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Department of Microbiology, Morrill IV North, University of Massachusetts, Amherst, MA 01003, USA
Correspondence
Tünde Mester
tmester{at}microbio.umass.edu
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ABSTRACT |
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Four supplementary figures are available with the online version of this paper.
Present address: Department of Geological Sciences, University of Idaho, Moscow, ID 83844 – 3022, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Micro-organisms growing via Fe(III) reduction in subsurface environments can simultaneously reduce toxic metals such as U(VI), Tc(VII), Co (III) and V(V), which may be a useful strategy for the bioremediation of metal-contaminated subsurface environments (Anderson & Lovley, 2002; Anderson et al., 2003; Caccavo et al., 1994; Finneran et al., 2002; Holmes et al., 2002; Lloyd & Lovley, 2001; Ortiz-Bernad et al., 2004). Furthermore, microbiological and geological evidence suggests that Fe(III) reduction was an important form of respiration on early Earth (Lovley et al., 2004; Tor et al., 2004; Vargas et al., 1998; Walker, 1987).
Molecular analysis has demonstrated that, with the exception of highly saline environments (Nevin et al., 2003), Geobacteraceae are the predominant Fe(III)-reducing micro-organisms in a diversity of sedimentary environments in which Fe(III) reduction is an important process (Anderson et al., 2003; Holmes et al., 2002; North et al., 2004; Röling et al., 2001; Rooney-Varga et al., 1999; Snoeyenbos-West et al., 2000; Stein et al., 2001). The primary form of Fe(III) in such environments is insoluble Fe(III) oxide (Lovley, 1991; Nevin & Lovley, 2002). The available evidence suggests that Geobacter species must directly contact Fe(III) oxides in order to reduce them (Childers et al., 2002; Lovley et al., 2004; Nevin & Lovley, 2000). Thus, electron transfer to Fe(III) oxides is expected to take place at or near the outer cell surface (Lovley et al., 2004).
Most studies on electron transfer to Fe(III) in Geobacter and other dissimilatory Fe(III) reducers, such as Shewanella species, have focused on the role of c-type cytochromes (Lovley et al., 2004; Richardson 2000). The genome of G. sulfurreducens reveals an exceptional number of putative c-type cytochromes (Methe et al., 2003), many of which are predicted to be membrane-bound, and some of which are required for Fe(III) reduction (Leang et al., 2003; Lloyd et al., 2003; Reguera et al., 2005; Mehta et al., 2005).
However, proteins aside from cytochromes may also be important in Fe(III) oxide reduction. For example, pili are expressed during growth on Fe(III) oxide, but not soluble Fe(III) citrate, and a pilus-deficient mutant could not reduce Fe(III) oxides (Childers et al., 2002; Reguera et al., 2005). Studies on the pili of G. sulfurreducens revealed the presence of a homologue for pilA, the gene for the structural pilin protein. A phylogenetic analysis placed the protein among the bacterial pseudopilins of type II secretion systems (Reguera et al., 2005).
In several Gram-negative bacteria, type II secretion systems are responsible for the translocation of proteins into or through the outer membrane (Arrieta et al., 2004; Pugsley, 1993a; Sandkvist, 2001a). Classical type II general secretion pathways (GSPs), such as the pullulanase system (pul) of Klebsiella oxytoca (d'Enfert et al., 1987), consist of 12–15 interacting proteins whose genes and genetic arrangement are well conserved among bacteria (Filloux, 1999; Pugsley, 1993b; Sandkvist, 2001b). Pseudopilins are proposed to form a pilus-like periplasmic structure that assists in the secretion of proteins either by providing a channel to the outside of the cell that proteins can pass through or by acting as a piston to push out proteins (Nunn, 1999; Vignon et al., 2003).
Here we report that the pseudopilin gene of G. sulfurreducens is required for reduction of insoluble Fe(III) and Mn(IV) oxide, but not the reduction of soluble Fe(III) in G. sulfurreducens. One of the proteins secreted by this putative type II secretion system is OmpB, a putative multicopper, outer-membrane protein that is specifically required for the reduction of Fe(III) oxide, but not soluble Fe(III).
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METHODS |
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[supE44
lacU169 (
80 lacZM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1] was used for DNA manipulations (Hanahan, 1983; Woodcock et al., 1989). Targeted gene disruption experiments were performed on Geobacter sulfurreducens strain DL1 to produce oxpG : : kan and ompB : : spec strains). G. sulfurreducens strain PCA (ATCC 51573) (Caccavo et al., 1994) was obtained from our laboratory culture collection. Cells were cultured with acetate as the electron donor and either fumarate (40 mM), Fe(III) citrate (50 mM) or poorly crystalline Fe(III) oxide (100 mM) as the electron acceptor as previously described (Coppi et al., 2001). Nitrilotriacetic acid (NTA) was added at a concentration of 4 mM or 1 mM. Kanamycin, streptomycin and spectinomycin were added at final concentrations of 200 µg ml–1 when required.
DNA manipulations and RT-PCR conditions for RNA expression analysis.
Genomic DNA was extracted with the Master Pure Complete DNA and RNA purification kit (Epicenter Technologies). Plasmid purification was carried out with the Mini Plasmid purification kit (Qiagen). Total RNA was purified from mid-exponential-phase cultures with RNeasy Mini kits (Qiagen) followed by treatment with RNase-free DNase (Ambion). Reverse transcription was performed with the Omniscript RT kit (Qiagen) according to the manufacturer's instructions. Total RNA (1 µg) served as template for cDNA synthesis and random hexamers were used as primers. The resultant cDNA was amplified with primers: OxpGF (5'-CTGCAGGAAAACCTCTACACC-3') and OxpGR (5'-GAACTGACAAGGTCTGAGCCG-3') for the oxpG gene.
Construction of an oxpG deletion mutant.
In order to create an oxpG mutant, recombinant PCR (Murphy et al., 2000) was used to construct a linear DNA fragment containing the first 0.038 kb of the oxpG gene preceded by upstream sequence and followed by a kanamycin resistance cassette, the last 0.1 kb of the hyaB gene, and 0.1 kb of downstream sequence. In creation of a oxpG deletion mutant, primers Gsp1 (5'-GCCTCTGGATATAATTCCTTAC-3') and Gsp2 (5'-GGGTAAACCCCTTGCGGTTACG-3') were used to amplify the sequence upstream of oxpG (576 bp; –572 to +38); primers Gsp3 (5'-GGGCTCGTGAGGCTGTCCTGCAGG-3') and Gsp4 (5'-CGTCGTTAAGGGCAGCCATGTCGC-3') were used to amplify the sequence downstream of oxpG (410 bp; from +115 to 113 bp downstream from the stop site). A kanamycin resistance cassette was amplified from pBBR1MCS-2 (Kovach et al., 1995) with primers KanF (5'-CGTAACCGCAAGGGGTTTACCCACCTGGGATGAATGTCAGCTA-3') and KanR (5'-GCAGGACAGCCTCACGAGCCCAAGCCCAACCTTTCATAGA-3'). Recombinant PCR was performed using primers Gsp1 and Gsp4. Electroporation, mutant isolation and genotype confirmation using Southern hybridization were performed as described by Coppi et al. (2001). Southern hybridization with G. sulfurreducens genomic DNA was performed according to Sambrook et al. (1989). Probes were labelled with [-32P]dCTP (Perkin Elmer) with the Multiprime DNA labelling system (Amersham Pharmacia Biotech). One of the correct genotype mutants, designated DLTM2, was chosen as the representative strain.
Construction of an ompB deletion mutant.
The ompB-deficient strain was created essentially the same way as for the oxpG mutant strain. Primers rgR1 (5'-GCTAGAATTCGGCGACGCGCTCCGAG-3') and rgR2 (5'-GCATGGATCCAGAACGGTTCCTGC-3') were used to amplify a 0.5 kb fragment located upstream of ompB. Primers rgR3 (5'-CAGGGTGTCGACGAATTTCCGCAT-3') and rgR4 (5'-GCATAAGCTTGTAAGCCGATTTGATG-3') were used to amplify a 0.5 kb fragment located downstream of ompB. The aadA spectinomycin resistance cassette was cloned from pSJS985 (Sandler & Clark, 1994) using primers RGspcRV1 (5'-CGATGATATCGCACAGGATGACGCCTAAC-3') and RGspcRV2 (5'-GCGATGATATCGAAGCGGCGTCGGCTTG-3'). The primers rgR1 and rgR4 were used to amplify the 1.9 kb fragment and used to perform single-step gene replacement according to the method of Leang et al. (2003). The mutation again was confirmed by Southern blot hybridization.
Expression of the oxpG and ompB genes in trans.
The mutant gsp : : kan was complemented using the expression vector pCDS as described previously (Leang et al., 2003). The primers used to amplify oxpG were: forward primer, 5'-CCCGGAATTCCCCGAAGTGGGTGAGCC-3'; and reverse primer, 5'-CCGGGATCCCGCGCTGGTATGACATGG-3' (with EcoRI and BamHI restriction sites underlined). The oxpG coding sequence was digested with EcoRI and BamHI and inserted into the EcoRI and BamHI sites of the expression vector pCDS to generate pCDS-oxpG. The oxpG gene was sequenced to screen for PCR artefacts. The oxpG deletion mutant was electroporated with pCDS-oxpG. Streptomycin was used as the selection marker to screen for the insert. The strain was designated DLTM2C. Successful transformants were screened for the insert in the correct orientation.
When ompB was expressed in trans, an expression vector derived from pCM66 (Marx & Lidstrom, 2001) was constructed. Primers rgG1 (5'-CCGAGACATGTCAACAGCTGATTGCCCTTCAC-3') and rgG2 (5'-CCGAGGACGTCGCGAAAACTCTCAAGGATCTTAC-3') were used to amplify a 1.8 kb fragment from pCD342 (Dehio et al., 1998). The PCR product was digested with AflII/AatIII and ligated to the 5.9 kb AatII/AflIII fragment from pCM66 to create pRG2. Primers rgR5 (5'-GCGATGAATTCTTACTGGATCGTCAGGTTGTTGAC-3') and rgR6 (5'-GCGATGAATTCCACAAAGGAGACATTTGATC-3') were used to amplify the ompB gene and the native ribosome-binding site. The 4 kb PCR product was digested with EcoRI and HindIII and ligated with EcoRI/HindIII pRG2 to create pRG2ompB. Further procedures were identical to those of oxpG expression.
Analytical techniques.
Cell suspension experiments were carried out as previously described (Leang et al., 2003). Fe(III) reduction was monitored by measuring the accumulation of HCl-extractable Fe(II) with ferrozine as previously described (Lovley & Phillips, 1986). Growth on fumarate was monitored spectrophotometrically by measuring the turbidity at 600 nm. Cell densities of other cultures were determined by acridine orange staining and epifluorescence microscopy (Lovley & Phillips, 1988).
Isolation of periplasmic proteins.
Cells grown with insoluble Fe(III) oxide as terminal electron acceptor supplemented with 4 mM NTA were grown to exponential phase. Cells were harvested after dissolving the insoluble Fe(III) oxide using oxalate solution (28 g ammonium oxalate l–1 and 15 g oxalic acid l–1). The cells were pelleted by centrifugation at 3000 g for 20 min, washed in PBS buffer and centrifugation was repeated. Cell pellets were resuspended in 25 mM HEPES (pH 7.5), 0.5 mM MgCl2 and 20 % sucrose containing protease inhibitor. Cells were stirred for 20 min on ice. EDTA (pH 8.0) was added to a final concentration of 10 mM and the suspension was stirred on ice for 5–10 min. Lysozyme was added at a final concentration of 0.5 mg ml–1 and stirring continued for another 30 min at room temperature to generate spheroplasts. The spheroplasts were pelleted by centrifugation at 5000 g at 4 °C for 15 min. The outer-membrane fraction was removed from this preparation by centrifugation at 50 000 g at 4 °C for 1 h. The supernatant was used as the periplasmic fraction.
Identification of OmpB.
The protein was excised from the SDS gel and digested with trypsin according Gharahdaghi et al. (1999). Digests were concentrated using ZipTip C18 pipette tips (Millipore) according to the recommended protocol with the exception of 1 % formic acid instead of trifluoroacetic acid. MALDI-MS (matrix-assisted-laser-desorption/ionization time-of-flight) and PSD (post-source decay) data were obtained on a Kratos Axima CFR (Kratos Analytical).
Gel electrophoresis and Western blot analysis.
Samples were electrophoresed on 10 % SDS polyacrylamide gels and visualized via Coomassie staining, with SeeBluePlus prestained proteins (Invitrogen) or subjected to Western analysis by transferring the proteins to Immuno blot PVDF membrane (Bio-Rad). The membrane was probed with a polyclonal antibody raised against a peptide of OmpB (KPDKTPIGPGDTPDC) (Sigma Genosys). Polyclonal alkaline phosphatase conjugated anti-rabbit antibody (Sigma) was used as secondary antibody, OmpB was visualized by SigmaFast 5-bromo-4-chloro-3-indolyl phosphatase/nitro blue tetrazolium tablets (Sigma).
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RESULTS |
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). This gene was designated oxide reducing pathway protein G (oxpG) because PSORT analysis (Nakai & Horton, 1999) and FingerPRINTScan (http://www.bioinf.man.ac.uk/fingerPRINTScan/) suggested that this ORF is similar to the genes for a general secretion pathway protein G related to type II general secretion pathways of Gram-negative bacteria. oxpG is not part of the classical type II secretion system, which has been described previously (Sandkvist, 2001b). The arrangement of the ORFs surrounding the putative G. sulfurreducens oxpG (see Supplementary Fig. S1, available with the online version of this paper) is like that around the gene xcm, part of a novel secretion system in Pseudomonas putida GB-1 that is required for secretion of a copper-containing manganese oxidase (De Vrind et al., 2003). The gene arrangement surrounding the G. sulfurreducens oxpG is also found in Geobacter metallireducens (http://www.jgi.doe.gov). RT-PCR analysis revealed that oxpG was expressed under all culture conditions studied, which included growth with Fe(III) citrate, fumarate, or insoluble Fe(III) or Mn(IV) oxides as the electron acceptor (see Supplementary Fig. S2).
Characterization of the oxpG deletion mutant
In order to identify the potential role of OxpG in Fe(III) respiration, an oxpG deletion mutant was constructed and was confirmed by Southern hybridization. Examination of the mutant by transmission electron microscopy demonstrated that it continued to produce pilin (data not shown).
The mutant reduced Fe(III) citrate (see Supplementary Fig. S3A) or fumarate (data not shown) as well as the wild-type. However, the oxpG deletion mutant was severely impaired in its ability to reduce insoluble Fe(III) (Fig. 1) and Mn(IV) oxides (data not shown). The mutant reduced Fe(III) in the Fe(III) oxide medium when the Fe(III) chelator NTA was added (Fig. 1). These results further suggested that the deletion of oxpG specifically affected the secretion of proteins required for reduction of insoluble electron acceptors. When oxpG was expressed in the deletion mutant in trans, the capacity for Fe(III) oxide reduction was restored (Fig. 1).
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). Therefore, in order to identify proteins secreted by the OXP-like system in G. sulfurreducens, the periplasmic protein fractions of mutant and wild-type cells were compared (Fig. 2). A protein with an approximate molecular mass of 140 kDa accumulated in the periplasm of the mutant cells (Fig. 2). The MALDI-MS analysis of this protein indicated that it was encoded by ORF GI-39996496 in the G. sulfurreducens genome (Methe et al., 2003). The protein was named outer-membrane protein B (OmpB).
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). The protein consists of 1303 aa, with a calculated molecular mass of 139.6 kDa, and a pI of 4.47. A 26 aa signal peptide was predicted, indicating the importance of the secretion pathway in the transport of this protein. The protein is predicted to be extracellular (PSORTB at http://www.expasy.org). However, when the various cell fractions were run on a SDS-PAGE, OmpB was identified in both the membrane fraction and the extracellular fluid, but not in the soluble fraction of the cells (Fig. 3), suggesting that OmpB is cell-surface exposed. Western blot analysis also indicated that OmpB is expressed at the same levels during growth with soluble electron acceptors, or insoluble Fe(III) oxide or Mn(IV) oxide (Fig. 3).
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) and Mn(IV) (Fig. 5). When the chelator NTA was added to the cultures containing Fe(III) oxide, there was no significant difference between the mutant and the wild-type in the reduction of Fe(III) (Fig. 4b). When the mutant was complemented with ompB in trans, the reduction of Fe(III) oxide was restored, but the rate was somewhat slower than that of the wild-type (Fig. 4a). The complementation of ompB in trans also restored the reduction of Mn(IV) (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Nevin & Lovley, 2002). Furthermore, the results demonstrate that, in addition to the c-type cytochromes that are widely regarded to be important outer-membrane proteins for electron transport and/or Fe(III) reduction, other proteins that are not cytochromes are also necessary for this process.
Role of OxpG in Fe(III) oxide reduction
The results suggest that the OxpG is involved in exporting OmpB, and possibly other proteins, specifically required for the reduction of Fe(III) and Mn(IV) oxides. OxpG is most homologous to XcmT3, a type II pseudopilin from Pseudomonas putida strain GB-1 (De Vrind et al., 2003) and the genomic region flanking the oxpG gene cluster also has similarity to the xcmT3 gene cluster. Genetic studies have demonstrated that the XcmX secretion system in P. putida strain GB-1 is involved in the secretion of a manganese-oxidizing enzyme (De Vrind et al., 2003), which is also a multicopper oxidase (Brouwers et al., 1999).
A type II secretion system is also required for Fe(III) reduction in the dissimilatory Fe(III) reducer Shewanella putrefaciens (DiChristina et al., 2002). When ferE, the gspE homologue in the S. putrefaciens secretion system, was deleted, Fe(III) and Mn(IV) reduction were inhibited. However, there are some significant differences between the systems required for Fe(III) reduction in S. putrefaciens and G. sulfurreducens. For example, the secretion system required for Fe(III) reduction in S. putrefaciens is more closely related to the classical type II GSP systems of Gram-negative bacteria that are involved in translocating toxins and hydrolases (Sandkvist, 2001b). In fact, the gene arrangement in S. putrefaciens is identical to that of Vibrio cholerae (Sandkvist, 2001b). In contrast, the arrangement of the genes surrounding oxpG in G. sulfurreducens is similar to that of the non-classical XcmX secretion system of P. putida (De Vrind et al., 2003). The genome of G. sulfurreducens contains the classical type II secretion system, showing more similarity to those in V. cholerae and S. putrefaciens (Sandkvist, 2001b), but its role in Fe(III) reduction has not been evaluated.
Another difference between the G. sulfurreducens and S. putrefaciens secretion systems required for Fe(III) reduction is that deleting ferE from S. putrefaciens inhibited the reduction of both soluble and insoluble Fe(III) (DiChristina et al., 2002), whereas deleting oxpG only inhibited Fe(III) oxide reduction in G. sulfurreducens. Preliminary evidence suggested that a haem-containing protein was not properly exported to the outer membrane in the secretion system mutant of S. putrefaciens, but the actual role of this cytochrome in Fe(III) reduction has yet to be determined (DiChristina et al., 2002). An outer-membrane c-type cytochrome, OmcB, is also required for reduction of both soluble and insoluble Fe(III) in G. sulfurreducens (Leang et al., 2003). OmcB was not observed to accumulate in the periplasm of the oxpG deletion mutant, and the fact that the oxpG deletion mutant could reduce soluble Fe(III) as well as the wild-type suggested that OmcB was properly positioned in the outer membrane. In contrast, the inhibition of Fe(III) and Mn(IV) oxide in the absence of a functioning secretion system in G. sulfurreducens could be attributed, at least in part, to the failure to export OmpB, which is not a c-type cytochrome.
Role of OmpB in Fe(III) and Mn(IV) oxide reduction
The results suggest that one reason why the oxpG-deficient mutant cannot reduce Fe(III) or Mn(IV) oxide is that OmpB is not exported across the outer membrane. Although previous studies on extracellular electron transport to Fe(III) and Mn(IV) oxides have primarily focused on the role of outer-membrane c-type cytochromes (Lovley et al., 2004; Richardson, 2000), it is becoming increasingly apparent that other types of proteins may also be important. For example, MtrB, which is a non-haem-containing outer-membrane protein required for Fe(III) reduction in S. putrefaciens (Beliaev & Saffarini, 1998), has the likely role of properly positioning cytochromes in the outer membrane (Myers & Myers, 2002). Pilin is required for Fe(III) oxide reduction in G. sulfurreducens, with the probable role of facilitating the final step in electron transfer to Fe(III) oxides (Reguera et al., 2005).
The protein most closely related to OmpB is MofA of the Mn(II) and Fe(II)-oxidizing micro-organism Leptothrix discophora (Brouwers et al., 2000a; Corstjens et al., 1997). Like MofA, OmpB contains four predicted copper-binding sites arranged in a manner commonly found in multicopper oxidases (Brouwers et al., 2000b). MofA is considered to be responsible for the oxidation of Mn(II). The partially purified MofA catalyses the oxidation of Mn(II) with oxygen as the electron acceptor (Corstjens et al., 1997). Mn(IV) is produced and accumulates in the sheath which surrounds the cells, where the putative manganese-oxidizing system is also found (Brouwers et al., 2000b). Mn(IV) oxide formation was also stimulated with the addition of copper to the medium, further suggesting the involvement of a copper protein.
Anaerobic functions for multicopper oxidases have not previously been proposed. It is not clear whether this protein is redox active, and what its substrates are. Further studies are needed to determine whether OmpB is directly involved in the reduction of Fe(III) oxide and Mn(IV), or whether it has a yet unknown function, which facilitates the reduction of these metals. The study of the amino acid sequence of OmpB reveals a unique feature of OmpB that is not found in other multicopper oxidases. It contains a fibronectin type III-like domain (see Supplementary Fig. S4), similar to those in bacterial hydrolases (Kataeva et al., 2002; Lin & Xiong, 2004). The fibronectin domain occurs most frequently in cellulases and chitinases in bacteria (Lin & Xiong, 2004; Watanabe et al., 1994). It is proposed that in those enzymes the fibronectin domain either facilitates the adhesion of the protein to polysaccharides or cell receptors, or it participates in the maintenance of the proper conformation at the binding and/or catalytic sites of the enzyme (Kataeva et al., 2002; Watanabe et al., 1994). OmpB also contains an EXXE motif, which may be a potential Fe-binding site. This motif was found to be responsible for Fe(III) binding in an Fe(III) transporter (FTR1) (see Supplementary Fig. S4) (Severance et al., 2004). A plausible explanation is that both the potential Fe(III)-binding and fibronectin motifs of OmpB might be involved in association of the cell with Fe(III) oxide. Additional studies to further elucidate these properties and the possible redox activity of the copper are warranted.
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ACKNOWLEDGEMENTS |
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Received 23 January 2006;
revised 19 April 2006;
accepted 4 May 2006.
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), the oxpG mutant strain (
) and the oxpG mutant strain complemented with oxpG in trans (
) in the absence (a) and presence (b) of 4 mM NTA. The results are the means of triplicate incubations; error bars represent SD.
