Dimeric Brucella abortus Irr protein controls its own expression and binds haem

Marcela Martínez, Rodolfo A. Ugalde and Marta Almirón

Instituto de Investigaciones Biotecnológicas, Instituto Tecnológico de Chascomús (IIB, INTECH), Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de General San Martín (CONICET-UNSAM), Ed 24, Av. General Paz 5445, San Martín 1650, Buenos Aires, Argentina

Correspondence
Marta Almirón
malmiron{at}iib.unsam.edu.ar


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Brucella abortus needs to synthesize haem in order to replicate intracellularly and to produce virulence in mice. Thus, to gain insight into the pathogenesis of the bacterium, regulatory proteins of the haem biosynthetic pathway were sought. An iron response regulator (Irr) from Bradyrhizobium japonicum, which is a close relative of Brucella, was previously described as being involved in the coordination of haem biosynthesis and iron availability. The Bru. abortus genome was searched for an irr orthologue gene, and the Bru. abortus irr gene was cloned, sequenced and disrupted. A null mutant was constructed that accumulated protoporphyrin IX under conditions of iron deprivation. This phenotype was overcome by a complementing plasmid carrying the wild-type irr. Purified recombinant Bru. abortus Irr behaved as a stable dimer and bound haem. Interestingly, in vivo, Irr was only detected in cells obtained from iron-limited cultures and the protein downregulated its own transcription. Through lacZ fusion, it was demonstrated that iron did not regulate irr transcription. The data reported show that Bru. abortus Irr is a homodimeric protein that is accumulated in iron-limited cells, controls its own transcription and downregulates the biosynthesis of haem precursors.


Abbreviations: ALA, {delta}-aminolaevulinic acid; DIP, 2,2'-dipyridyl; HRM, haem regulatory motif

The GenBank/EMBL/DDBJ accession number for the sequence of the Brucella abortus irr gene is AY243456.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Brucellae are Gram-negative facultative intracellular bacteria that are responsible for brucellosis, a chronic zoonotic disease. Brucella abortus is the aetiological agent of bovine brucellosis, which induces abortion and sterility in cattle, and undulant fever in humans. The pathogenesis of this organism resides in its ability to invade and replicate within professional and non-professional phagocytes (Smith & Ficht, 1990).

The genus Brucella belongs to the {alpha}-2 group of proteobacteria, along with plant pathogens and symbionts. Recent analysis of the Brucella and Rhizobium genomes revealed extensive sequence homology and synteny (Paulsen et al., 2002). This genomic conservation is reflected in common strategies for intracellular survival (Lestrate et al., 2000; Paulsen et al., 2002; Ugalde, 1999). For example, the absence of the haem biosynthetic enzyme HemH affects the virulence of Bru. abortus (Almiron et al., 2001) and the development of nodules in Bradyrhizbium japonicum (Frustaci & O'Brian, 1992). Haem is required as a prosthetic group in many physiological processes, such as electron transfer, hydroxylation and the sensing of diatomic gases (O'Brian & Thony-Meyer, 2002). It is also involved in the transcriptional and post-transcriptional regulation of different genes (Ogawa et al., 2001; Pfeifer et al., 1989). Haem is synthesized from the precursor {delta}-aminolaevulinic acid (ALA) through six enzymic reactions that lead to protoporphyrin IX. In the last step, ferrochelatase (HemH) catalyses the incorporation of ferrous iron into the protoporphyrin IX ring to yield haem. The formation of haem through this pathway could also be compromised by iron limitation, which is a condition faced by the bacteria during infection. Even though Bru. abortus has the ability to synthesize haem, as well as to incorporate it from the medium, we showed previously that the absence of hemH abolished Bru. abortus virulence and intracellular survival (Almiron et al., 2001). Therefore, it is of interest to study the regulation of haem biosynthesis under conditions of iron deprivation.

In Brad. japonicum, which is a member of the family Rhizobiaceae, the iron response regulator (Irr) has been described as a repressor of haem biosynthesis when iron is scarce, thus avoiding the accumulation of protoporphyrin (Hamza et al., 1998). Irr belongs to the ferric-uptake regulator (Fur) family, the members of which are involved in metal-dependent regulation in bacteria (Escolar et al., 1999).

Bru. abortus genome mining revealed the presence of two putative irr genes. Here we describe the characterization and disruption of the gene with the highest similarity to the Brad. japonicum irr gene, including the flanking regions. The haem biosynthesis under iron limitation in the Bru. abortus irr mutant was analysed, as well as the effects of iron on irr transcription and Irr protein levels. A recombinant Bru. abortus Irr was investigated for its ability to bind haem, dimerize and regulate its own transcription.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
Bru. abortus strains 2308 and 2308C (2308dhbC : : lacZ) were obtained from the laboratory stock. Escherichia coli DH5{alpha} (Woodcock et al., 1989) competent cells were used for plasmid manipulation. All bacterial liquid cultures were carried out at 37 °C in a rotary shaker at 250 r.p.m., in Luria–Bertani medium (LB) or Brucella Broth (BB) (Difco). LB with 0·45 mM of the iron chelator 2,2'-dipyridyl (DIP) and modified Gerhardt's medium (MG) were used as iron-deficient media. In the latter medium, iron was removed by exhaustive treatment with 8-hydroxyquinoline and the oxime was further extracted with chloroform (Lopez-Goñi et al., 1992). For solid media, Bacto agar (1·6 %, w/v) was added. When required, the media were supplemented with 100 µg ampicillin ml–1 or 50 µg kanamycin ml–1. Procedures using live brucellae were performed in a Biosafety Level 3 laboratory. All the reagents were purchased from Sigma, unless otherwise stated.

Gene cloning and sequencing.
Classic recombinant DNA techniques were performed according to standard protocols (Sambrook et al., 1989). The putative irr gene was obtained by colony PCR from Bru. abortus 2308 using the primers Upirr (5'-TGGATTTTAGTGGTTCAGG-3') and Loirr (5'-GACTTCCTTCACCTTTTCCA-3'), both obtained from the putative Brucella melitensis irr sequence in GenBank (AE009628). The amplified product (1527 bp) was cloned into pGEM-T Easy (Promega), following the instructions from the manufacturer. The new plasmid was named pGEM-Tirr. Nucleotide sequencing of the amplified fragment was performed using dye-labelled universal terminators on an ABI Prism 377 sequencer (Applied Biosystems) with the primers T7 and Sp6 (Promega). The 1·5 kb EcoRI fragment obtained from pGEM-Tirr was ligated to pBBR1MSC-4 (Kovach et al., 1995) digested with EcoRI. The resulting plasmid, named pBBRirr, was employed for complementation experiments.

Construction of mutants.
(i) Bru. abortus 2308IK. In order to disrupt the Bru. abortus irr gene, the plasmid pGEM-Tirr was linearized with NruI (47 bp downstream of the ATG) and ligated to a 1·3 kb HincII fragment containing a kanamycin-resistance cassette (Oka et al., 1981). The new plasmid, named pGEM-TIK, was electroporated into Bru. abortus 2308, where it is unable to replicate. Thus, double-homologous recombinants were selected as Bru. abortus Kmr Aps. PCR and Southern-blot analysis were performed to confirm the mutant.

(ii) Bru. abortus 2308IL. To construct a transcriptional lacZ fusion to the irr gene, the plasmid pGEM-Tirr was digested with NruI and ligated to a 4·5 kb SmaI fragment containing the lacZ cassette (Becker et al., 1995), resulting in plasmid pGEM-TIL. The transcriptional fusion was confirmed by PCR and sequence analysis; {beta}-galactosidase activity was determined as described by Miller (1992). pGEM-TIL was electroporated into Bru. abortus 2308. Double recombinants were selected by their antibiotic resistance and confirmed using the methodology indicated above.

Detection of protoporphyrin IX in supernatants of Bru. abortus cultures.
Accumulation of porphyrins was assayed as described elsewhere (Cox & Charles, 1973). Briefly, Bru. abortus cells grown in BB were centrifuged (4500 g for 15 min), washed twice with phosphate-buffered saline (PBS) and diluted into 50 ml MG or MG supplemented with 50 µM ferric citrate. Cells from those cultures, grown to saturation, were harvested and the porphyrins from the supernatants were extracted with 12·5 ml ethyl acetate/glacial acetic acid (3 : 1, v/v) at 18 °C overnight with shaking. Then, the ethyl acetate layer was washed once with MilliQ water and concentrated at low temperature in a vacuum system. The absorption spectrum of this extract was recorded between 700 nm and 325 nm (Spectronic GENEYS 5). Known dilutions of purchased protoporphyrin in a mixture of ethyl acetate and glacial acetic acid were used as standards.

Expression, purification and detection of Irr.
To express Irr, the sense primer (5'-ATCGGATCCGATGCATTCTTCACATACCCAC-3'; BamHI site underlined) and the antisense primer Loirr (see above) were used to amplify a 741 bp fragment from Bru. abortus 2308 chromosomal DNA. The PCR product was digested with BamHI and HindIII (located 101 bp downstream of the stop codon) and directionally cloned into pTrcHis B (Invitrogen). The new plasmid, pTrcHisirr, was transformed into and expressed in E. coli DH5{alpha}. The His6-tagged Irr was purified from the soluble fractions following the instructions from the manufacturer. It was further used to obtain antibodies against Irr by immunization of mice, following standard procedures.

To detect the levels of the Irr protein in cells that were grown in iron-sufficient or iron-deficient media, Bru. abortus 2308IK(pBBRirr) was first grown twice in MG in order to deplete the cells' iron content. Iron-limited cells from late exponential phase were diluted 1/20 either in MG or in MG supplemented with 50 µM ferric citrate. Exponential-phase cells from each culture were harvested, washed with PBS, and resuspended in lysis buffer (10 mM Tris/HCl, pH 8·0; 10 mM NaCl; 1 mM sodium citrate; 1·5 % SDS) for sonication in an ice-water bath (one cycle of 30 s and two cycles of 20 s). Proteins from the soluble extracts were quantified by the Lowry method using BSA as standard. Further, equal amounts of protein from different samples were resolved using 15 % SDS-PAGE and transferred onto a nitrocellulose membrane to detect Irr by chemiluminescence (Pierce).

Haem-binding assays.
The interaction of Irr with haem was studied through the spectral properties of haem (Zhang & Guarente, 1995). Haemin was dissolved in a 50 : 48 : 2 mixture of ethanol/water/5 M NaOH and binding studies were carried out using an appropriate dilution in 50 mM Tris/HCl pH 8·0. The absorption spectrum of 10 µM haem was recorded in the presence or absence of 10 µM Irr. Negative and positive controls were carried out using lysozyme and BSA respectively.

Molecular mass determination.
The molecular mass of the recombinant Irr was determined by gel filtration using a Superdex 75 10/30 HR column (Amersham Pharmacia Biotech). The molecular mass standards used were BSA (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12·4 kDa). The samples were resolved at a flow rate of 0·7 ml min–1 using 50 mM Tris/HCl as eluent. The column profile was monitored by following the absorbance at 215 nm. The fractions from the major peaks were pooled, concentrated by precipitation with TCA, dissolved in loading buffer (Sambrook et al., 1989) and resolved using 15 % SDS-PAGE.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Search for an Irr orthologue in Bru. abortus
To investigate whether an Irr protein was present in Bru. abortus, we used the Brad. japonicum Irr sequence to carry out a BLAST search (Altschul et al., 1990) against the translated genome of Bru. abortus (R. A. Ugalde, unpublished). Two homologous sequences were found in chromosome I. This study was performed with the paralogue (Ba12233) that displays the highest similarity (56 % identity) and is located, similar to Brad. japonicum irr, downstream of genes involved in lipid biosynthesis.

Bru. abortus irr encodes a protein of 145 amino acids that, according to a TBLASTX search (Altschul et al., 1990), is confined to the {alpha}-2 group of proteobacteria. In this group, most of the proteins lack the haem regulatory motif (HRM) that is associated in Brad. japonicum with its iron-conditional stability (Qi et al., 1999), and they form a highly homogeneous group with 68–71 % identity among its members (Fig. 1). The amino acid sequences of these proteins display conserved domains that are characteristic of Fur-like proteins, thus suggesting a role in the transcription of metal-ion-responsive genes.



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Fig. 1. Amino acid alignment of Irr from the {alpha}-2 group of proteobacteria. The Brucella abortus Irr sequence (AAO89498) is compared with the sequences from its close relatives Mesorhizobium loti (BAB51996), Rhizobium leguminosarum (CAD37806) and Agrobacterium tumefaciens (AAK85974). It is also aligned with sequences from the less related species Bradyrhizobium japonicum (AAC32183) and Rhodopseudomonas palustris (ZP00010023). Sequences were from GenBank. Conserved amino acids are shaded in grey. The haem-binding domains HRM and HXH are outlined in black. Comparison was performed with the CLUSTAL method using the MegAlign program.

 
Analysis of protoporphyrin accumulation in Bru. abortus cultures
To investigate whether Bru. abortus Irr is involved in the regulation of haem biosynthesis, a Bru. abortus 2308irr-null mutant was constructed, as described in Methods. The mutant showed the same growth phenotype as wild-type cells in the media used (data not shown). The wild-type Bru. abortus 2308, the mutant Bru. abortus 2308IK and the complemented Bru. abortus 2308IK(pBBRirr) were tested for porphyrin accumulation after being grown in iron-sufficient or iron-deficient media. Only the mutant strain that was grown under conditions of iron deprivation showed a brownish-red colour. This pigmentation appeared in both the cell pellet and the supernatant of the culture. The pigment was further identified as protoporphyrin IX by spectrophotometry (Fig. 2). No protoporphyrin IX accumulation was detected in the mutant strain grown under iron-sufficient conditions (data not shown). Therefore, the absence of Irr induced the accumulation of protoporphyrin IX under iron-limited growth conditions. These results suggest that, as was described in Brad. japonicum, Bru. abortus Irr is involved in the downregulation of haem biosynthesis under conditions of iron deprivation.



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Fig. 2. (a) Protoporphyrin accumulation in iron-restricted Bru. abortus 2308IK. Strains 2308 (1), 2308IK(pBBRirr) (2) and 2308IK (3) were cultured in iron-deficient and iron-sufficient media. Supernatants from cultures were extracted with a mixture of ethyl acetate and glacial acetic acid and absorption spectra of concentrated extracts were recorded. The figure shows the recorded scans from iron-restricted cultures. (b) Standard protoporphyrin IX. Absorption peak wavelengths are indicated.

 
Regulation of irr expression by iron
As Irr is involved in the regulation of haem metabolism when iron is limited, we investigated whether the iron concentration affects irr expression. Bru. abortus 2308irr : : lacZ (strain 2308IL) was constructed and {beta}-galactosidase activity was assayed in cells grown in iron-sufficient (LB) or iron-deprived (LB-DIP) media. As shown in Fig. 3(a), the {beta}-galactosidase activities were similar in both media. Moreover, when the assays were repeated with bacteria grown in MG medium or MG supplemented with iron, 1919±28 and 1856±77 Miller units were obtained, respectively. These results indicate that the transcription of irr was not affected by the presence of iron or by the composition of the growth media. As a control for the experimental conditions, the {beta}-galactosidase activity of a dhbC–lacZ transcriptional fusion strain (2308C) in LB and LB-DIP media was measured (Fig. 3a). dhbC is the first gene of the Bru. abortus siderophore operon and is known to be strongly derepressed under iron-deficient growth conditions (Bellaire et al., 2003); it can be seen from Fig. 3(a) that dhbC transcription is inhibited by the addition of iron to the medium.



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Fig. 3. Expression of Bru. abortus irr according to the external iron concentration. (a) Effect of iron on the expression of the irr promoter. Bru. abortus 2308IL was grown in iron-deficient (–) or iron-sufficient (+) media and assayed according to Miller (1992). {beta}-Galactosidase activity of the iron-responsive dhbC gene from strain 2308C was simultaneously assayed as a control. Activities (means±SD of four determinations made in duplicate) are shown. The results are representative of at least three independent experiments. (b) Effect of iron on the Irr levels. Cells carrying the medium-copy plasmid harbouring wild-type irr were grown in low-iron medium (lane 1). Aliquots from this culture were used to inoculate fresh iron-deficient (lane 2) or iron-sufficient (lane 3) media. After growth, cells from each culture were processed as described in Methods. From each processed culture, 40 µg samples of proteins were resolved using 15 % SDS-PAGE and analysed by Western blotting using polyclonal anti-Irr serum.

 
The effect of iron on the intracellular level of the Irr protein was also investigated. The experiment was carried out with the 2308IK(pBBRirr) strain expressing Irr from a medium-copy-number plasmid, as we failed to detect Irr by immunoblotting analysis in the wild-type strain. The cells were grown under conditions of iron limitation and were diluted into iron-deficient or iron-sufficient media. The cultures were allowed to grow to the late exponential phase and were further processed in order to analyse the Irr levels by Western blotting analysis. As shown in Fig. 3(b), Irr was only detected in cells grown under our experimental iron-restrictive conditions (lanes 1 and 2). Irr was undetectable in cells that were regrown under iron-sufficient conditions, whereas it was increased in cells grown under conditions of iron limitation. Thus, the presence of iron reduced the amount of Irr that was present in the cells. As mentioned above, irr–lacZ activity was not affected by the iron concentration, so it is possible that iron affects Irr protein stability.

Assays of haem binding to Irr
Bru. abortus Irr lacks the HRM that is associated with binding to haem and the turnover of the protein. However, as haem can interact with proteins lacking this motif, we analysed its binding to Irr. The absorption spectrum of haem changes upon interaction with peptides or proteins (Zhang & Guarente, 1995). Thus, the absorption spectrum of haem in the presence or absence of purified recombinant Irr was recorded. As shown in Fig. 4, the 382 nm absorption peak of haem shifted to 394 nm in the presence of Irr. This shift is characteristic of haemoproteins, in which the Soret bands shift to longer wavelengths due to changes of electron distribution in the porphyrin ring.



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Fig. 4. Effect of Irr on the absorption spectrum of haem. (a) Absorption spectrum of 10 µM haem was recorded in the absence (1) or in the presence (2) of 10 µM recombinant Irr. A scan of 10 µM Irr alone (*) is also shown. (b) Absorption spectra of 5 µM haem with 5 µM lysozyme (3) or 5 µM BSA (4) were also recorded as negative and positive controls, respectively. Absorption peak wavelengths are indicated.

 
Analysis of oligomerization of Irr
Irr belongs to the Fur family, and Fur was previously reported to act as a dimer (Stojiljkovic & Hantke, 1995). Therefore gel-filtration chromatography of a purified recombinant protein was used to determine the possible presence of Irr oligomers. The molecular mass of the protein estimated by SDS-PAGE was about 20 kDa. The elution profile from the chromatography revealed the presence of a major molecular form of 43 kDa and a less-abundant molecular form of 81·9 kDa (Fig. 5). The fractions were pooled and resolved using SDS-PAGE. As shown in Fig. 5(b), the 43 kDa peak yielded only a 20 kDa protein. The minor peak showed high-molecular-mass proteins that co-purified in the affinity chromatography. Therefore, we concluded that Irr was present exclusively as a dimer of 43 kDa. Moreover, this homodimeric complex was highly stable, as it was obtained even when the sample was heat treated for 1 min before the chromatographic analysis.



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Fig. 5. Analytical gel filtration chromatography of Bru. abortus Irr. (a) Elution profile of 75 mg of the purified recombinant Irr from a Superdex 75 HR 10/30 HPLC column. A major peak at 43 kDa (2) and a minor one at 81·9 kDa (1) were detected. (b) Proteins from both peaks were resolved using 15 % SDS-PAGE together with molecular mass markers (M) and stained with Coomassie blue.

 
Effect of Irr on its own expression
We then investigated whether Irr regulated its own expression, as other members of the Fur family do. For this purpose, we analysed the {beta}-galactosidase activity of the 2308IL strain transformed with pBBRirr, which carries a chromosomal irr–lacZ fusion and plasmid-borne wild-type irr under the control of its own promoter. The strain that was complemented with the vector alone was used as a control. The assay was carried out in LB-DIP medium, as Irr was only detected under conditions of iron deprivation. The {beta}-galactosidase activity of the chromosomal fusion (2425±224 Miller units) was reduced by about one-half in the presence of Irr (1034±72 Miller units). A similar result was obtained with the 2308IL isogenic mutant carrying an additional irr copy in the chromosome (data not shown), indicating that the downregulation was not an artefact of the plasmid-copy number.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We have demonstrated the existence of a functional protein in Bru. abortus that regulates the synthesis of haem. The analysis of the Bru. abortus genome showed a strong homology to Brad. japonicum Irr (Hamza et al., 1998), thus revealing that Bru. abortus has a putative irr gene. A chromosomal mutation in irr of the wild-type Bru. abortus strain led to protoporphyrin IX accumulation in the cells when grown under iron-limited conditions. This phenotype, which is based on the repression of haem biosynthesis when iron availability is low, was also recently assigned to an Irr protein described in Rhizobium leguminosarum (Wexler et al., 2003).

In both Brad. japonicum and R. leguminosarum, the regulation of haem biosynthesis by the Irr proteins was observed at the transcription level of hemB, which encodes the second enzyme of haem biosynthesis from ALA. Irr represses the expression of hemB. As this phenotype was observed in all three species, it is possible that Bru. abortus Irr also exerts hemB repression. We have observed that wild-type Bru. abortus transformed with a pBBR1MSC4 derivative encoding a hemB–lacZ fusion yielded approximately 10-fold less {beta}-galactosidase activity than an isogenic irr mutant strain transformed with the same plasmid (data not shown). Whilst this is consistent with Irr-mediated repression of hemB transcription, we observed that growth of the transformed wild-type bacteria was impaired in iron-limited medium. Thus further studies will be needed to confirm that Irr regulates hemB transcription and to determine the site of DNA binding by Irr and whether it corresponds to a consensus sequence.

The Irr protein belongs to the Fur family and is confined to the {alpha}-2 proteobacteria, according to an analysis of the available bacterial genome databases. Interestingly, the genomes of Bru. abortus and Brad. japonicum contain more than one putative sequence encoding Irr-like proteins, without homology to perR and besides the fur and zur genes. This might reflect the importance of keeping iron under restrictive control in these organisms. It would be interesting to know whether these putative Irr-like proteins are expressed simultaneously and have similar functions.

Hence, Irr is a regulator protein that responds to iron concentrations in a particular way. In the present work, we present evidence that the rate of irr transcription in Bru. abortus is high, regardless of the iron level in the growth medium. Nonetheless, the protein was only detected in cells carrying many copies of the wild-type irr gene when grown under iron-limited conditions. The fact that Irr could not be detected by Western blotting analysis when expressed from the chromosomal irr gene under conditions of iron limitation could be an indication that it is generally poorly expressed. In this regard, the transcription of the homologous irr genes in Brad. japonicum and R. leguminosarum has been reported to be upregulated under low iron concentrations. Although in Brad. japonicum, irr transcription depends on Fur-Fe (Hamza et al., 2000), it does not involve Fur in R. leguminosarum (Wexler et al., 2003).

Even though there is constitutively high transcription of irr in Bru. abortus, observations with Brad. japonicum suggest that the Irr protein may be degraded when the intracellular iron levels are increased (Qi et al., 1999). Bru. abortus cells that were grown in iron-deficient media accumulated Irr at a certain level. This Irr protein was undetectable when the same cells were diluted into iron-containing media, whereas an increased concentration was observed when the cells were diluted into iron-deficient media. These results suggest an efficient iron-dependent mechanism for the protein degradation. Another possibility is that the intracellular concentration of Irr is lowered as a consequence of the cell dilution, and that this protein level is under the limit of the detection system used in the current work. However, two lines of evidence support the former hypothesis: first, the cell mass of the cultures did not increase substantially, as the cells were growing in the minimal medium for only a relatively short time; and second, similar results were obtained when the experiments were repeated with more protein loaded onto the SDS-polyacrylamide gel for Irr detection. On the other hand, it is evident that intracellular iron depletion prompts the synthesis of a stable Irr protein.

Of the two other Irr homologues described in the species mentioned above, Brad. japonicum Irr is the best studied. For the stability of this protein, a mechanism based on two haem-binding sites in the Irr sequence has been proposed (Yang et al., 2005). The Brad. japonicum Irr has an HRM that binds ferric haem and a second motif of three histidines (H117, H118 and H119) that interacts with ferrous haem. It has been shown that changing one of these histidines to another amino acid reduces the binding efficiency (Yang et al., 2005). The Bru. abortus Irr does not contain the HRM and conserves only two histidines (HXH) of the second motif. In spite of this, we report here that the recombinant Bru. abortus Irr protein is able to bind haem. There is a possibility that this haem-binding domain tested in vitro could be related to the third uncharacterized ferric haem-binding site in the Brad. japonicum Irr. The red shift of the haem spectrum displayed by the Bru. abortus Irr is characteristic of haemoproteins that retain a methionine/histidine or bis-histidine coordination to the haem iron (Zhang & Guarente, 1995). It has also been reported that direct iron binding to histidine-rich proteins affects the stability of these proteins (Guo et al., 1995; Pohl et al., 2003), and Irr itself is a histidine-rich protein. Even though the haem-binding site of the Bru. abortus Irr remains unknown, it is possible that, in this micro-organism, post-transcriptional regulation is being exerted by binding to the haem molecule, as was established in Brad. japonicum. Studies involving a ferrochelatase mutant have suggested that the Brad. japonicum Irr degradation occurs at the site of haem biosynthesis via an interaction between Irr and ferrochelatase (Qi & O'Brian, 2002). This type of study cannot be easily carried out in Bru. abortus, due to the high demand for exogenous haemin of Bru. abortus cells lacking ferrochelatase.

An increase in the intracellular Irr concentration downregulates its own transcription, thereby allowing the cells to maintain Irr at a suitable level. This kind of regulation was observed when the experiments were performed with a chromosomal copy and was also demonstrated in the strain that carried many copies of the wild-type irr in trans, in order to confirm the presence of Irr in the cells under our experimental conditions. This autoregulation is characteristic of members of the Fur family, as is the ability to dimerize. Here we present evidence that the Bru. abortus Irr is able to form homodimers in vitro and that this conformation is highly stable.

As far as we know, this is the first report to describe the presence of an Irr in Bru. abortus. Furthermore, we conclude that this protein behaves as a regulator with the ability to dimerize and is present in cells only when the iron concentration is limited, thereby repressing haem biosynthesis. Under iron-sufficient conditions, the protein becomes less stable, probably due to its ability to bind iron from haem. As low iron availability is one of the conditions imposed by a host during bacterial infection, it will be of interest to evaluate whether Irr regulates other genes that are involved in Bru. abortus pathogenesis. To this end, studies are currently being developed in our laboratory.


   ACKNOWLEDGEMENTS
 
We thank Andrea Merás for technical assistance with the use of HPLC, and Pablo Briones for computational assistance. We are grateful to Elena Fabiano for kindly providing protoporphyrin IX. This work was supported by grants PICT 01-6580 to M. Almirón and PICT 01-6565 to R. A. Ugalde, both from Agencia Nacional de Promoción Científica y Tecnológica, Argentina.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 23 May 2005; revised 25 July 2005; accepted 25 July 2005.



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