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Journal of Bacteriology, September 1999, p. 5843-5846, Vol. 181, No. 18
Department of Biochemistry, School of
Medicine and Biomedical Sciences, State University of New York at
Buffalo, Buffalo, New York 14214
Received 4 May 1999/Accepted 7 July 1999
The recent identification of the iron response regulator (Irr) in
Bradyrhizobium japonicum raised the question of whether the
global regulator Fur is present in that organism. A fur
gene homolog was isolated by the functional complementation of an
Escherichia coli fur mutant. The B. japonicum
Fur bound to a Fur box DNA element in vitro, and a fur
mutant grown in iron-replete medium was derepressed for iron uptake
activity. Thus, B. japonicum expresses at least two
regulators of iron metabolism.
Studies on the regulation of iron
homeostasis in bacteria have focused on Fur, a global transcriptional
regulator that represses target genes when bound to iron. The Irr (iron
response regulator) protein from the bacterium Bradyrhizobium
japonicum mediates iron control of heme biosynthesis and affects
iron transport (9). Irr has low homology to Fur, and
although it is not a functional homolog (9), its
identification led us to ask whether B. japonicum also
expresses a functional fur gene.
A B. japonicum genomic expression library was screened for
clones that repressed the fiu::lacZ
reporter gene fusion in Escherichia coli fur strain H1780 as
discerned by the formation of white or light blue colonies in the
presence of 40 µg of
5-bromo-4-chloro-3-indolyl-
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Copyright © 1999, American Society for Microbiology. All rights reserved.
Identification of a Functional fur Gene
in Bradyrhizobium japonicum
ABSTRACT
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-D-galactopyranoside (X-Gal) per ml and 100 µM FeCl3. Three overlapping
library clones, pSKBJF1, pSKBJF9, and pSKBJF22, complemented the
fur strain both on plates and in liquid cultures (Fig.
1). pSKBJF22 repressed less well, perhaps
because the complementing gene (see below) was more distal from the
plasmid-borne lacZ promoter. A 830-bp ApaI-Sau3AI subclone on pSKBJF800 including the
overlapping region was also sufficient to complement the fur
strain (Fig. 1). This fragment contained an open reading frame encoding
a protein with homology to characterized Fur proteins from numerous
organisms (Fig. 2). Among the proteins
shown experimentally to be Fur, the B. japonicum protein was
most homologous to that of E. coli, showing 39% identity
and 49% similarity to E. coli Fur. Southern blot analysis
indicated a single fur gene in the B. japonicum
genome (data not shown). The complementation and homology indicate that the cloned B. japonicum DNA encodes a structural and
functional homolog of Fur. A fur-like gene of
Rhizobium leguminosarum was isolated (6) encoding
a product with 77% similarity to B. japonicum Fur,
indicating that the R. leguminosarum gene is a functional fur gene as well. Previous work indicates that the B. japonicum Irr is functionally distinct from bacterial Fur despite
their modest homology to each other (9). Consistent with
this, the plasmid-borne irr gene did not functionally
complement the E. coli fur strain as did the B. japonicum fur gene (Fig. 1B).
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FIG. 1.
Complementation of E. coli fur strain H1780
for in vivo repression of fiu::lacZ
gene fusion by B. japonicum library clones. (A) B. japonicum genomic region represented by library plasmids pSKBJF1,
pSKBJF9, and pSKBJF22, which complemented the fur strain on
plates as described in the text. pSKBJF800 is an
ApaI-Sau3AI-digested subclone of pSKBJF22. The
arrow represents the open reading frame encoding the fur
homolog. Restriction sites: A, ApaI; B, BglII; C,
ClaI; E, EcoRI; Sa, SacI; S,
SphI; St, StyI. (B) Repression of
-galactosidase activity in fur strain H1780 by B. japonicum library clones pSKBJF1 (F1), pSKBJF9 (F9), pSKBJF22
(F22), and pSKBJF800 (F800). Cells containing the library vector
pBluescript SK(+) (pSK) were the control for unrepressed activity. The
positive control was pMH15fur (EcFur), which contained the E. coli fur gene. Also shown is the result for B. japonicum
irr gene on pSKSBIrr (Irr).
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FIG. 2.
Deduced amino acid sequence of the B. japonicum Fur protein (bottom sequence) and alignment with other
Fur proteins. Asterisks and colons denote amino acids that are
identical or similar, respectively, at that position for all sequences.
The sources of the proteins used, along with GenBank accession numbers,
are as follows: E. coli (X02589), Klebsiella
pneumoniae (L23871), Yersinia pestis (Z12101),
Vibrio cholerae (M85154), Vibrio anguillarum
(L19717), Vibrio parahaemolyticus (AB003752), Vibrio
vulnificus (L06428), P. aeruginosa (L00604),
Pseudomonas putida (X82037), Neisseria
gonorrhoeae (L11361), Neisseria meningitidis (L19777),
Bordetella pertussis (U11699), Haemophilus
ducreyi (U37224), Campylobacter jejuni (AF052056),
Campylobacter upsaliensis (L77075), Synechococcus
sp. PCC 7942 (L41065), Legionella pneumophila (U06072), and
R. leguminosarum (Y13657).
Complementation analysis showed that the B. japonicum Fur (BjFur) had repressor activity in vivo (Fig. 1); thus, we examined its DNA-binding activity in extracts from cells that overexpressed Fur by gel mobility shift assays with a double-stranded DNA probe that includes a "Fur box" consensus sequence recognized by E. coli Fur (EcFur) and other characterized Fur proteins (4, 5, 11). In addition, the assays were carried out in the presence or absence of Mn2+, which has been shown to substitute for Fe2+ as a cofactor of Fur in vitro (5). Mn2+ is used because Fe2+ is readily oxidized in air to Fe3+, which is not functional as a Fur cofactor. The results show that BjFur bound to the Fur box consensus element in the presence of metal (Fig. 3A) but not in its absence (Fig. 3B). EcFur showed metal-dependent DNA-binding activity as well (Fig. 3). However, in the absence of metal, the DNA-binding activity of EcFur produced a complex that was different from that observed in the presence of metal. Binding of E. coli apo-Fur to operator DNA with lower affinity in a gel mobility shift assay has been documented previously (1). Unlike BjFur, the B. japonicum Irr protein did not bind to the iron box element under any condition (Fig. 3), consistent with the conclusion that Irr is not functionally equivalent to Fur.
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E. coli fur has a Fur box element in its promoter and is modestly autoregulated, whereas the Pseudomonas aeruginosa fur gene does not (11). No recognizable Fur box element was found upstream of the fur gene, and an ApaI-StyI fragment that included 268 bp of DNA upstream of the fur open reading frame did not bind to BjFur in a gel mobility shift assay (data not shown). Thus, the B. japonicum fur gene does not appear to be autoregulated.
Many bacterial species, including B. japonicum (8,
9), express an inducible high-affinity iron uptake activity in
response to iron limitation as a means of scavenging the metal. Fur
proteins repress bacterial iron transport genes under iron-replete
conditions; thus, a fur mutant of E. coli is
derepressed for iron transport (for a review, see references
2 and 10). A B. japonicum
fur null mutant of wild-type strain USDA I110 was constructed by
the insertional inactivation of the fur gene of parent
strain I110 with an 
cassette to construct strain GEM4. Ferric
citrate uptake activity was carried out as described previously
(9). The fur strain showed similar iron uptake
activity as the wild type when grown in iron-limited medium (Fig.
4). However, the mutant expressed a
three- to fourfold higher initial rate of iron uptake activity than the
parent strain when grown in high-iron medium. This modest derepression
was similar to that observed for an E. coli fur mutant (10). A phenotype for the mutant strain showed that the
cloned fur gene is functional in B. japonicum.
The uptake activity of the fur strain grown in high-iron
medium reached saturation by 10 to 15 min (Fig. 4 and data not shown),
whereas activity induced in the wild type by iron deprivation is
unchanged for at least 90 min (9). Irr is not expressed in
high-iron medium (9); thus, it is possible that the iron
transport system normally induced under iron limitation in the wild
type was not fully expressed in the fur strain grown in
high-iron medium.
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The identification of a functional fur gene in B. japonicum shows that Irr does not replace Fur, but rather both regulators function in that organism to regulate iron metabolism. Genes encoding Fur-like proteins in addition to bona fide Fur have been recently identified in E. coli and Bacillus subtilis; these proteins are involved in the maintenance of zinc homeostasis (Zur) (7, 12), or in a manganese-dependent response to oxidative stress (PerR) (3). Thus, there appears to be a family of functionally diverse Fur-like proteins, including Zur, PerR, and Irr, that are all involved in metal-dependent regulation but are distinct from Fur. These findings underscore the need for experimental evidence to correctly identify fur homologs obtained by nucleotide sequence information.
Nucleotide sequence accession number. The nucleotide sequence of the ApaI-SauIIIA insert of pSKBJF800 was deposited in GenBank under accession no. AF052295.
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ACKNOWLEDGMENTS |
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We thank K. Hantke and M. Vasil for E. coli strains and plasmids.
This work was supported by National Science Foundation grant MCB-9722974 to M.R.O.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biochemistry, 140 Farber Hall, State University of New York at Buffalo, Buffalo, NY 14214. Phone: (716) 829-3200. Fax: (716) 829-2725. E-mail: mrobrian{at}buffalo.edu.
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Journal of Bacteriology, September 1999, p. 5843-5846, Vol. 181, No. 18
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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