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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), San Martín 1650, Argentina
Correspondence
Marta Almirón
malmiron{at}iib.unsam.edu.ar
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-2 group of Proteobacteria only, where it negatively regulates haem biosynthesis when iron is scarce. Additional evidence that Irr also regulates the synthesis of both siderophores is presented here. Transcriptional lacZ fusion and chemical determinations revealed that Irr induced the transcription of the operon involved in the synthesis of the catecholic siderophores, which were consequently secreted under conditions of iron limitation. Irr was able to bind the upstream region of the operon, as shown by electrophoretic mobility shift assay. A B. abortus irr mutant showed higher intracellular haem content, catalase activity and resistance to hydrogen peroxide than the wild-type strain. The mutation also improved the replication and survival of iron-depleted bacteria within cultured mammalian cells. Although the pathogenesis of Brucella correlates with its ability to replicate intracellularly, pathogenicity was not attenuated when assayed in a murine model.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This iron acquisition mechanism is frequently linked to the virulence of bacteria because it contributes to the establishment of a successful infection (Litwin & Calderwood, 1993). In contrast, a high concentration of iron is toxic, due to its participation in reactions that generate reactive oxygen species, which can lead to cellular damage. In these conditions, bacteria usually induce the synthesis of storage proteins and oxidative stress enzymes that remove toxic molecules. According to this scenario, the tight regulation of iron homeostasis is fundamental for bacterial life.
Brucella abortus is the aetiological agent of bovine brucellosis, which is an infection of humans and cattle. The organism lives mainly intracellularly, and its pathogenesis correlates with the ability to invade and replicate within professional and non-professional phagocytes (Smith & Ficht, 1990). During infection, iron availability is reduced as part of the host defence against micro-organisms, implying that B. abortus faces iron limitation in this process (Bullen & Griffiths, 1999). Under conditions of iron depletion, B. abortus secretes the two catecholic siderophores brucebactin and 2,3-dihydroxybenzoic acid (2,3-DHBA), which have been associated with virulence of the species in ruminants, suggesting the importance of this mechanism for iron acquisition in the natural host (Bellaire et al., 2003). Brucebactin, whose structure remains unknown, has recently been reported to be the most active siderophore of B. abortus (Gonzalez Carrero et al., 2002); it is produced from 2,3-DHBA through a poorly understood pathway. Biosynthesis of 2,3-DHBA requires genes organized in the dhbCEBA operon, whose expression is regulated by iron (Bellaire et al., 2003). Even though two Fur boxes have been identified in the promoter region, the ferric-uptake regulator Fur does not repress transcription of this operon (Roop et al., 2004). No additional data on this regulation have been reported heretofore.
We have recently characterized the iron response regulator Irr from B. abortus, and it belongs to the Fur family (Martínez et al., 2005). Irr downregulates haem biosynthesis when iron is insufficient. This biosynthesis involves six sequential enzymic reactions leading to protoporphyrin IX. In the last step of the pathway, ferrochelatase inserts ferrous iron into the porphyrin ring to yield haem. The interruption of this step is detrimental for B. abortus virulence (Almirón et al., 2001). Hence, considering the role of Irr in the regulation of an iron-demanding pathway implicated in B. abortus virulence, we decided to investigate whether Irr controls iron-uptake systems, and whether it plays any role during infection. Here, we present evidence that Irr directly induces the biosynthesis of 2,3-DHBA and brucebactin, but that it is not required for virulence in mice. Interestingly, under conditions of iron limitation, a mutant lacking Irr displayed increased resistance to hydrogen peroxide due to higher levels of haem and catalase activity than the wild-type strain. Compared with the wild-type, the mutant showed improved intracellular replication and survival inside HeLa and J774 cells, thus implicating Irr in the pathogenesis of B. abortus.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). A double mutant 2308IC (2308irr : : km, dhbC : : lacZ), which carries the chromosomal mutations of 2308IK and 2308C, was constructed for this study by gene replacement (Martínez et al., 2005). All bacterial liquid cultures were incubated at 37 °C in a rotary shaker at 250 r.p.m. Luria–Bertani medium containing the iron chelator 2,2'-dipyridyl (LB-DIP; 0.45 mM), and modified Gerhardt's (MG) medium, were used as iron-deficient media (Lopez-Goñi et al., 1992). 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.
-Galactosidase enzyme assay.
Levels of -galactosidase were measured from liquid iron-depleted cultures as described by Miller (1992).
Siderophore detection
The chrome azurol S (CAS) assay.
This assay was used to determine brucebactin secretion only, since different amounts of commercial 2,3-DHBA had no effect on the absorbance of the CAS reagent, either in plate or in liquid cultures. CAS agar plates were prepared according to the method of Schwyn & Neilands (1987), with the following modifications: (i) MG was used instead of MM9 medium, (ii) the K2HPO4 concentration was reduced to 0.3 g l–1, and (iii) glucose was added in place of glycerol and lactic acid to avoid interference of these compounds with CAS (Gonzalez Carrero et al., 2002). Cells grown for 24 h in 1 ml MG were washed and resuspended in 10 µl PBS, which was applied to a sterile filter disk on the CAS plate. The halo produced around the spot was observed after 72 h incubation at 37 °C. For detection in liquid cultures, the supernatants were mixed with an equal volume of CAS reagent, and the absorbance was read at 630 nm. Percentage siderophore activity was calculated by using the formula [(X–Y)/X]x100, where X and Y are the absorbance values of the growth medium and the sample supernatant, respectively. In order to set up the experimental conditions for catechol detection, measurements were taken at different time points during the incubation of cells in MG medium. Maximal differences were obtained when the cultures reached OD600 
1. No catechol was detected when determinations were made from supernatants of MG cultures supplemented with 200 µM ferric citrate.
HPLC analysis.
This was performed to detect 2,3-DHBA. Supernatants (10 ml) from the bacterial cultures assayed for brucebactin were acidified to pH 2.0 with 6 M HCl. Acidified samples were extracted with ethyl acetate [20 ml (100 ml supernatant)–1]. The catechol-containing extracts were concentrated in a vacuum system, and 0.2 ml of each extract was fractionated on a C18 Sephasil peptide reverse-phase column (12 µM, 4.6x250 mm; Pharmacia Biotech). The procedure was performed using a gradient of 10–50 % acetonitrile in water, with 0.1 % trifluoroacetic acid at a constant flow rate of 1 ml min–1. The column profile was monitored by following the absorbance at 254 nm. Commercial 2,3-DHBA dissolved in ethyl acetate was employed as a standard.
Haem determination.
The intracellular concentration of haem in B. abortus strains was determined as described by Frustaci et al. (1991). Commercial haemin was used as a standard in the range 10–200 nM, and it was assayed at the same time as the B. abortus samples.
Catalase assay.
B. abortus strains were grown to OD600 1.0 in LB-DIP. Cells were centrifuged, washed, and resuspended in PBS. Suspensions were sonicated, and further centrifuged (20 000 g for 10 min at 4 °C). Aliquots from the supernatants were assayed for catalase activity, which was determined by following the decomposition of 18 mM hydrogen peroxide at 240 nm (Beers & Sizer, 1952). One unit of activity was defined as the amount of enzyme that catalysed the decomposition of one micromole of hydrogen peroxide per minute. Protein concentration was determined as described by Bradford (1976).
Hydrogen peroxide sensitivity assay.
Cells were grown in either LB or LB-DIP to the exponential or stationary phase of growth. Cells were centrifuged (20 000 g for 10 min), washed, and diluted 1 in 10 in PBS. Aliquots from the logarithmic- and stationary-phase suspensions were challenged with 33 and 100 mM hydrogen peroxide, respectively. Assays were carried out at room temperature, without shaking. At different time points, samples were taken, and these were serially diluted, and plated on LB plates. Colonies were counted after 48 h at 37 °C. Survival rates at different time points were determined as percentages of the number of colonies in the original inoculum.
DNA-binding assay.
The ability of the recombinant Irr protein to bind to the upstream dhbCEBA region was determined by an electrophoretic mobility shift assay (EMSA). A 0.62 kb DNA fragment was PCR-amplified with the sense primer (5'-GCTCTAGACCTGTCCCGGCGCAG-3') and the antisense primer (5'-TTCTGCAGTTGCCGCTGGCCGCCA-3'). The PCR product was digested with HindIII, and the 0.24 kb fragment containing the two promoter regions was purified from an agarose gel. Samples containing 0.7 µg of this DNA were mixed with 0, 0.6 or 0.6 nmol of the recombinant Irr in 1x binding buffer (20 mM Tris, pH 7.8, 5 % glycerol, v/v, 1 mM DTT, 50 µg BSA ml–1, and 5 µg salmon sperm DNA ml–1). The samples were then incubated at room temperature for 15 min. When needed, 3 µl polyclonal mouse anti-Irr serum was added to the reaction mix, which was incubated for another 15 min. As a control, a 0.76 kb chromosomal DNA fragment was used; this was amplified by PCR with the sense primer (5'-CGGGATCCTCGTGTTCGCAGGCTCA-3') and the antisense primer (5'-TGCACTGCAGTTCGGGAGGACGAAT-3'). EMSA reactions were analysed on 1.5 % agarose gel in 0.5x Tris/borate/EDTA buffer. After the electrophoresis, the gel was stained with 0.5 µg ethidium bromide ml–1, and visualized under UV light.
Intracellular Brucella survival experiment.
Infection of HeLa and murine-macrophage-like J774 cell lines was performed as previously described (Almirón et al., 2001), with the following modifications: (i) bacterial strains were grown in either LB or LB-DIP; (ii) during infection, no fetal bovine serum was added to the cell medium in order to avoid any possible iron acquisition; and (iii) at 1 h post-infection (p.i.), when non-adherent bacteria had been eliminated, infected eukaryotic cells were incubated with the appropriate medium supplemented with fetal bovine serum.
In vivo experimental infection.
Eight-week-old female BALB/C mice were injected intraperitoneally with 0.1 ml of a bacterial suspension prepared in PBS (about 104 c.f.u. grown in LB-DIP). At 1 and 3 weeks p.i., an excess of ether anaesthesia was administered, and mice were bled to death by cardiac puncture. The spleen was aseptically dissected, weighed, and then homogenized in PBS. The number of viable bacteria was determined by plating serial dilutions on LB agar.
Statistical analysis.
All statistical analysis was performed using Student's two-tailed t test. P values 
0.05 were considered significant. Results are expressed as means±SD.
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RESULTS |
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(a), after 3 days incubation, the mutant B. abortus 2308IK secreted less brucebactin than the wild-type 2308, as judged by the size of the halo. Brucebactin production of the mutant was restored when complemented with the wild-type irr, thus confirming that Irr was responsible for the observed phenotype.
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, the B. abortus wild-type and the complemented strain secreted three- to fivefold more 2,3-DHBA than the mutant B. abortus 2308IK. To compare secretion of the catecholic compounds in the same medium, the relative activity of brucebactin was determined in liquid samples, as described in Methods. In agreement with the above result, the parental strain secreted over twofold more brucebactin than the irr mutant. The relative siderophore activities determined in the supernatants of 2308 and 2308IK from three independent experiments performed in duplicate were 87±1 and 29±6 %, respectively (P<0.05). The mutant phenotype was also reverted by complementation with the wild-type irr gene to give 76±11 % activity, against 1 % obtained in the supernatant of 2308C. Therefore, active Irr increased the amount of catecholic siderophores secreted by B. abortus.
Effect of Irr on the expression of the dhbCEBA operon
In order to determine whether the higher amounts of the secreted siderophores were the consequence of induced transcription of the genes involved in their synthesis, a chromosomal dhbC–lacZ fusion was analysed in the background of the parental and irr mutant strains, 2308C and 2308IC, respectively. The -galactosidase activity of the 2308C strain (1252±140 Miller units) was about twofold higher than that of the strain 2308IC carrying the irr mutation (609±72 Miller units) (P<0.05). These results were obtained from three independent experiments performed in duplicate. No -galactosidase activity was obtained when the experiments were done either with cultures at OD600 <0.4, or with MG cultures supplemented with ferric citrate. Maximal induction of transcription was observed during the stationary growth phase of iron-limited cultures. These data corresponded to the decreased secretion of siderophores in the absence of Irr, suggesting that Irr contributes to positive transcriptional regulation of the operon when cells are growing under conditions of iron limitation.
To investigate the interaction of Irr with DNA, gel mobility shift assays were performed, as shown in Fig. 2. The fragment of DNA containing the two promoters of the dhbCEBA operon was mixed with the recombinant Irr protein, as indicated in Methods. The DNA mobility was retarded in the presence of increasing concentrations of Irr (Fig. 2b, lanes 3 and 4), and also when the anti-Irr serum was added to the reaction mix (Fig. 2b, lane 5). Even though the shift observed in the DNA mobility was slight, it was highly reproducible. This binding seems to be specific for two reasons. First, the non-specific binding of Irr was minimized by the addition of salmon-sperm DNA to the binding buffer. Second, the mobility of a DNA fragment without the ICE motif was not retarded in the presence of the maximal amount of Irr protein used in our experimental conditions (Fig. 2b, lanes 6 and 7). The extent of retardation could not be further improved by use of different binding buffers, native PAGE, or manipulation of the agarose concentration.
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), logarithmic B. abortus 2308 and 2308IK cells were challenged with 33 mM hydrogen peroxide, and stationary-phase cells with 100 mM hydrogen peroxide. As shown in Fig. 3, mutant cells were more resistant than the wild-type when grown in iron-deficient medium. Between 40 and 60 % of the mutant population survived after 35 min treatment when cells were in exponential phase (Fig. 3a), and after 15 min when they were in stationary phase (Fig. 3b). At similar time points, less than 10 % of the wild-type population survived. After 60 min exposure, while both logarithmic and stationary B. abortus 2308 cells could not recover from the imposed stress condition, mutant cells remained viable. Non-viable cells were recovered after 15 min exposure to 33 or 100 mM hydrogen peroxide when the experiment was done with logarithmic- or stationary-phase cells grown in LB medium (data from four independent experiments).
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). Thus, we determined the specific activity of catalase in the B. abortus strains. The B. abortus irr mutant expressed significantly more catalase activity than the wild-type. Values obtained from three independent experiments performed when the cultures reached OD600 1 were 562±85 and 47±5 units mg–1 for 2308IK and 2308, respectively (P<0.05). These data support the survival phenotype shown in Fig. 3.
As catalase is a haemoprotein, and we have previously shown that the B. abortus irr mutant accumulates haem precursors under iron limitation (Martínez et al., 2005), we investigated whether there was a higher level of haem in mutant cells that could account for the higher catalase activity observed. Increases of more than twofold in the haemin intracellular concentration of B. abortus 2308IK were obtained in comparison with 2308 when the cells were grown in MG. The data obtained from three independent experiments were 15.4±3.6 and 40.2±8.1 nM (P<0.05) for the wild-type and the mutant cells, respectively; data obtained from three independent experiments repeated in LB-DIP medium were in agreement with these results: 48.2±1.1 and 80.8±3.1 nM for wild-type and mutant cells, respectively; P<0.05.
Intracellular survival
The capacity to invade and replicate inside HeLa and J774 cell lines was assayed with 2308 and 2308IK grown in iron-deficient and iron-sufficient media, and 2308IK(pBBRirr) grown in iron-deficient medium. Cell invasion of HeLa cells (Fig. 4a) and the macrophage-like J774 cells (Fig. 4b) was similar among the strains tested. However, at 24 h p.i. of bacteria grown in iron-deficient medium, it was observed that while 2308IK was able to replicate inside HeLa cells, 2308 showed a decrease in the number of viable intracellular bacteria. At 48 h p.i., both strains were replicating. As shown in the inset, when bacteria were grown in iron-sufficient medium, this decline was not observed for the wild-type. Thus, to investigate whether the enhanced replication of 2308IK at 24 h p.i. was due to the absence of Irr, 2308IK(pBBRirr) was assayed under the same conditions. This complemented strain behaved in the same way as the wild-type, suggesting that the phenotype was a consequence of the mutation in the irr gene. When J774 cells were infected with 2308 or 2308IK, an initial reduction in the number of viable intracellular bacteria was detected at 10 h p.i. Whilst 2308IK showed replication at 24 and 48 h p.i., the viability of 2308 cells was seriously affected; replication of intracellular 2308 was detected at 48 h p.i. only. Both 2308 and 2308IK behaved similarly when the cells were infected with bacteria grown in iron-sufficient medium (inset). The complemented 2308IK(pBBRirr) strain was able to invade J774 cells; however, we were not confident of the accuracy of data obtained, since the infected cells were unstable. We are currently unable to explain the reason for this behaviour.
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DISCUSSION |
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). In this work, we demonstrate that B. abortus Irr upregulates siderophore production in B. abortus under conditions of iron limitation. The B. abortus irr secreted significantly less brucebactin than the wild-type strain. Further, detection of 2,3-DHBA from iron-limited bacterial cultures revealed that secretion of this siderophore was also reduced in the mutant. The deficiency in siderophore secretion in the mutant was restored by genetic complementation with a plasmid harbouring wild-type irr, indicating the involvement of Irr in the higher amounts of siderophores that accumulated in the extracellular medium of the wild-type cells. This could be the result of an induction in the biosynthesis of the catechols, and/or an induction of unknown secretion machinery. When the transcription of the B. abortus dhbC was investigated, gene expression was decreased about twofold in the irr mutant compared with the wild-type under conditions of iron limitation. Therefore, Irr positively regulates 2,3-DHBA and brucebactin production; however, these results do not exclude regulation of secretion. Since the factor by which the secretion of the siderophores was affected was higher than that for transcription alone, it can be supposed that secretion was also induced. At this point, it must be considered that transcription and secretion are two different processes that do not necessarily respond with equal intensity to the same regulation.
In general, iron-uptake systems have been reported to be negatively regulated by Fur-like proteins that use ferrous ion as a cofactor. Thus, when iron is scarce, those regulated genes are de-repressed (Wandersman & Delepelaire, 2004). The dhbCEBA operon is expressed under conditions of iron limitation from two promoters containing Fur boxes (Bellaire et al., 2003); no evidence of its repression has yet been described. However, the fact that siderophores were secreted in the absence of the positive regulation in the irr mutant strongly suggests that the dhbCEBA operon is under dual regulation: it is repressed by an unknown regulator in the presence of iron, and induced by Irr in the absence of this metal. Using a gel retardation assay, we demonstrated that Irr is able to bind the upstream region of the operon. This region contains two putative Irr boxes (ICE motifs) (Rudolph et al., 2006), which are located just overlapping each of the two –10 promoters described in the dhbCEBA operon. The DNA–Irr complexes were better resolved from naked DNA in agarose than they were in native polyacrylamide gels, possibly because they were large assembled complexes. In this regard, it must be kept in mind that Irr is able to dimerize in vitro, and the target DNA used in our gel mobility retardation assays presents two putative DNA-binding motifs. However, further analyses are needed to identify the DNA sequence that interacts with Irr, and whether it binds DNA as a dimer.
Irr is also implicated in oxidative stress resistance. While almost no B. abortus wild-type cells were recovered after exposure to hydrogen peroxide, more than 40 % of B. abortus irr cells survived this oxidative stress, regardless of the physiological iron-deficient bacterial state. The B. abortus irr showed higher catalase activity than the wild-type. B. abortus catalase is transcriptionally regulated by OxyR, increasing its synthesis in response to hydrogen peroxide (Kim & Mayfield, 2000; Kim et al., 2000). Beyond the induction of the gene, its product needs haem as a cofactor to be an active enzyme. Thus, the oxidative stress resistance of the irr mutant may be the result of extra haem molecules available to facilitate rapid formation of haemoproteins. It is also possible that Irr regulates the expression of catalase. In this regard, Irr could be a functional homologue of Bacillus subtilis Per (peroxide regulon regulator), which is a Fur-like protein that regulates both katA and a haem biosynthesis enzyme (Bsat et al., 1998). Similarly, a Campylobacter jejuni perR mutant has been described as being hyper-resistant to oxidative stress, with the katA and the ahpC genes being under derepression (van Vliet et al., 1999; Harvie et al., 2005). Per does not seem to be encoded in the genomes of B. abortus (Halling et al., 2005; Chain et al., 2005), Brucella melitensis (DelVecchio et al., 2002) and Brucella suis (Paulsen et al., 2002). A BLAST search (Altschul et al., 1990) using these translated genomes showed sequence homologies with very low values with a protein that belongs to the Irr family (Martínez et al., 2005), and with Fur. On the other hand, Irr is not present in Bac. subtilis or C. jejuni. In contrast, Bac. subtilis Per does not participate in the regulation of the Bacillus catecholic siderophores, while Bacillus Fur does (Chen et al., 1995).
The pathogenicity of Brucella spp. is based on their ability to survive and replicate inside macrophages. As one of the main bactericidal mechanisms used by macrophages is to produce hydrogen peroxide (Jiang & Baldwin, 1993), it can be expected that a B. abortus strain with high catalase activity will offer more resistance to being killed by the macrophage-like J774 cells. The results presented here support this hypothesis: the B. abortus irr showed a better adaptation to the intracellular environment, recovering sooner than the wild-type from the initial loss of viability. The mechanisms against bacteria used by non-phagocytic cells are different; nonetheless, the irr mutant was able to successfully replicate during the first 24 h p.i. Wild-type cells lost viability during the same period; nevertheless, they replicated to the same extent as the irr mutant cells. It is noteworthy that the infection of HeLa and J774 cell lines with wild-type iron-starved B. abortus in the absence of fetal bovine serum was less efficient than when the cell lines are infected in non-limiting conditions (Almirón et al., 2001); nonetheless, the invasion capability of the bacteria was not affected.
Previous reports have shown that the absence of catalase does not attenuate the virulence of B. melitensis in goats (Gee et al., 2004) or mice (Sangari & Aguero, 1996). In a different way, 2,3-DHBA is not required for B. abortus virulence in mice (Bellaire et al., 1999), while it is considered a virulence factor in its natural host (Bellaire et al., 2003). Here, we present evidence that the combined effects of an increment in catalase activity and haem content, together with a decline in siderophore synthesis, do not alter the virulence of B. abortus in mice. This observation suggests that the observed in vitro phenotype of the irr mutant is not relevant in this animal model.
When the results are analysed together, it can be observed that differences between the B. abortus wild-type and B. abortus irr phenotypes are evident after short time periods, as if they are reflecting a delay in the wild-type response to the imposed stress condition. This lag may correspond with the time needed for B. abortus to increase its haem level through iron uptake and haem biosynthesis, or through haem-uptake systems. Once the goal of increasing the haem level is achieved, the stability of Irr is affected (Martínez et al., 2005; Qi & O'Brian, 2002; Qi et al., 1999). Thus, the wild-type phenotype in the iron-limited condition will resemble that of the irr mutant, resulting in an improved stress response.
To conclude, Irr directly induces iron assimilation to ensure haem biosynthesis in B. abortus. Haem biosynthesis is essential for the intracellular survival of this animal pathogen (Almirón et al., 2001). In a similar way, Bradyrhizobium japonicum Irr coordinates high-affinity iron-uptake systems with the synthesis of haem, a molecule indispensable to efficiently nodulate soybeans (Hamza et al., 1998). Whilst Brucella and Bradyrhizobium belong to the -2 Proteobacteria, and Irr is confined to this group, it is possible that the regulation exerted by Irr represents a common strategy for intracellular adaptation among plant symbionts and animal pathogens (LeVier et al., 2000; Ugalde, 1999).
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ACKNOWLEDGEMENTS |
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Received 19 December 2005;
revised 31 May 2006;
accepted 31 May 2006.
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) and 2308IK (
) cells were grown in LB-DIP, and treated with 33 mM during the exponential phase of growth (a), and with 100 mM hydrogen peroxide during stationary phase (b). The number of c.f.u. ml–1 recovered at different time points was related to the initial number of viable cells (about 1x108 c.f.u. ml–1), and expressed as a percentage. Data are means±SD of three independent experiments. *P<0.05.
) at an m.o.i. of 100 after growth under iron limitation. At different times p.i., eukaryotic cells were lysed, and the number of viable intracellular bacteria was determined. The inset shows, using the same units, the infection with B. abortus 2308 and 2308IK grown without iron limitation. (b) J774 cells were infected at an m.o.i. of 50 with B. abortus 2308 (