keywords: ECF factor, fhu genes, iron-mediated regulation, pseudogene, rhizobia, siderophores
Abbreviations: CAS, chrome azurol sulphonate; ECF, extracytoplasmic factor; NTA, nitrilotriacetate; X-Gluc, 5-bromo-4-chloro-3-indolyl ß-D-glucuronide
The GenBank/EMBL/DDBJ accession number for the sequence determined in this work is AJ238208.
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INTRODUCTION |
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Rhizobium leguminosarum bv. viciae, the symbiont of peas, lentils, vetches and some beans, makes vicibactin, a cyclic trihydroxamate siderophore with three residues each of N2-acetyl-N5-hydroxy-D-ornithine and D-hydroxybutyrate (Dilworth et al., 1998 ). Different rhizobia make other hydroxamates (Persmark et al., 1993
), catechols (Roy et al., 1994
), citrate (Guerinot et al., 1990
) or anthranilate (Barsomonian et al., 1992
). In some cases, rhizobial mutants defective in siderophore synthesis fix N2 normally (Reigh & OConnell, 1993
; Fabiano et al., 1995
), but in others, Sid- mutants fail to fix N2 symbiotically (Barsomian et al., 1992
). Yeoman et al. (1997)
found that cyc (ccm) mutants of R. leguminosarum bv. viciae which are defective in cytochrome c maturation were, for reasons that are not clear, also compromised for vicibactin synthesis. Such mutants were also Fix- on peas, due to the defect in electron transport.
Stevens et al. (1999) identified some of the fhu genes of R. leguminosarum. These are homologues of the corresponding genes in (for example) Escherichia coli which are involved in the uptake of hydroxamate siderophores. In E. coli, FhuA is an outer-membrane receptor, FhuD a periplasmic transporter, FhuB an integral cytoplasmic membrane protein and FhuC an ATPase (Braun et al., 1998
). In R. leguminosarum, fhuCDB are in one operon whose expression is enhanced in cells grown in low concentrations of iron. Mutations in fhuCDB caused cells to make larger haloes on plates containing the universal siderophore indicator chrome azurol sulphonate (CAS) (Schwyn & Neilands, 1987
) and were defective for vicibactin and iron uptake (Stevens et al., 1999
). These mutants nodulated and fixed N2 normally on peas, indicating that vicibactin is not important in iron nutrition in bacteroids. It is not known if these bacteria make another, bacteroid-specific siderophore. It may also be the case that bacteroids acquire iron in the ferrous form; although bacteroids can take up both Fe2+ and Fe3+ iron, the efficiency is greater with the former (LeVier et al., 1996
; Moreau et al., 1998
).
In E. coli, fhuA is in the fhuACDB operon. In R. leguminosarum strain 8401pRL1JI, there is a different arrangement in which there is a version of fhuA oriented divergently from fhuCDB. However, this copy of fhuA appears to be a pseudogene; it has many stop codons and is not detectably expressed (Stevens et al., 1999 ). LeVier & Guerinot (1996)
identified a gene, fegA, in Bradyrhizobium japonicum which was a homologue of fhuA and which specified an outer-membrane protein, made in response to iron deprivation.
A functional fhuA gene of R. leguminosarum has now been discovered and is described here. The effects of iron availability and of the regulatory genes rpoI, fur and feuQ on its transcription are described as are its expression in pea root nodules. We also looked for the presence of fhuA and its pseudogene version, fhuA, in a number of field isolates of R. leguminosarum.
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METHODS |
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In vivo genetic manipulations.
Plasmids were transferred by conjugation into R. leguminosarum using the helper plasmid pRK2013 (Figurski & Helinski, 1979 ). Strain 8401 was mutagenized with Tngus by using it as a recipient in a conjugational cross with E. coli strain MM294, which contains a derivative of the plasmid pRK600 into which the transposon Tngus has been inserted (Sharma & Signer, 1990
). Since pRK600 is mobilizable into Rhizobium but fails to replicate in that host, it acts as a suicide plasmid. Thus, by selecting Kanr transconjugants (specified by Tngus), derivatives of strain 8401 with random insertions of the transposon into the genome were obtained, at frequencies of approximately 10-6. Kanr colonies were picked to minimal (Y) medium containing CAS. Transduction of Tngus from mutant A691 was done, using rhizobiophage RL38, as described by Buchanan-Wollaston (1979)
.
In vitro DNA manipulations.
Routine transformations, restriction digestions, ligations, Southern blotting and hybridization were done essentially as described by Downie et al. (1983) . R. leguminosarum genomic DNA was isolated using a Promega genomic preparation kit. Sequencing was done by the dideoxy chain-termination method, in some cases by MWG Ltd, Germany. Data were analysed with the DNA-Star package. Searches of databases used BLAST in the EGCG package. The primers used to amplify the two fhuA genes were: fhuA, 5'-TCCATAGGTTCCGCCCGCATCCGT-3' and 5'-TTTCGACGATGTGATAGGCGACCG-3';
fhuA, 5'-GGAGCAGATCGGCAAGGTCGGCGTG-3' and 5'-CGCCGATCGCCGTAATATTCTGTGC-3'. The primers used to amplify the fhuA promoter region were: 5'-CGCAGATCTTCGCAGCCATCGAGGGGGC-3' and 5'-CGCGCATGCCGTAATTGATATAGGGCTGGC-3'.
Iron uptake.
Uptake of iron from 55Fe-NTA (prepared with 55FeCl3 and sodium nitrilotriacetate) was measured as described by Yeoman et al. (1997) . Cells were grown in minimal (Y) medium with FeCl3 (20 µM) or in the absence of added iron but with 2,2'-dipyridyl (20 µM). Vicibactin was identified by electrospray mass spectroscopy as described by Yeoman et al., 1999
.
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RESULTS |
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It was shown that A775 was defective in iron uptake. Cells were grown in low-iron medium and exposed to 55Fe-NTA. No detectable uptake of this substrate was observed (<2% of the wild-type value), over a period of 15 min. It was also confirmed that the extra CAS-staining material was vicibactin, not some novel siderophore. Cells of A775 were grown and the cell-free growth medium assayed for hydroxamate. Strain A775 contained approximately four times more hydroxamate than did the wild-type and it was confirmed by electrospray mass spectroscopy that this hydroxamate corresponded to vicibactin.
Cloning and analysis of the fhuA gene
To locate the Tngus insertion precisely, DNA was isolated from strain A775 and digested with EcoRI. Fragments were ligated to pBluescript and used to transform E. coli, selecting Kanr transformants. By such means, part of Tngus, containing Kanr and gus, together with Rhizobium genomic DNA immediately upstream of the gus reporter, was cloned to form pIJ9116. The DNA adjacent to gus was sequenced; this indicated that the transposon was located in DNA whose deduced protein product had similarity to the C-terminus of FhuA of E. coli and other bacterial hydroxamate receptors (Table 2; and see below). Significantly, this 3' end of an fhuA homologue was similar in sequence to the corresponding region of the pseudogene version,
fhuA, identified by Stevens et al. (1999)
in the same strain of R. leguminosarum. However the sequences were not identical (see below). Sequencing also showed that in A775, the gus reporter Tngus was in the same orientation as fhuA.
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In the region 5' of fhuA there were no obvious regulatory sequences (e.g. a fur box) and the promoter of this gene has not yet been identified. A gap of 1·5 kb separates the 3' end of fhuA and the start of rpoI. Within this DNA there was no ORF larger than 300 bp and there was no homology of this DNA nor any potential peptide products to sequences in databases. However, this is a rather large intergenic space, and it may be that it does contain a short gene of unknown function. In the intergenic region, 57 bp 3' of fhuA is a perfect inverted repeat, (5'-CCGTCGCCCACCAGGCCCGTCGACCTCGACGGCCTGGTGGGCGACGG-3'), which might act as a -independent transcriptional terminator.
Expression of fhuA: effects of iron, rpoI, feuQ and fur
The fhuCDB operon of R. leguminosarum is expressed at higher levels in cells that are depleted for iron than in those that are replete for the metal (Stevens et al., 1999 ). A derivative of the fhuA::gus mutant strain A775 containing pBIO1097 (to correct the defect in iron uptake of strain A775 itself), was grown in high- and low-iron media (see Methods) and assayed for ß-glucuronidase. As with fhuCDB, expression was higher in the latter than the former medium, the activity being 54±12 and 858±67 Miller units, respectively.
We had previously identified two R. leguminosarum genes, feuQ and rpoI, which are believed to be regulatory and affect iron uptake. Yeoman et al. (1997) showed that a mutation in feuQ (which is likely to be a sensor in another representative of the two-component family of transcriptional regulators) severely affected iron uptake, although siderophore production appeared to be unaltered. Mutations in rpoI, which is downstream of fhuA, nearly abolish vicibactin production and the presence of cloned rpoI enhances siderophore production in R. leguminosarum.
To measure the effects of feuQ and rpoI on fhuA expression, an fhuA::lacZ transcriptional fusion plasmid was made as follows. A 1 kb PCR fragment, containing 470 bp of the N-terminal coding region of fhuA plus 530 bp upstream of fhuA was made, using the primers shown in Methods, and with pBIO1096 as template. This fragment was cloned first into pUC18 and thence into the EcoRISphI sites of the wide host-range promoter-probe plasmid pMP220 to form pBIO1111. This plasmid was then mobilized into wild-type strain 8401pRL1JI and the feuQ and rpoI mutant derivatives, J100 and J256, respectively. The transconjugants were grown in high- and low-iron media and were assayed for ß-galactosidase. As with the fhuA::gus fusion, it was found that addition of Fe3+ to the growth medium reduced expression of the fusion. This was true for the wild-type background (211 Miller units in high-iron and 1555 units in low-iron medium) and in the two mutants J100 (216 and 1455 units) and J256 (188 and 1318 units). These results showed that neither rpoI (strain J256) nor feuQ (strain J100) is required for transcription of fhuA, nor do they mediate the iron-dependent control of its expression. It had also been shown previously that neither rpoI nor feuQ had any detectable effect on expression of an fhuB::lacZ fusion in either high- or low-iron media (Stevens et al., 1999 ; Yeoman et al., 1999
).
deLuca et al. (1998) described a homologue of the global regulator fur in R. leguminosarum but could not obtain a knockout mutation in it, suggesting that this gene is essential. Nevertheless, since fur regulates expression of the fhu genes of other bacteria in an iron-dependent way (Crosa, 1997
), we examined the effects of fur on expression of the fhuA::gus fusion in strain A775 by conjugating into it plasmid pBIO929, which contains fur of R. leguminosarum. This derivative was grown in high- and low-iron media and assayed for ß-glucuronidase; it had no effect on the fhuA::gus expression in either medium. However, in the absence of a knockout mutant, we cannot be sure whether fur has a role in regulating fhuA transcription or not.
fhuA and symbiotic nitrogen fixation
The original fhuA mutant, A775, was derived from R. leguminosarum strain 8401, which lacks a symbiotic plasmid and so fails to nodulate. To examine the effects of the fhuA::gus mutation on nodulation, peas were inoculated with strain J253, a derivative of A775 into which plasmid pRL1JI had been introduced by conjugation. Judged by the numbers and sizes of the nodules, their time of appearance and the levels of C2H2 reduction, J253 appeared to be unaffected in symbiotic N2 fixation. Bacteria isolated from these nodules were found to retain the large-halo phenotype of the input fhuA inoculant and were all Kanr. This Nod+ Fix+ phenotype is similar to what is seen with fhuCDB mutants (Stevens et al., 1999 ) and points to the fact that the fhu system of Fe3+ uptake is unimportant in N2-fixing bacteroids. Consistent with this were the observations obtained with nodules stained with X-Gluc. The only part of the nodule with significant ß-glucuronidase activity was near the meristem, where non-differentiated bacteria, some still in infection threads, are located. In the zone containing mature, N2-fixing bacteroids, no staining was seen (Fig. 2
).
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Pseudogene fhuA occurs in different R. leguminosarum strains
Since a mutation in the version of fhuA described here causes a defect in iron uptake and a large-halo phenotype on CAS, it is clear that this gene is functional. In contrast, the fhuA pseudogene that is adjacent to fhuCDB, but which is unlinked to the functional fhuA gene identified here, is a non-functional pseudogene and is not expressed (Stevens et al., 1999
).
A comparison of parts of the C-terminal regions of FhuA and FhuA is shown in Fig. 3
. The similarity is no greater than that between R. leguminosarum and members of the FhuA family of proteins in other bacteria (Table 2
). We wished to see if genes corresponding to
fhuA were widespread in strains of R. leguminosarum. Probes corresponding to parts of fhuA and
fhuA were made by PCR using as templates pBIO1096 and pBIO400, which respectively contain the two versions of the gene. The locations of the primers used for
fhuA are shown in Fig. 3
; those used for fhuA are given in Methods and are located in the 5' half of that gene.
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DISCUSSION |
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Using transcriptional fusions to gus and to lacZ, we found that fhuA of R. leguminosarum was transcribed at elevated levels in cells starved of iron. Stevens et al. (1999) had similarly found that the unlinked fhuCDB operon of R. leguminosarum was Fe3+ regulated. We do not know, however, what regulatory gene mediates this iron-dependent expression. It is apparent from the results obtained here that two regulatory genes, rpoI and feuQ, which affect iron uptake in R. leguminosarum, are not involved. Given the adjacent locations of rpoI and fhuA, together with the facts that mutations in rpoI abolish siderophore synthesis and that overexpression of rpoI causes enhanced production of vicibactin (Yeoman et al., 1999
), we were surprised at the lack of interaction between fhuA and rpoI. At present, the target gene(s) that require the RpoI
factor for their transcription remains to be identified.
An fhuA::gus fusion was used to show that although fhuA is expressed in undifferentiated bacteria in the nodule, the mature N2-fixing bacteroids do not transcribe this gene at detectable levels. The basis of this switch-off regulation is not known. Several genes that are expressed in free-living rhizobia are quiescent in bacteroids; these include the pss (exo) genes for polysaccharide synthesis (Latchford et al., 1991 ), the amtB gene that is involved in ammonium transport (Tate et al., 1999
), and some nod genes required for the early steps in the infection process (Schlaman et al., 1991
; Marie et al., 1992
). The mechanisms involved in the down-regulation of genes in bacteroids have received little attention and it remains to be seen if there is some global control or if individual genes have specific shut-down systems. The finding that fhuA is not expressed in bacteroids is consistent with the lack of symbiotic defects found with various fhu mutants (this study; Stevens et al., 1999
). To date, the only R. leguminosarum mutants as yet identified that are defective both in siderophore synthesis and in N2 fixation are the cyc (ccm) mutants described by Yeoman et al. (1997)
. In these cases, it seems almost certain that it is the respiratory defect rather than that in siderophore synthesis that is responsible for the symbiotic phenotype (Delgado et al., 1995
). The negative results with defined fhu mutants strongly indicate that vicibactin is not used for iron uptake in R. leguminosarum bacteroids. It may be that there is another, unknown bacteroid-specific siderophore system. Alternatively, there is circumstantial evidence that bacteroids acquire iron in the reduced, ferrous form. Bacteroids of B. japonicum in soybean nodules can import both Fe3+ and Fe2+, but the uptake of the latter is more efficient (Moreau et al., 1998
). However, in the absence of (feo) mutants that are defective in Fe2+ uptake, it is impossible to know the relative importance of the two forms of iron in bacteroid nutrition.
Stevens et al. (1999) identified a pseudogene version of fhuA, next to the functional fhuCDB genes. In that study, no hybridization to any other DNA was observed when a probe spanning
fhuA was used. It is clear, though, that R. leguminosarum strain 8401pRL1JI does contain a functional gene that is unlinked to
fhuA; however, a comparison of the sequences of fhuA and
fhuA in this strain shows that there is DNA and protein homology in only limited areas. The similarity of the potential products of the fhuA and
fhuA sequences in R. leguminosarum strain 8401pRL1JI is no greater than that found between the FhuA of this strain and those of other bacteria. This suggests that these two genes did not arise via recent gene duplication followed by limited divergence. Rather, it points to fhuA and
fhuA having had quite different origins, one, perhaps, having been acquired by gene transfer.
It is apparent that other different field isolates of R. leguminosarum contain homologues of both the functional and the pseudogene versions of fhuA. However, the two strains that contained a close homologue of fhuA appeared to be closely related to each other and to strain 8401pRL1JI as judged from their RFLP patterns at other loci (Rigottier-Gois et al., 1998
; this study). In these two strains, the sequenced regions of the
fhuA homologues were identical to each other and differed in only one base pair from the allele in 8401pRL1JI. We were surprised that a pseudogene version of the gene differed so little in different strains since, by definition, such genes are not subject to the constraints that are required to maintain gene function. It will be of interest to know the precise sequences of the regions around the pseudogene versions of fhuA in different strains of R. leguminosarum. Are they in the same relative positions in the chromosomes? How large are the regions that distinguish these pseudogene regions from those in the strains that do not harbour them (or which, perhaps, contain different versions of fhuA pseudogenes)?
Pseudogenes are relatively rare in prokaryotes. It was noted by Stevens et al. (1999) that in bacteria, the deduced original products of several pseudogenes, including R. leguminosarum
fhuA, are located at the cell surface. FhuA of E. coli is a receptor for several coliphages, colicins and at least one antibiotic (Killman & Braun, 1992
; Killman et al., 1995
). It may be that there is particularly strong selection pressure to lose versions of such cell-surface proteins that can act as targets for such antimicrobial agents. The finding here that pseudogenes exist in only a minority of strains of R. leguminosarum suggests that such selection pressure may be intermittent and not uniform in different populations.
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ACKNOWLEDGEMENTS |
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Received 9 November 1999;
revised 16 December 1999;
accepted 10 January 2000.