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ch Sedlá
ek1era1
1 Department of Biochemistry, Faculty of Science, Masaryk University, Czech Republic, CZ-611 37 Brno, Czech Republic
2 Department of Molecular Cell Physiology, Faculty of Earth and Life Science, VU University Amsterdam, NL-1081 HV Amsterdam, The Netherlands
Correspondence
Igor Kuera
ikucera{at}chemi.muni.cz
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). However, due to a relatively high chemical reactivity of Fe(III) complexes, it is often difficult to establish a unique inter-relationship between iron assimilation on the one hand and the Fe(III)-reducing activity of a particular enzyme on the other. The situation seems relatively clear-cut for systems where the reductive step precedes the import of iron into cells. Saccharomyces cerevisiae, for example, uses two externally directed, plasma-membrane ferric reductases, FRE1 and FRE2, to generate ferrous iron that can be accessed by the uptake proteins (Dancis et al., 1990; Georgatsou & Alexandraki, 1994). Deletion of the FREI and FRE2 genes from the yeast genome not only completely abolishes membrane-associated ferric reductase activity, but also renders the cell incapable of growing for an extended period of time in iron-deficient media. Regarding cytoplasmic Fe(III) reductases, only one analogous observation has been reported to date. FhuF, a cytoplasmic 2Fe–2S protein of Escherichia coli, has been shown to be indispensable for growth on ferrioxamine B as the sole iron source. FhuF in its [Fe2+–Fe3+] state can directly reduce ferrioxamine B-bound Fe(III); this suggests its function as a specific reductase for this type of siderophore (Matzanke et al., 2004; Muller et al., 1998).
Paracoccus denitrificans is a Gram-negative, strictly respiring, facultative anaerobic bacterium that is found in soil, sewage and sludge (for a review on its metabolism and genetics, see Baker et al., 1998). When the cells experience iron deficiency, they produce a mixture of catechol-containing compounds (Tait, 1975). One of them, now called parabactin, was identified as N-[3-(2,3-dihydroxybenzamido)propyl]-N-[4-(2,3-dihydroxybenyamido)butyl]-2-(2-hydroxyphenyl)-trans-5-methyloxazoline-4-carboxamide (Person & Neilands, 1979). Fe(III) from the environment binds to parabactin via the four oxygens of the two catechol groups and two additional ligands (N and O) associated with the 2-(2-hydroxy)oxazoline group. The ferric parabactin–chelate complex formed then targets to a highly specific receptor in the outer membrane (Bergeron et al., 1988). After internalization, further intracellular processing may involve hydrolysis of the parabactin oxazoline ring to give an open structure with a lower affinity for Fe(III) and/or enzymic reduction of Fe(III) to Fe(II) (Robinson & McArdle, 1981; Tait, 1975). It should be noted that P. denitrificans also acquires iron independently of siderophore production via a poorly defined low-affinity uptake pathway (Wee et al., 1988).
P. denitrificans possesses two soluble enzymes, FerA and FerB, capable of reducing a number of Fe(III) complexes at the expense of NADH (Mazoch et al., 2004). Both of them interact with flavins, but in a different manner. FerA can be classified as an NADH : flavin oxidoreductase with a sequential reaction mechanism. It requires the addition of FMN or riboflavin for activity on Fe(III) substrates. In these reactions, the apparent substrate specificity of FerA seems to originate exclusively from different chemical reactivities of Fe(III) compounds with the free reduced flavin produced by the enzyme. In contrast to FerA, FerB contains a non-covalently bound redox-active FAD coenzyme, uses NADPH as well as NADH, does not reduce free FMN, and displays a ping-pong type kinetic pattern with NADH and Fe(III)-nitrilotriacetate as substrates.
In the present study we have cloned and characterized the genes encoding FerA and FerB and have undertaken phenotypic analyses of strains mutated in these genes in order to get a fundamental understanding of their physiological role in bacterial iron metabolism.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. P. denitrificans strains were grown in Brain Heart Infusion (BHI) broth (Oxoid) or in minimal medium (Tait, 1975) containing 50 mM succinate. When appropriate, the media were rendered iron-deficient by the addition of 2,2'-bipyridyl at a minimum concentration that still fully prevented bacterial growth without an externally added iron source. For cultivation of Escherichia coli strains, Yeast-tryptone (YT) broth (Oxoid) was used. Selection for resistance to antibiotics was performed with rifampicin (20 or 80 µg ml–1), kanamycin (25 µg ml–1), streptomycin (25 µg ml–1) and tetracycline (12.5 µg ml–1) (all Sigma).
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Preparation of ferric parabactin.
Parabactin was isolated from cell-free supernatants of P. denitrificans cultures by extraction with ethyl acetate (Neilands, 1983). The 1 : 1 Fe(III) complex was formed by mixing a 40 mM solution of parabactin in 1 M NaOH with an equal volume of 40 mM FeCl3 in 1 M HCl.
PCR and DNA sequencing.
Oligonucleotide primers (Table 2) were designed using Oligo 4.0 (MBI) and synthesized commercially by Invitrogen (via KRD, Prague, Czech Republic). Primers were based on sequence data from the P. denitrificans Pd1222 sequencing project (http://genome.ornl.gov/microbial/pden/). Standard PCR amplifications utilized Taq DNA polymerase (TopBio). Successful PCR amplification and digestion by restrictases were confirmed by agarose gel electrophoresis. DNA sequencing was carried out commercially by Genomac.
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). Restriction endonucleases and T4 ligase were from New England BioLabs. Purification of DNA following PCR or restriction was achieved using the QIAEX II Gel Extraction kit (Qiagen). Plasmid DNA was purified from E. coli using the QIAprep Spin Miniprep kit (Qiagen). Chemically competent E. coli TG1 cells were transformed by heat-shock in accordance with the manufacturer's instructions.
Construction of ferA and ferB mutant strains.
Internal 350 bp fragments of the ferA and ferB genes were PCR-amplified using Taq DNA polymerase with the primer pairs 1/3 and 2/4 (Table 2
). The resulting fragments were ligated into pGEM-T Easy and these constructs were then transferred to E. coli. Once the identity of the insert had been confirmed by sequencing across the junction sites, the BamHI–SacI fragment was excised and cloned into the BamHI/SacI sites of suicide vector pRVS3 (van Spanning et al., 1995). The resulting plasmids pRVS3121 (for inactivation of ferA) or pRVS3021 (for inactivation of ferB) were mobilized from E. coli TG1 to Pd1222 via triparental mating using E. coli HB101(pRK2020) as the helper strain (de Gier et al., 1994). Clones of P. denitrificans that inserted the suicide vector successfully into their chromosome by homologous recombination were selected on medium with rifampicin and kanamycin. The mutant strains obtained in this way were checked for correct integration of the respective vectors by PCR using primer pairs 1/9 and 7/9 for ferA inactivation, and 3/10 and 8/10 for ferB inactivation.
Construction of a complemented strain of the ferA mutant.
The ferA gene was amplified with primers 5 and 6 (Table 2). The 800 bp product of this amplification was digested with HindIII and BamHI, and cloned into broad-host-range vector pEG400 (Gerhus et al., 1990), yielding pEG4122. Plasmid pEG4122 was transferred from E. coli TG1 to the ferA mutant strain Pd20121 via triparental mating. The resulting strain was designated Pd20122. As a control, the same plasmid was also transferred to the wild-type strain Pd1222, resulting in strain Pd12122.
Construction of promoter-lacZ fusions.
DNA regions upstream of the coding sequences of the ferA and ferB genes were PCR-amplified using Taq DNA polymerase with the primer pairs 11/13 and 12/14, respectively (Table 2). The fragments obtained were ligated into the pGEM-T Easy vector (Promega) and transferred to E. coli TG1. The EcoRI–BamHI fragments of the pGEM derivatives were then excised and inserted into the EcoRI/BamHI-restricted vector pBK11 (Kessler et al., 1992). The resulting promoter-lacZ reporter vectors, designated pPr121 and pPr021, were individually transferred into P. denitrificans Pd1222 via triparental mating. Clones of P. denitrificans that inserted the vector successfully into their chromosome were selected on plates with rifampicin and streptomycin. Colonies were checked for proper integration of the promoter-lacZ fusion by site-specific PCR using the forward primers 11 or 12 in combination with the reverse primer 15, specific for the lacZ gene. The strains kept for further studies were designated Pd30121 and Pd30021.
Growth kinetics.
Growth parameters of P. denitrificans strains were quantified using a Bioscreen Microbiological Growth Analyser (Bioscreen C; Labsystems). Overnight cultures in BHI were inoculated 1 : 300 in triplicate into microtitre plates containing 0.3 ml of appropriate medium. The plates were then sealed and incubated for 5 days at 30 °C with continual shaking. The optical density at 600 nm was measured every 20 min. The lag phase duration and specific growth rates were calculated from plots of log(OD600) versus time, using a method similar to that described by Warringer & Blomberg (2003).
Assays.
Ferric reductase activity was measured spectrophotometrically in 25 mM Tris/HCl, pH 7.4, at 30 °C, with either 0.15 mM NADH or NADPH as electron donor, 0.2 mM ferric nitrilotriacetate as electron acceptor, and 0.8 mM ferrozine as a chromogenic Fe(II)-chelating agent (Mazoch et al., 2004). Additionally, where indicated, 0.05 mM FMN was also included. β-Galactosidase activity was assayed by the method described by Miller (1972). The concentration of catecholate-derived compounds was determined by Arnow's assay (Arnow, 1937) using 2,3-dihydroxybenzoic acid as a standard. Total protein was determined by the Lowry method.
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RESULTS |
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) were used as probes to search the P. denitrificans strain Pd1222 genome database (http://genome.ornl.gov/microbial/pden/). Significant matches (90 and 96 %) were found to the putative coding regions 2689 and 4071, respectively. At the predicted N terminus of the product from the 546 bp gene fragment, the FerA sequence is preceded by a methionine, which is probably cleaved off after translation. The mature protein is expected to be 180 aa long and have a relative molecular mass (Mr) of 18 898 and a pI of 6.2. This corresponds well with our earlier experimental estimates of Mr (18 814 and 18 917) and pI (6.9). The FerB protein encoded by the 549 bp gene fragment starts with a sequence identical to that of the published FerB, and appears to consist of 182 aa residues, has an Mr of 20 222 and a pI of 6.5. Again, these values are in agreement with those found previously for the purified FerB protein (20 196 and 5.5, respectively). Minor differences may be due to the employment of a different strain of P. denitrificans compared to the original source for ferric reductase purification.
The genetic organization of the regions surrounding the ferA and ferB genes is shown in Fig. 1. ferA is located downstream of three ORFs, two of which (ORF2 and ORF3) are predicted to encode inositol monophosphatase and a modulator of DNA gyrase, i.e. proteins with no obvious relationship to iron metabolism. Inspection of the 5' region of ferA revealed the sequence 5'-TTGATG-N17-TAGAAA-3' that conforms closely (8 of 12 bases) to the canonical E. coli 
70 motif 5'-TTGACA-N(15–19)-TATAAT-3' (Hawley & McClure, 1983). This finding may indicate that ferA has its own promoter. All ORFs located in the vicinity of ferB have the opposite transcriptional polarity, arguing against ferB being a part of an operon. Upstream of the ferB gene, only a Shine–Dalgarno sequence (5'-AGGAGA-3') was apparent from –7 to the translation start; no consensus sequences typical for E. coli promoters were identified at the appropriate positions.
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Disruption of the ferric reductase genes
The available nucleotide sequences allowed us to design primers specific for inner parts of the ferric reductase genes (primer pairs 1/2 and 3/4; see Table 2). These were then used in PCRs with genomic DNA of P. denitrificans Pd1222 as template to generate internal ferA and ferB fragments for the directed plasmid integration procedure outlined in Methods. The cell extracts of ferA (Pd20121) and ferB (Pd20021) mutant strains were tested for ferric reductase activity with Fe(III) nitrilotriacetate as electron acceptor, and NADH or NADPH as electron donors (Table 3). In the extract of the ferA mutant strain, both NADH and NADPH supported the reaction, but FMN had no effect, clearly demonstrating the absence of FerA. In the extract of the ferB mutant strain, the reaction rates with NADH or NADPH were about half compared to the control, and there was an approximately threefold further stimulation of NADH-dependent activity upon addition of 50 µM FMN. To confirm that the activity in the absence of FMN was not due to residual FerB, the extracts from the wild-type cells and from the mutant were chromatographed on a HP10/10 Mono Q column (Pharmacia) with a salt gradient from 0 to 1 M NaCl. While the elution profile for Fe(III) reductase activity of the wild-type cell extract consisted of a peak corresponding to FerA (elution at 0.05–0.15 mM NaCl) and a peak corresponding to FerB (elution at 0.25–0.35 mM NaCl), the latter peak was missing from the extract of the ferB mutant. In summary, these data confirm that both insertional inactivations had resulted in the exclusive inability to express FerA (in the ferA mutant strain) or FerB (in the ferB mutant strain).
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). A full growth response of the ferA mutant strain was observed at a concentration of ferric sulfate that was almost 10-fold higher as compared to the wild-type and ferB mutant strains. At subsaturating initial concentrations of Fe, the growing wild-type and ferB mutant cells excreted catechol-containing compounds in amounts up to about 30 µM, whereas only traces of catechols were detectable in culture medium of the FerA-deficient cells. Differences in utilization of various iron sources are shown in Table 4. The ferA mutant strain failed to grow in the presence of 50 µM ferric 2,3-dihydroxybenzoate, and grew only poorly on other ferric complexes of the same concentrations. Contrary to what was seen in the synthetic medium (Fig. 2, Table 4), the growth characteristics of both mutant strains growing in brain heart infusion (BHI) broth were indistinguishable from those of the wild-type strain (results not shown).
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, Table 4). In confirmation of the expected expression of active FerA, the total NADH-linked ferric reductase activity in the cell-free extract from the complemented ferA mutant cells became more responsive to further stimulation by FMN (Table 3).
Complementation of ferA : : Kanr with the ferA gene expressed in trans had to be performed in the presence of streptomycin to maintain the pEG4122 plasmid. Since the antibiotic itself may adversely affect cellular function even in resistant strains, it is not very meaningful to directly compare physiological responses of the complemented mutant strain with those observed for the wild-type strain when streptomycin was absent (Table 4). To make this comparison as valid as possible, we introduced pEG4122 also into the original, unmutagenized strain Pd1222. The resulting strain, Pd12122, grew on medium containing streptomycin with kinetics virtually identical to that of the complemented ferA mutant strain (data not shown). Taken together, these complementation data clearly demonstrated that the defective phenotype of ferA : : Kanr was solely caused by the absence of FerA.
Search for revertants
Growth recovery of the ferA cells observed after a prolonged period of incubation with no selective antibiotic pressure (kanamycin absent) could in principle result from a spontaneous reversion to the wild-type. To check this possibility, Pd20121 was grown in mineral medium without antibiotics and reinoculated three times from stationary-phase cultures. The growth pattern with a prolonged lag phase continued to occur during these growth cycles. The resulting culture was spread and grown on BHI agar plates to give single colonies; ten of them were randomly picked for PCR analysis. In all cases, amplification of a PCR product of 446 bp was obtained with the primer pair 7/9, revealing the presence of the suicide plasmid in the bacterial genome at the proper insertion site. These data showed that the observed behaviour of the ferA mutant cultures was due to the mutant itself and not to the appearance of a true or pseudo-wild-type revertant.
Effect of added chorismate
Because the synthesis of catecholate siderophores starts with the formation of isochorismic acid from chorismic acid (Knaggs, 1999), we wondered whether the severely impaired production of parabactin in the ferA mutant might be attributable to a reduced availability of chorismate. We therefore repeated the growth experiments on the ferA cells under the same conditions as before (Table 4), but in the presence of 1 mM chorismate. There was, indeed, a partial reversal of the mutant phenotype as manifested by a decreased lag phase (10±5 h), elevated growth rate (0.17±0.03 h–1) and production of a significant level of catechols (9±1 µM) (mean±SD, data from three parallel cultivations). In contrast, neither cell growth nor siderophore accumulation was affected by the addition of chorismate to the wild-type cell cultures. The above hypothesis of a limited supply of chorismate in ferA cells thus appears to be substantiated.
Parabactin reduction catalysed by FerA
Having recognized the potential importance of FerA for iron assimilation, we decided to examine the activity of the purified enzyme towards the Fe(III) complex of parabactin. The assay mixture contained ferrozine to trap the released Fe(II) in the form of a coloured complex. When the initial concentration of Fe(III) parabactin was varied at 150 µM NADH, 50 µM FMN, 800 µM ferrozine, 3.9 µg FerA, a saturation curve was obtained from which non-linear regression gave an apparent half-saturation constant of 6±1 µM and a limiting rate of 2.1±0.1 nmol s–1 (mg protein)–1. The reaction did not proceed when the enzyme or any of the reagents was omitted. These observations clearly demonstrated the capability of FerA to deal with a reductive release of iron from ferriparabactin under the mediation of a free flavin.
Sequence homology of FerA
The amino acid sequence of FerA was subjected to a BLASTP search against the non-redundant protein database (Swiss-Prot/TREMBL). This resulted in the identification of 10 sequences with 40–45 % identity and 50–60 % similarity to FerA. Most of them are oxidoreductases with known participation of flavins. A multiple sequence alignment of the most homologous regions of four representatives of these proteins is shown in Supplementary Fig. S1, available with the online version of this paper.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Since P. denitrificans exclusively synthesizes siderophores of the catechol type (Tait, 1975), we considered FerA as a better candidate than FerB to be a member of the iron assimilatory pathway. This hypothesis is now verified by the results of our disruption-complementation experiments, which demonstrate a marked growth deficit of the ferA mutant strain in minimal medium containing ferric salts. The observation that the ferA mutant grows normally in BHI medium probably means that P. denitrificans possesses more than one pathway for iron uptake, one of which is operative under nutrient-rich growth conditions and does not rely on FerA.
It seems of interest to note that in E. coli there is an NAD(P)H : flavin oxidoreductase (Fre) with catalytic properties similar to those of P. denitrificans FerA (Coves & Fontecave, 1993). An E. coli mutant lacking the active fre gene was found to grow at the same rate as wild-type cells, both in rich or minimal media. Consequently, Fre was apparently dispensable under the experimental conditions used (Coves et al., 1993). This behaviour differs from that of P. denitrificans and may be related to a relatively high level of an additional flavin/Fe(III) reductase activity that apparently resides in the 
-subunit of the E. coli sulfite reductase. In the case of P. denitrificans, chromatographic analyses of their cytoplasmic fraction have given no clear hint of the presence of a third Fe(III) reductase besides FerA and FerB.
Based on the sequence analysis (Fig. S1), FerA is most similar to component B of the enzyme complex that oxidatively splits nitrilotriacetate (NTA) into iminodiacetate and glyoxylate. This complex, first purified from the bacterium Aminobacter aminovorans (formerly Chelatobacter heintzii) (Uetz et al., 1992), has a heterodimeric AB structure. Component A is an NTA monooxygenase that uses FMNH2 and O2 to oxidize NTA, and component B is an NADH : FMN oxidoreductase that provides FMNH2 for NTA oxidation. The two components are only loosely linked to one another, and B can easily be replaced by flavin reductases from other sources (Xu et al., 1997). It remains uncertain whether these features relate in any way to FerA, since P. denitrificans does not seem to be able to utilize NTA as sole nitrogen source (V. Sedláek, unpublished observations), and we currently have no evidence for a physical coupling between FerA and any other enzyme.
We postulate that FerA provides reducing equivalents for Fe(III) reduction in vivo. We base this postulation on the fact that we have demonstrated that FerA effectively catalyses an FMN-mediated reductive release of iron from the ferric chelate of the authentic P. denitrificans siderophore parabactin, even when the latter complex is present at only micromolar concentrations. FerA thus can be added to the list of flavin reductases known to use bacterial siderophores as substrates (Schroder et al., 2003). From cyclic voltametric data, the reduction potential of Fe(III)-parabactin was calculated to be –0.673 V, versus a standard hydrogen electrode, at pH 7.0 (Robinson & McArdle, 1981), i.e. well below the range of physiological reductants. Nevertheless, as discussed by Boukhalfa & Crumbliss (2002), reduction of even very stable ferric complexes can still proceed provided that the ferrous ion formed subsequently binds to an appropriate ligand. Most of the in vitro ferric reductase assays use ferrozine as a specific Fe(II) chelator to pull the redox reaction forward. The nature of the ligand exchange reactions within the bacterial cell remains to be established, although there is some evidence that Fe(II) in E. coli can transiently interact with a phosphorylated sugar derivative (Bohnke & Matzanke, 1995).
We also postulate that the absence of FerA negatively affects the production of siderophores. In many micro-organisms, the expression of genes involved in siderophore biosynthesis is regulated via the ferric uptake regulator (Fur) protein, which acts as an iron-responsive, DNA-binding repressor protein. Binding of Fur-Fe(II) to operator sequences results in repression of these genes, while under low-iron conditions, Fur is released and transcription takes place (Lee & Helmann, 2007). If the same holds true for P. denitrificans, then it might be predicted that deletion of FerA would promote derepression of siderophore formation as a consequence of decreased intracellular concentration of FMNH2 and hence also of Fe(II). Our failure to observe this derepression may indicate that reduced flavins take part directly in the reaction(s) of the aromatic biosynthesis pathway. Especially noteworthy in this respect is the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate, because virtually all chorismate synthases studied to date require FMNH2 for their activity (Macheroux et al., 1999). It is therefore conceivable that in the ferA mutant strain, chorismate is still synthesized, but at a rate insufficient to sustain a massive build-up of siderophores. In line with the foregoing interpretation is our finding that the provision of extra chorismate has the effect of increasing catecholate production.
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ACKNOWLEDGEMENTS |
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Edited by: Pierre Cornelis
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Xu, Y., Mortimer, M. W., Fisher, T. S., Kahn, M. L., Brockman, F. J. & Xun, L. (1997). Cloning, sequencing, and analysis of a gene cluster from Chelatobacter heintzii ATCC 29600 encoding nitrilotriacetate monooxygenase and NADH : flavin mononucleotide oxidoreductase. J Bacteriol 179, 1112–1116.
Received 28 July 2008;
revised 18 December 2008;
accepted 31 December 2008.
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, Parental strain (Pd1222); 
, ferA mutant (Pd20121); 
, complemented ferA mutant (Pd20122); 
, ferB mutant (Pd20021).