East Carolina Brody University School of Medicine, Dept of Microbiology and Immunology, 600 Moye Blvd, Greenville, NC 27858-4354, USA
Correspondence
Edson R. Rocha
rochae{at}mail.ecu.edu
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ABSTRACT |
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INTRODUCTION |
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Ferritin has the capacity to accommodate a large amount of iron, about 30004000 iron atoms per molecule, which is important for its role in linking cellular iron metabolism and oxidative stress homeostasis (Touati, 2000; Arosio & Levi, 2002
; Andrews et al., 2003
; Theil, 2003
). In bacteria, this link is well studied in Escherichia coli FtnA. By scavenging intracellular free iron, FtnA stores about 50 % of total cellular iron upon entry into stationary phase and it protects against Fe(II)-mediated formation of hydroxyl radicals via Fenton chemistry (Abdul-Tehrani et al., 1999
; Touati, 2000
). This is accomplished through the ferrooxidase centre, which is essential for iron incorporation (Arosio & Levi, 2002
; Andrews, 1998
). Iron stored in FtnA may become available for metabolism when E. coli is shifted from iron-rich to iron-limiting conditions (Abdul-Tehrani et al., 1999
). In this regard, iron-storage proteins are generally synthesized in response to iron-replete conditions in order to prevent excess accumulation of intracellular iron and simultaneously there is a down-regulation of the high-affinity iron uptake mechanism. The ferric uptake regulator, Fur, which uses Fe(II) as corepressor, is the primary regulator controlling iron metabolism (Ratledge & Dover, 2000
; Andrews et al., 2003
).
In anaerobic bacteria little is known about metal/oxidative stress interactions and little consideration has been given to the fact that iron in the anaerobic environment is presumed to be present in the reduced form as Fe(II). This is probably because free Fe(II) can predominate over Fe(III) in the absence of oxygen, and being soluble even at neutral pH it is likely to be readily available. Furthermore, the absence of oxygen lowers the risk of toxic radical generation. Nevertheless, this may be an oversimplification since a ferritin-like homologue which harbours iron under anaerobic conditions is present in Bacteroides fragilis (Rocha et al., 1992). Recently, a ferritin from the closely related periodontal pathogen Porphyromonas gingivalis and a bacterioferritin from the sulfate-reducing bacterium Desulfovibrio desulfuricans have been cloned and characterized (Ratnayake et al., 2000
; Romao et al., 2000
). This provides evidence that mobilization and storage of non-haem iron by anaerobic bacteria may be more complex than previously thought. It has been assumed that in anaerobic organisms ferritins are an important component of an oxygen defence mechanism that protects against the deleterious effect of uncontrolled ferrous iron oxidation (Rocha et al., 1992
; Romao et al., 2000
). Moreover, the presence of both oxygen-detoxifying enzymes and iron-storage mechanisms in anaerobes is essential for their survival in an adverse oxygen-rich environment (Romao et al., 2000
; da Costa et al., 2001
).
When shifted from anaerobic to aerobic conditions, the growth of Bacteroides spp. is immediately halted and the bacteria enter a stationary-phase-like mode (Rocha et al., 1996, 2003
; Pan & Imlay, 2001
). Simultaneously, an extensive response is immediately induced, and a significant shift in protein expression patterns has been shown to be important for protection and survival under aerobic conditions (Rocha et al., 1996
). Control of this complex oxidative stress response is not yet fully understood but an important component of it, the peroxide-inducible response, has been shown to be regulated by the redox transcriptional regulator OxyR (Rocha et al., 2000
). OxyR-independent oxygen-dependent control of the response also has been demonstrated, indicating that there are additional levels of control of the oxidative stress response (Rocha et al., 2003
). In aerobic and facultative bacteria, it has been shown that the control of iron metabolism and the oxidative stress response are linked via a number of transcriptional regulators (Chen et al., 1995
; Bereswill et al., 2000
; Touati, 2000
; Massé & Gottesman, 2002
; Fong et al., 2003
) but the influence and role of iron in the B. fragilis oxidative-stress response has not been given much attention. Iron transport and mobilization in the cell are absolutely key to controlling the levels of free iron and subsequent damage from oxygen radicals. It is known that B. fragilis responds to iron limitation with an iron-induced expression of several proteins on the cell surface (Otto et al., 1988
) and it produces an iron-storage protein, ferritin, that can incorporate radiolabelled iron in vivo (Rocha et al., 1992
). Thus it appears that B. fragilis has a well-developed iron-responsive regulatory network, but this system has not yet been elucidated. In this study we present data showing that B. fragilis ferritin, FtnA, is up-regulated at the transcriptional level in the presence of excess iron in an oxidative environment but not in reduced anaerobic conditions, and that the peroxide regulator OxyR is involved in B. fragilis ferritin regulation.
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METHODS |
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Cloning, DNA sequencing and construction of ftnA insertion mutant.
All DNA modifications and manipulations were carried out according to standard protocols (Sambrook et al., 1989). To amplify the B. fragilis ftnA homologue the following two oligonucleotide primers were designed based on conserved amino acid sequence residues of bacterial ferritins available from GenBank. The sense and antisense oligonucleotide sequences are as follows: 5'-GAR CAR ATH WSI GCI GAR ATG TGG-3' and 5'-YTC YTC YTC IWS YTG YTC-3'. A 375 bp fragment was then amplified from the chromosome by Taq polymerase using a PCR amplification kit. The amplified fragment was extracted from an agarose gel and ligated into the HincII site of the suicide vector pFD516 (Smith et al., 1995
), resulting in pFD775. The new construct, pFD775, was mobilized from E. coli DH10B into B. fragilis 638R (Privitera et al., 1979
) by aerobic triparental filter mating protocols (Shoemaker et al., 1986
). The transconjugants were selected on BHIS agar plates containing 20 µg rifampicin ml1, 100 µg gentamicin ml1 and 10 µg erythromycin ml1. The B. fragilis ftnA : : pFD516 insertion mutant, strain IB335, was subjected to Southern blot hybridization analysis to confirm the single cross-over disruption of the target gene. A dps oxyR double mutant strain was constructed by mobilizing pFD759 from E. coli DH10B into B. fragilis 638R by triparental mating protocol as described above. pFD775 was inserted into the oxyR and dps oxyR strains by triparental mating to create oxyR ftnA and dps oxyR ftnA mutant strains respectively. The strategy to isolate the entire ftnA gene region was to rescue the suicide vector pFD775 from the chromosome of strain IB335 using procedures previously described (Rocha & Smith, 1999
). Automated nucleotide sequencing was performed on double-stranded DNA templates (Molecular Biology Resource Facility, University of Tennessee, Knoxville). Additional oligonucleotide primers were designed based on available sequence information to extend and confirm existing sequence. Computer analysis of nucleotide and amino acid sequence data was performed using the University of Wisconsin Genetics Computer Group (GCG) DNA sequence analysis software version 10.3 (Devereux et al., 1984
; Accelrys Inc.).
RNA extraction, Northern blot hybridization and primer extension.
Total RNA extraction and Northern blot analysis of mRNA were carried out as previously described (Rocha & Smith, 1997) and an internal fragment of ftnA was used as specific probe. Densitometry analysis of the autoradiograph was normalized to the relative intensity of total 23S and 16S rRNA detected on the ethidium-bromide-stained agarose gel to correct for any loading differences.
Primer extension analysis was performed on total RNA obtained from mid-exponential-phase cells of B. fragilis 638R grown anaerobically and then subjected to oxidative stress conditions as described previously (Rocha & Smith, 1997). ftnA-specific oligonucleotides, 5'-GCTTCTTCATCCAGTGTGC-3' complementary to nucleotides 117135 and 5'-GGCATAAGCATGCCCCATCTCTTCC-3' complementary to nucleotides 143 to 167 of the ftnA coding region, were used to map the 5'-end of the extended product. The oligonucleotides were labelled with [
-32P]ATP and used as primers for the reverse transcriptase reaction as described previously (Rocha & Smith, 1997
). The extended labelled products were electrophoresed on 8 % polyacrylamide gels containing urea. A nucleotide sequence ladder was prepared with Sequenase 2.0 (USB) using a template covering the transcription start site region, with the same oligonucleotide that was used for the reverse transcription reactions.
Construction of ftnA' : : xylB transcriptional fusions.
A 300 bp fragment encompassing 249 bp upstream and 51 bp downstream of the ftnA translational start codon was PCR amplified using High-fidelity Taq DNA polymerase with the oligonucleotides 5'-GCGTGGGGATCCGATAGCC-3' and 5'-CTCGGCTGAATTCTGCTCG-3', containing restriction sites for BamHI and EcoRI (in italic) respectively. The 300 bp BamHI/EcoRI fragment was cloned into the BamHI/EcoRI sites of pFD700 (Rocha et al., 2000) and the 1·2 kb EcoRI fragment from a promoterless
-xylosidase/
-arabinosidase bifunctional reporter gene (Whitehead, 1997
) was cloned into the unique EcoRI site of the new construct pFD812. Plasmid pFD812 was mobilized from E. coli DH10B into B. fragilis strains by aerobic triparental filter mating as mentioned above and integrated into the B. fragilis chromosome bglA locus (Rocha et al., 2000
).
Enzyme assays.
The -xylosidase assays in crude extracts were performed using p-nitrophenyl
-D-xylopyranoside as substrate as previously described (Rocha et al., 2000
). One unit of
-xylosidase is the amount of enzyme which releases 1 µmol p-nitrophenol min1 at 37 °C.
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RESULTS |
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ftnA regulation under anaerobic and aerobic conditions
To investigate the effect of iron availability on the expression of ferritin, Northern hybridization analysis was performed with RNA extracted from mid-exponential-phase cultures grown under iron-replete and iron-limiting conditions. Anaerobic cultures grown under the iron-limiting conditions showed a lower growth rate than the iron-replete cultures (Fig. 2b). ftnA was transcribed as a monocistronic message of approximately 600 nt. Under anaerobic conditions, the basal level of ftnA mRNA was not dependent on the ferrous iron content in the growth medium (Fig. 2a
). In contrast, ftnA mRNA was induced nearly 10-fold following oxygen exposure in iron-replete conditions compared to anaerobic culture controls. However, in iron-limiting cultures, the induction of ftnA mRNA by oxygen was lower (about fourfold) compared to anaerobic culture controls. This suggests that the oxidative stress induction of ftnA mRNA induction following a shift of the culture from anaerobic to aerobic conditions is dependent on the effect of oxygen and Fe(II) in concert. The effect of the H2O2 was less pronounced than that of oxygen exposure, possibly due to the decomposition of H2O2 by Fe(II) in the culture medium (Abdul-Tehrani et al., 1999
).
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Free thiol groups play an important metabolic role in anaerobic bacteria, and anaerobic oxidation of thiols by diamide mimics the effect of oxygen on these organisms (Morris, 1975). Thus, to investigate the participation of thiol oxidation in the regulation of ftnA mRNA expression, cultures were treated with diamide, and the ftnA mRNA levels were analysed by Northern hybridization. In the absence of oxygen, ftnA expression was induced following diamide treatment in a dose-dependent manner up to 500 µM (Fig. 4
). There was about twofold induction when cultures were treated with 50 µM diamide, fivefold after addition of 100 µM diamide and 12-fold after treatment with 500 µM diamide compared to anaerobic untreated control culture.
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DISCUSSION |
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In facultative and aerobic bacteria control of iron metabolism and the oxidative stress response are linked via several transcriptional and post-transcriptional regulators (Chen et al., 1995; Bereswill et al., 2000
; Horsburgh et al., 2001
; Hirosue et al., 2001
; Massé & Gottesman, 2002
). In E. coli, a Fur-regulated small RNA, RyhB, controls the expression of FtnA and Bfr (Massé & Gottesman, 2002
) while in Helicobater pylori, Fur activates the expression of Pfr (ferritin) by binding directly to the pfr promoter sequence (Bereswill et al., 2000
; Delany et al., 2001
). Recently, the dual ferritins genes, ftnAB, of Actinobacillus actinomycetemcomitans were shown to be induced by oxygen with complex transcriptional regulation controlled by the aerobic/anaerobic sensor ArcB, the quorum-sensing autoinducer LuxS (Fong et al., 2003
) and possibly FNR, an oxygen-sensing transcriptional regulator of aerobic and facultative bacteria (Hirosue et al., 2001
). The importance of a co-integrated regulation of iron homeostasis and oxidative stress has been demonstrated in vivo for Staphylococcus aureus. The Fur-like repressor of the peroxide response, Per, regulates the iron-storage protein ferritin in addition to several other oxidative stress proteins (Horsburgh et al., 2001
). In this report we showed that the B. fragilis oxidative stress response is also linked to iron metabolism through the thiol/disulfide redox regulator of the peroxide response, OxyR. However, we believe that the OxyR-mediated control is in fact indirect, since analysis of the ftnA promoter region did not show an OxyR nucleotide-binding motif sequence present in the promoter regions of B. fragilis peroxide-response genes ahpCF, katB, dps, tpx, rbr-2 and rpbA (data not shown). Perhaps the effect of OxyR on ftnA expression occurs via its interaction with other regulatory mechanisms involved in iron homeostasis, as has been demonstrated to occur in E. coli (Zheng et al., 1999
).
Although the E. coli ftnA mutant has no apparent redox-stress sensitivity, a strong indication that disturbing iron homeostasis leads to increased sensitivity to oxidative stress was demonstrated in an ftnA fur double mutant, which is more sensitive to hydroperoxides than are the single mutant strains (Abdul-Tehrani et al., 1999). In addition, overexpression of FtnA compensates for the deleterious effect of fur deletion (Touati, 2000
). In B. fragilis we also found that a mutation in the ftnA gene alone showed no loss of viability upon oxygen exposure, in agreement with data reported for ftn and dps ftn double mutants in the closely related organism P. gingivalis (Ueshima et al., 2003
). However, a role for iron detoxification during aerotolerance in B. fragilis was demonstrated when an ftnA mutation in conjunction with a disruption in the dps and oxyR genes amplified the sensitivity of this anaerobe to oxygen exposure (Fig. 7
). Additional evidence that ferritin or bacterioferritin participate in protection against iron-mediated oxygen radical cellular damage was obtained in other bacteria such as H. pylori, Campylobacter jejuni and Neisseria gonorrhoeae, where disruption of the iron-storage mechanism increased bacterial sensitivity to oxidative stress in vitro (Wai et al., 1996
; Chen & Morse, 1999
; Waidner et al., 2002
) and the ability to colonize tissues in vivo (Waidner et al., 2002
).
Ferritin isolated from B. fragilis grown under anaerobic conditions contains about three iron atoms per molecule (Rocha et al., 1992). The bacterioferritin from the strict anaerobe D. desulfuricans contains two iron atoms per monomer (Romao et al., 2000
). This low iron content does not correlate with their role in iron-storage but it is presumed that they may play a role in detoxification during transient exposure to oxygen (Rocha et al., 1992
). This may be related to the fact that in anaerobic conditions, ferrous iron is readily available and toxicity of excess iron is likely not a problem. On the other hand, in the presence of oxygen, free ferrous iron will be unavoidably converted to its ferric form, with potential toxicity through generation of oxygen-derived cell-damaging free radicals. Thus induction of ferritin synthesis upon oxygen exposure correlates with the need for B. fragilis to maintain excess iron in a non-toxic form.
In conclusion, this study presents evidence that regulation of B. fragilis ftnA expression occurs at the transcriptional level. In addition, it was demonstrated that iron, oxygen and thiol oxidation are involved in the modulation of B. fragilis ftnA expression; however, the molecular mechanisms involved in the transcriptional regulation are not yet known and will be the subject of further investigation. This study confirms and adds to previous reports indicating that the oxidative stress response in this aerotolerant anaerobe involves a complex genetically regulated response.
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ACKNOWLEDGEMENTS |
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Received 28 November 2003;
revised 8 March 2004;
accepted 26 March 2004.
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