MtrC, an outer membrane decahaem c cytochrome required for metal reduction in Shewanella putrefaciens MR-1

Authors

  • Alex S. Beliaev,

    1. 1 Department of Microbiology, University of Massachusetts, Amherst, MA , USA. 2Department of Biological Sciences, University of Wisconsin–Milwaukee, 3209 N. Maryland Ave., Milwaukee, WI 53211, USA.
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  • 1 Daad A. Saffarini,

    1. 1 Department of Microbiology, University of Massachusetts, Amherst, MA , USA. 2Department of Biological Sciences, University of Wisconsin–Milwaukee, 3209 N. Maryland Ave., Milwaukee, WI 53211, USA.
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  • 2 Jennifer L. McLaughlin,

    1. 1 Department of Microbiology, University of Massachusetts, Amherst, MA , USA. 2Department of Biological Sciences, University of Wisconsin–Milwaukee, 3209 N. Maryland Ave., Milwaukee, WI 53211, USA.
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  • and 1 David Hunnicutt 2

    1. 1 Department of Microbiology, University of Massachusetts, Amherst, MA , USA. 2Department of Biological Sciences, University of Wisconsin–Milwaukee, 3209 N. Maryland Ave., Milwaukee, WI 53211, USA.
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*For correspondence. E-mail daads@ uwm.edu; Tel. (+1) 414 229 2964; Fax (+1) 414 229 3926.

Abstract

Shewanella putrefaciens is a facultative anaerobe that can use metal oxides as terminal electron acceptors during anaerobic respiration. Two proteins, MtrB and Cct, have been identified that are specifically involved in metal reduction. Analysis of S. putrefaciens mutants deficient in metal reduction led to the identification of two additional proteins that are involved in this process. MtrA is a periplasmic decahaem c-type cytochrome that appears to be part of the electron transport chain, which leads to Fe(III) and Mn(IV) reduction. MtrC is an outer membrane decahaem c-type cytochrome that appears to be required for the activity of the terminal Fe(III) reductase. Membrane fractions of mutants deficient in MtrC exhibited a decreased level of Fe(III) reduction compared with the wild type. We suggest that MtrC may be a component of the terminal reductase or may be required for its assembly.

Introduction

Bacterial metal reduction is a common respiratory process in anaerobic environments. A large number of bacterial species have been identified that are capable of metal reduction. These include strict anaerobes, such as Geobacter sulfurreducens (Lovley, 1993), and facultative anaerobes, such as Shewanella putrefaciens (Myers and Nealson, 1988), Aeromonas hydrophila (Knight and Blakemore, 1998) and Pantoea agglomerans (Francis et al., 2000). S. putrefaciens, which belongs to the γ-group of the Proteobacteria, has been isolated from a number of freshwater and marine environments, oil fields, spoiled fish and other sources (Nealson and Saffarini, 1994). S. putrefaciens can use a large number of terminal electron acceptors, including oxygen, nitrate, nitrite, fumarate and insoluble metal oxides or oxyhydroxides such as Mn(IV) and Fe(III). When grown anaerobically in the presence of metals such as Fe(III) or Mn(IV) oxides, S. putrefaciens cells are found almost exclusively on the surface of the metal particles. Bacterial cell contact with the oxide particles is required for reduction to occur (Arnold et al., 1988; Little et al., 1997). This contact is thought to be mediated by proteins found on the outer surface of the cells (Caccavo, 1999). We have recently described an outer membrane protein, MtrB, which contains a putative metal binding site (CXXC) and is required for Fe(III) and Mn(IV) reduction (Beliaev and Saffarini, 1998). It is possible that this protein plays a role in metal binding during reduction.

Cell fractionation studies suggest that the majority of the terminal Fe(III) reductase activity of S. putrefaciens MR-1 is located in the outer membrane (Myers and Myers, 1993). The Fe(III) reductase activity of the strict anaerobe G. sulfurreducens has also been shown to be associated with the outer membrane (Gaspard et al., 1998). These results are surprising, as terminal reductases of Gram-negative bacteria are typically located in the cytoplasmic (inner) membrane. c-type cytochromes, which are usually found in the cell membrane or periplasmic space of Gram-negative bacteria, have also been localized to the outer membrane of S. putrefaciens MR-1 (Myers and Myers, 1992; 1997). The location of reductases on the outer membrane is predicted to be an advantage for metal-reducing bacteria and may be a way of dealing with insoluble electron acceptors.

The mechanisms of electron transfer leading to metal reduction are poorly understood. Cct, a cytochrome c3 isolated from Shewanella frigidimarina, that is specifically involved in metal reduction has been identified recently (Gordon et al., 2000). In this paper, we describe two c-type cytochromes, MtrA and MtrC, that are involved in Fe(III) and Mn(IV) reduction. Western blot analyses and cell fractionation studies indicated that MtrC is located in the outer membrane of S. putrefaciens MR-1, whereas MtrA appears to be periplasmic. Loss of MtrC leads to decreased levels of Fe(III) reductase activity in vitro, suggesting that this cytochrome may be required for the activity of the terminal Fe(III) reductase or is one of its components. A model describing Fe(III) reduction by S. putrefaciens is presented.

Results

Isolation and sequence analysis of mtrC

S. putrefaciens mutants deficient in Fe(III) reduction were generated by Tn5 insertional mutagenesis as described previously (Beliaev and Saffarini, 1998). One mutant, SR-8, was tested for both anaerobic growth and reduction of the terminal electron acceptors used by the wild-type strain MR-1. SR-8 was able to use fumarate, sulphite, thiosulphate, nitrate, nitrite, trimethylamine oxide (TMAO) and dimethyl sulphoxide (DMSO) as terminal electron acceptors, but was deficient in both Fe(III) and Mn(IV) reduction (Fig. 1).

Figure 1.

Metal reduction by wild-type and mutant strains of S. putrefaciens.

A. Fe(III) reduction by wild-type (MR-1) and mutant (SR-8) strains of S. putrefaciens. Fe(II) production was monitored at 562 nm using the ferrozine assay.

B. Mn(IV) reduction. Clearing of the black precipitate is an indication of Mn(IV) reduction, as seen in the wild-type strain MR-1, but not in the mutant strain SR-8.

The DNA region interrupted by Tn5 insertion in SR-8 was isolated by cloning the BamHI–EcoRI fragment containing the kanamycin resistance gene from Tn5 and adjacent S. putrefaciens DNA. This plasmid was used to isolate lambda clones that contain the intact DNA region. Sequence analyses of the wild-type and interrupted DNA regions revealed that Tn5 insertion in SR-8 interrupted a 2013 bp open reading frame (ORF), which we designated mtrC. Two consecutive ATG codons are located 6–9 bases downstream of a putative ribosome binding site. It is not clear which one serves as the initiation codon. mtrC encodes a protein of 671 amino acids if we assume that the first AUG serves as the initiation codon. The N-terminal region contains a hydrophobic stretch that is typical of signal sequences. The predicted cleavage site of this leader peptide lies between amino acids 21 and 22 (Nielson et al., 1997). This cleavage site is followed by a lipid modification consensus sequence, LXXC, suggesting that MtrC may be a lipoprotein (Hayashi and Wu, 1990). Based on the presence of 10 putative haem binding sites (CXXCH), MtrC was predicted to be a c-type cytochrome. This was confirmed by haem staining of cell extracts separated by SDS–PAGE. A band of 75 kDa, which corresponds to the predicted mass of the mature MtrC including 10 haems, was detected in the wild-type but not in the mutant cell extracts (Fig. 2).

Figure 2.

Haem staining of anaerobically grown MR-1 and SR-8 cell extracts. The band that corresponds to MtrC is indicated by an arrow. Numbers correspond to protein molecular weight markers.

Gene organization of mtrC, mtrA and mtrB

Sequence analysis of mtrC and adjacent DNA revealed that mtrC lies upstream of mtrA and mtrB. mtrB was shown to encode an outer membrane protein that is required for metal reduction (Beliaev and Saffarini, 1998). The organization of the three genes is shown in Fig. 3. A potential hairpin–loop structure was found between mtrC and mtrA. We suggested previously that this structure might function as a transcriptional terminator and that mtrA and mtrB form an operon (Beliaev and Saffarini, 1998). In this study, we analysed the regions upstream of mtrC and mtrA for transcription start sites. We used primers complementary to regions downstream of the start codon of each gene and the 5′ rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR) kit to synthesize cDNAs that correspond to the 5′ end of the mRNA. The resulting fragments were sequenced, and only one promoter, upstream of mtrC, was identified. This suggested that the DNA immediately upstream of mtrA lacks a promoter and that mtrA, mtrB and mtrC may form an operon. This was confirmed using a modified version of the previously described plasmid pSC52. This plasmid contains mtrA and mtrB downstream of the lac promoter (Fig. 3) and was used to complement the mtrB mutant SR-21 (Beliaev and Saffarini, 1998). We inserted a 2.3 kb fragment that contains the kanamycin resistance gene and transcriptional terminators from the interposon pHP45ΩKm (Fellay et al., 1987) into the HindIII site that lies between the lac promoter and mtrAB. The resulting plasmid, pSC53, was introduced into SR-21 to generate SR-523. The levels of Fe(III) reduction by SR-21C (SR-21 containing pSC52) and SR-523 (SR-21 containing pSC53) are shown in Table 1. The ability of pSC52 to complement SR-21 coupled to the failure of pSC-53 to do so suggests that transcription of mtrAB in pSC52 was driven by the lac promoter, which appears to be functional in S. putrefaciens MR-1. These results confirm the absence of a promoter sequence upstream of mtrAB. The transcription start site of mtrCAB was determined to be 119 bases upstream of the first AUG codon in mtrC. The identified promoter region was weakly similar to known promoter sequences. The −35 region (TAGAAG) matched the S. putrefaciens etrA sequence (TTGGGT; Saffarini and Nealson, 1993) in two out of six positions and matched the Escherichia coli consensus sequence (TTGACA) in three out of six positions. The −10 element of the promoter (GGTTAG) matched the etrA sequence (GATTAT) in four out of six positions and matched the E. coli consensus (TATAAT) in two out of six positions.

Figure 3.

Genetic map and organization of mtrC, mtrA and mtrB. The inverted repeats of putative hairpin–loop structures are marked by arrows. The 5.2 kb HindIII–EcoRI, cloned downstream of the lac promoter and used to complement SR-21, is also shown.

Table 1. Fe(III) reductase activity by whole cells of wild-type (MR-1) and mutant S. putrefaciens strains.
Strain (phenotype)% Fe(III) reductase activitya
  1. a . Fe(III) reductase activity was calculated as a percentage of wild-type activity. 100% activity corresponds to 2.78 µmol of Fe(II) produced min −1. The results are representative of three different experiments performed in duplicate.

MR-1 (MtrA+, MtrB+, MtrC+)100
SR-8 (MtrA, MtrB, MtrC)13.7
SR-A1 (MtrA, MtrB, MtrC+)1.3
SR-21 (MtrA+, MtrB, MtrC+)1.3
SR-522 (MtrA+, MtrB+, MtrC)21.6
SR-523 (MtrA+, MtrB, MtrC+)1.3
SR-A11 (MtrA, MtrB+, MtrC+)3.1
SR-21C (MtrA+, MtrB+, MtrC+)94

Analysis of mtrA and mtrC functions

As mentioned above, because the three mtr genes form an operon, insertional inactivation of mtrC was expected to be polar on the downstream genes. This was confirmed by Western blot analysis as shown in Fig. 4. Anaerobically grown SR-8 cells lacked MtrA, MtrB and MtrC. Therefore, the phenotype observed in SR-8 may be complicated by the inactivation of the downstream gene, mtrB, which was shown to be required for both Fe(III) and Mn(IV) reduction (Beliaev and Saffarini, 1998). To determine the function of MtrC in metal reduction, we transformed SR-8 with pSC52, which contains mtrAB and complements an mtrB mutant, to generate SR-522. Western blotting analysis indicated that SR-522 produced MtrA and MtrB at approximately wild-type levels, but did not produce MtrC (data not shown). SR-522 was tested for its ability to use the terminal electron acceptors used by the wild type and was found to be deficient in both Fe(III) and Mn(IV) reduction. The rates of Fe(III) reduction in cultures of SR-522 (21.6% of wild-type levels) were slightly higher than those observed in cultures of SR-8 (13.7% of wild-type levels; Table 1).

Figure 4.

Analysis of MtrA, MtrB and MtrC in wild-type and mutant cell extracts of S. putrefaciens.

A. Western blot analysis of the wild-type and mutant strain whole-cell extracts using antisera raised against MtrC. A band that corresponds to MtrC is indicated.

B. Western blot analysis of whole-cell extracts using antisera raised against MtrB. A band that corresponds to MtrB is indicated.

C. Western blot analysis of MR-1 (1), SR-A1 (2), SR-8 (3) and SR-21 (4) soluble fractions using antisera raised against MtrA.

To determine the function of mtrA in metal reduction, we generated the mutant SR-A1, as described in Experimental procedures. This mutant has the suicide vector pVIK165 inserted into mtrA. Western blotting analysis determined that the mutation in SR-A1 was polar on mtrB, as shown in Fig. 4B. We introduced pSC26 (Table 2), which contains mtrB expressed from the lac promoter, into SR-A1 to generate SR-A11. The synthesis of MtrB by this strain was confirmed by Western blotting. SR-A11, which lacked MtrA only, was found to be deficient in both Fe(III) and Mn(IV) reduction. The rates of Fe(III) reduction by SR-A11 cells were only 3.1% of wild-type levels (Table 1).

Table 2. Bacterial strains and plasmids used in this study.
Strain or plasmidDescriptionReference
S. putrefaciens
 MR-1Lake Oneida isolate Myers and Nealson (1988)
 MR-1RRifampicin-resistant strain of MR-1 Beliaev and Saffarini (1998)
 SR-21MR-1R but mtrB::Tn5, MtrB Rifr, Kmr Beliaev and Saffarini (1998)
 SR-21CSR-21 carrying pSC52, MtrC+, MtrA+, MtrB+ Rifr, Kmr, Tcr Beliaev and Saffarini (1998)
 SR-8MR-1R but mtrC::Tn5, MtrC, MtrA, MtrB Rifr, KmrThis work
 SR-522SR-8 transformed with pSC52, MtrC, MtrA+, MtrB+This work
 SR-523SR-21/pSC53, Kmr, Tcr, MtrC+, MtrA+, MtrBThis work
 SR-A1MR-1R but mtrA::pVIK165, Rifr, Kmr, MtrC+, MtrA, MtrBThis work
 SR-A11SR-A1 transformed with pSC29, MtrC+, MtrA, MtrB+This work
 SR-B2MR-1R; mtrB–lacZ transcriptional fusionThis work
 SR-C2MR-1R; mtrC–lacZ transcriptional fusionThis work
Plasmids
 pSPORT1Cloning and sequencing vector, AmprBRL Life Technologies
 pRK415IncP broad-host-range cloning vector, Tcr Keen et al. (1988)
 pSR8pSPORT1 carrying 0.8 kb EcoRI–BamHI fragment of mtrCThis work
 pSC52pRK415 carrying mtrAB downstream of the lac promoter Beliaev and Saffarini (1998)
 pSC29pRK415 carrying mtrB downstream of the lac promoterThis work
 pSC53pSC52 derivative carrying ΩKm insertion between the lac promoter and mtrABThis work
 pVIK165pGP704 derivative, R6K ori, RP4 oriT, gfp, Kmr Kalogeraki and Winans (1997)
 pAB258pVIK165 carrying 258 bp mtrA fragmentThis work
 pVIK112pGP704 derivative, R6K pir-dependent replication,RP4 oriT, lacZ, Kmr Kalogeraki and Winans (1997)
 pAB717pVIK112 carrying 717 bp mtrB fragmentThis work
 pAB463pVIK112 carrying 463 bp mtrC fragmentThis work
 pETC2pET30 carrying 1723 bp mtrC fragmentThis work
 pETA6pET30 carrying 734 bp mtrA fragmentThis work
 pETB4pET30 carrying 1794 bp mtrB fragmentThis work

Although SR-21 and SR-A1 (which lack MtrB and MtrA respectively) were deficient in Fe(III) reduction in vivo, this deficiency was not detected when membrane fractions were used to measure Fe(III) reductase activity in vitro. The rates of Fe(III) reduction by SR-21 and SR-A1 membrane fractions, using formate as the electron donor, were similar to those of the wild-type strain MR-1 (Table 3). However, membrane fractions from strains that lacked MtrC exhibited lower Fe(III) reductase activity compared with the wild-type strain (Table 3), suggesting that MtrC may play a role in the final step of Fe(III) reduction.

Table 3. Specific iron reductase activity in total membrane fractions of wild-type and mutant strains.


Strain
Iron reductase activity
(nmol of Fe(II) produced 
min−1 mg−1 protein)
MR-1 (MtrC+, MtrA+, MtrB+)265.28 ± 16.7
SR-8 (MtrC, MtrA, MtrB)101.37 ± 24.2
SR-21 (MtrC+, MtrA+, MtrB)313.50 ± 87.3
SR-A1 (MtrC+, MtrA, MtrB)343.02 ± 51.8
SR-522 (MtrC, MtrA+, MtrB+)90.06 ± 11.7

Localization of MtrA and MtrC

S. putrefaciens MR-1 soluble, cell membrane, and outer membrane fractions were obtained as described previously (Beliaev and Saffarini, 1998). These fractions were analysed by Western blotting using antisera raised against MtrA and MtrC. MtrA was found in the soluble fraction (Fig. 4C). Sequence analysis of MtrA predicts the presence of a signal sequence, suggesting that this cytochrome is a periplasmic protein. A second cross-reacting protein band was observed in all samples examined (Fig. 4C). This band may represent MtrD, which shares a high degree of sequence similarity with MtrA (see below).

MtrC was found primarily in the outer membrane fraction of S. putrefaciens MR-1 (Fig. 5). In this regard, MtrC is similar to OmcA, a c-type cytochrome from S. putrefaciens MR-1 that has been shown to be an outer membrane protein (Myers and Myers, 1997). Although OmcA is an outer membrane cytochrome that shares amino acid sequence similarity with MtrC, insertional inactivation of omcA does not appear to affect either Fe(III) or Mn(IV) reduction (A. S. Beliaev and D. A. Saffarini, unpublished)

Figure 5.

Localization of MtrC. SR-8 soluble (S), cell membrane (C) and outer membrane (O) fractions were analysed by Western blotting using antisera raised against MtrC.

MtrCAB expression

To measure the levels of expression of mtrCAB under different growth conditions and to determine the role of the apparent hairpin–loop structure that lies between mtrC and mtrA, we constructed strains that carry lacZ transcriptional fusions. SR-B2 and SR-C2, which carry mtrB–lacZ and mtrC–lacZ fusions, respectively, were grown anaerobically with different electron acceptors. The levels of β-galactosidase were measured in both strains. The levels of β-galactosidase in each strain were the same regardless of the electron acceptor provided in the medium (Table 4). This is consistent with earlier observations, which indicated the presence of Fe(III) reductase activity in S. putrefaciens cells grown under different conditions (Myers and Nealson, 1990). SR-C2 exhibited approximately 12 times more β-galactosidase activity than SR-B2 under all growth conditions tested. The presence of the hairpin–loop structure between mtrC and mtrAB may explain the apparent high level of expression of mtrC. Hairpin–loops may protect upstream mRNA regions from nuclease digestion. Hairpin–loop structures may also play a role at the transcriptional level. A stem–loop may destabilize RNA polymerase interaction with DNA, resulting in reduced transcription of downstream genes. It is not clear at present whether the potential hairpin–loop between mtrC and mtrAB affects transcription or mRNA stability.

Table 4. β-Galactosidase activity (mUnits) of lacZ transcriptional fusions
 Terminal electron acceptora
Strain (fusion)NitrateFumarateFe(III)
  1. a . Cells were grown anaerobically to mid-log phase with nitrate, fumarate or Fe(III) as terminal electron acceptors. Cells were harvested and resuspended in assay buffer to an O.D. 600 of 0.5.

SR-B2 (mtrB–lacZ)3.033.083.42
SR-C2 (mtrC–lacZ)40.8837.1241.99

Discussion

Metal-reducing bacteria, in contrast to other microorganisms, have to deal with the problem of using insoluble electron acceptors. Although the mechanisms of metal reduction are not yet well understood, available data suggest that the enzymes required for this process are located in the outer membrane. Outer membrane fractions from S. putrefaciens and other metal reducers have been shown to contain c-type cytochromes and to have the majority of the ferric reductase activity. We recently identified an outer membrane protein, MtrB, that is required for metal reduction (Beliaev and Saffarini, 1998). In this paper, we describe two putative decahaem c-type cytochromes, MtrA and MtrC, that are also required for metal reduction in S. putrefaciens MR-1. MtrC, a decahaem c-type cytochrome, appears to be located in the outer membrane. Mutants that lack MtrC exhibit low levels of Fe(III) reduction both in vivo and in vitro. The fact that MtrC is required for wild-type levels of Fe(III) reductase activity in membrane fractions suggests that this cytochrome may be a component of the terminal ferric reductase, assuming that the enzyme is a multisubunit protein. Alternatively, MtrC may be required for the assembly or stability of the reductase. The fact that SR-8 retains some reductase activity may indicate the presence of more than one ferric reductase in wild-type cells, as has been suggested previously (Arnold et al., 1986; 1990).

The gene encoding MtrC is part of an operon that includes mtrA and mtrB. MtrA is a soluble decahaem c-type cytochrome that appears to be a periplasmic protein. Loss of MtrA leads to a deficiency in metal reduction by whole cells. Membrane preparations of mtrA mutants, however, retain the ability to reduce Fe(III) at rates similar to those of the wild type. These results imply that MtrA, although required for metal reduction by intact cells, is not necessary for the activity of the terminal reductase. The location of this cytochrome in the periplasmic space also suggests that MtrA is part of the electron transport chain that leads to Fe(III) reduction and is not involved in the final reduction step.

The mutants SR-A11, SR-21 and SR-A1, which lack MtrA, MtrB or both, respectively, exhibit negligible levels of Fe(III) reduction in vivo compared with the wild-type strain MR-1 (≈1.3–3%). SR-8, which lacks MtrA, MtrB and MtrC, exhibits slightly higher levels of Fe(III) reduction in vivo (13.7% of the wild-type rate) compared with the previously mentioned mutants. These results are surprising. We have shown that mtrC, mtrA and mtrB are co-transcribed, and insertional inactivation of mtrC leads to the loss of all proteins encoded by this operon. Therefore, the mtrCAB mutant SR-8 was expected to have similar levels of Fe(III) reduction to SR-21, which lacks only MtrB. One explanation for this discrepancy is the possible presence of another ferric reductase, as has been suggested earlier by Arnold et al. (1986; 1990). It is possible that the lack of MtrC leads to the activation or induction of a second ferric reductase. This hypothesis is consistent with the observed ferric reductase activity of membrane fractions of strains that lack MtrC. The activity of these membrane fractions is reduced compared with the wild type, but is not completely abolished. We have identified three genes, mtrDEF (GenBank accession no. AF083240), whose amino acid sequences exhibit similarities to MtrA, MtrB and MtrC respectively. It is possible that mtrDEF encodes a second, lower activity ferric reductase. Work is in progress to determine the functions of MtrD, MtrE and MtrF, if any, in metal reduction.

Based on the observations described above, we suggest a model for electron transfer leading to Fe(III) reduction (Fig. 6). Menaquinones, which are intermediates in metal reduction (S. Blumerman and D. A. Saffarini, unpublished), are reduced by a dehydrogenase. Oxidation of menaquinones may lead to the reduction of CymA, a c-type cytochrome that has been shown to be required for fumarate, nitrate and metal reduction (Myers and Myers, 2000). According to our hypothesis, reduced CymA transfers electrons, directly or indirectly, to MtrA. Recently, Gordon et al. (2000) identified a cytochrome c3, Cct, that is involved in Fe(III) reduction in S. frigidimarina. This cytochrome is similar to a tetrahaem cytochrome c3 that has been identified in S. putrefaciens (Tsapin et al., 1996). Cct may act to shuttle electrons between CymA and MtrA. MtrA, a periplasmic c-type cytochrome, may interact with and transfer electrons to MtrC, another putative decahaem c-type cytochrome that is located in the outer membrane. Finally, MtrC, by itself or as part of an enzyme complex, may reduce Fe(III). MtrB, which has been shown to be an outer membrane protein (Beliaev and Saffarini, 1998), contains a putative metal binding site, CXXC, at amino acids 42–45. This protein may play a role in the binding of bacterial cells to the metal particles during anaerobic reduction. Membrane fractions of SR-21, which lack MtrB, are still able to carry out Fe(III) reduction at wild-type levels. Biochemical studies are under way to determine the validity of the model described above. It is important to note that the mutants described in this paper, in addition to their inability to reduce Fe(III), have also lost the ability to reduce Mn(IV). Very little is known about the mechanisms of Mn(IV) reduction. Our results suggest that MtrA, MtrB and MtrC are also involved in Mn(IV) reduction.

Figure 6.

A model that depicts a possible electron transport pathway (arrows) that leads to Fe(III) reduction.

Experimental procedures

Bacterial strains, growth conditions and DNA manipulations

A list of the bacterial strains and plasmids used in this study is given in Table 2. Growth conditions for E. coli and S. putrefaciens strains have been described previously (Beliaev and Saffarini, 1998). DNA isolation, digestion, labelling and other manipulations were performed using standard protocols (Sambrook et al., 1989).

Isolation and analysis of SR-8

Mutagenesis of S. putrefaciens MR-1 using Tn5 and phenotypic analysis of the mutants have been described previously (Beliaev and Saffarini, 1998). A spontaneous rifampicin-resistant strain of S. putrefaciens, MR-1R, was used in the mating experiments to generate the transposon mutants. MR-1R is capable of anaerobic growth with the different electron acceptors used by the wild type. We isolated one mutant, SR-8, based on its inability to reduce Fe(III) and Mn(IV) under anaerobic conditions. SR-8 genomic DNA was digested with BamHI and EcoRI and ligated into pSPORT1 (Gibco BRL Life Technologies). The ligated DNA was used to transform E. coli DH5αMCR, and clones containing SR-8 DNA adjacent to the Tn5 insertion site were selected by plating the transformants on LB agar plates supplemented with 50 µg ml−1 kanamycin (Beliaev and Saffarini, 1998). The initial nucleotide sequence of DNA adjacent to the transposon insertion site was obtained using a 17 base primer complementary to the 5′ end of Tn5 (Beliaev and Saffarini, 1998). Further sequence data were obtained by primer walking using the dideoxy sequencing procedure (Sanger et al., 1977) either manually or using an ABI automated sequencing apparatus (Applied Biosystems). Sequences were analysed using macvector 6.0 and assemblylign software (Oxford Molecular Group). Comparisons with database sequences were made using the blast and fasta algorithms (Altschul et al., 1990; 1997).

Disruption of mtrA

The suicide vector pVIK165 (Kalogeraki and Winans, 1997) was used to disrupt mtrA. An internal fragment of mtrA (258 bp) was amplified by PCR using the primers 258A-F and 258A-R (Table 5). The amplified fragment was cloned into the EcoRV site of pVIK165, and the resulting plasmid, pAB258, was introduced into E. coli S17-1λ(pir) by electroporation. The plasmid was transferred to MR-1R, a rifampicin-resistant strain of S. putrefaciens MR-1, by conjugation. Disruption of mtrA was confirmed by PCR using a primer complementary to the pVIK165 gfp reporter gene (primer GFP-R) and two external primers (258A-F1/258A-R1) flanking the plasmid insertion sites (Table 5).

Table 5. List of primers used in this study.
258A-F5′-TGCGATTGACTCCTCTAAGAG-3′258A-R5′-AGGATCTTTTGCGACGTG-3′
258A-F15′-GCGTCGAAGTGGGATGAG-3′258A-R15′-CATAGCTGTGGCGCACGG-3′
GFP-R5′-CTCATCTTTGTTTCCTCCG-3′LAC-R5′-AGGTCGACCATGGTCATAGC-3′
212F65′-GAGGAGTCAATCGCACC-3′212F155′-GATGCTAACCTGCTGGCT-3′
1723C-F5′-GTGGTGAGCCAGCAGGTAG-3′1723C-R5′-GTTGCAGTGATCGGCGTAG-3′
734A-F5′-GCGTCGAAGTGGGATGAG-3′734A-R5′-CATAGCTGTGGCGCACGG-3′
1794B-F5′-GCGTGAAGAGAAAACCGG-3′1794B-R5′-TTCTGACTGAATGTGCAG-3′
717B-U5′-ACCGTGTCGCTGATGGG-3′463C-U5′-GGCAAAAAACTGGTCACG-3′
717B-F5′-AGAATTCCGGTAACCGTGACGGCTCAC-3′  
717B-R5′-AGGTACCGTAACTCATGCTCAGCATCAG-3′  
463C-F5′-AGAATTCACTAAGTATACCGGCATG-3′  
643C-R5′-AGGTACCGTTGCAGTGATCGGCGTAG-3′  

The broad-host-range plasmid pRK415 (Keen et al., 1988) was used for complementation studies of S. putrefaciens mutants. A 2.9 kb fragment carrying mtrB was cloned into pRK415 to generate pSC29. This plasmid was introduced into the mutant SR-A1 by conjugation. Recombinant colonies were selected on LB agar plates supplemented with tetracycline (17 µg ml−1) and kanamycin (25 µg ml−1).

Construction and assay of lacZ transcriptional fusions

Transcriptional fusions in MR-1R were constructed using the suicide vector pVIK112. Fragments that correspond to the 3′ end of mtrB and mtrC, 717 and 463 bp, respectively, were amplified by PCR. The primers, 717B-R/717B-F and 463C-R/463C-F, were designed to include restriction sites for EcoRI and KpnI. The amplified fragments were digested with EcoRI and KpnI, then ligated into pVIK112 to yield pAB717 (containing the 3′ end of mtrB) and pAB463 (containing the 3′ end of mtrC). The plasmids were transferred into S. putrefaciens MR-1R by conjugation, and kanamycin-resistant colonies were selected. Two recombinant S. putrefaciens strains were isolated and designated SR-B2 and SR-C2. The integration of pAB717 in SR-B2 and of pAB463 in SR-C2 was confirmed by PCR using primers complementary to the pVIK112 lacZ reporter gene (primer LAC-R) and to regions upstream of the plasmid insertion sites (primers 717B-U and 463C-U; Table 5).

To determine the levels of β-galactosidase activity, SR-B2 and SR-C2 were grown anaerobically to mid-log phase in LB supplemented with 10 mM fumarate, 10 mM ferric citrate or 2 mM sodium nitrate. Cells were harvested by centrifugation and resuspended in RLB buffer (Promega) to an OD600 of 0.5. Cells were lysed by the addition of Triton X-100 to 0.1% followed by sonication. β-Galactosidase activity was measured using the β-galactosidase assay system (Promega) according to the manufacturer's instructions.

Promoter analysis

Total RNA from S. putrefaciens was isolated using the Totally RNA kit (Ambion). RNA was treated with 100 units ml−1 RNase-free RQ1 DNase (Promega) for 30 min at 37°C to remove contaminating DNA. The samples were determined to be DNA free when a product was not detected after 35 cycles of PCR using primers 258A-F and 258A-R (Table 5) and total RNA as a template.

Promoter analysis was performed by the ‘anchored’ PCR technique using the 5′ RACE system (Frohman, 1993; Gibco BRL Life Technologies). Primers complementary to mtrC and mtrA, 212F15 and 212F6, respectively (Table 5), were used to prime cDNA synthesis. 212F6 lies 450 bases downstream of the mtrA AUG start codon, whereas 212F15 lies 160 bases downstream of the mtrC initiation codon. PCR was used to amplify the cDNA, and the fragments were sequenced to determine the transcription start sites.

Anaerobic reduction and enzyme assays

Anaerobic growth with, and reduction of, TMAO, DMSO, nitrate, nitrite, thiosulphate, sulphite, fumarate, Fe(III) and Mn(IV) by S. putrefaciens strains were assayed as described previously (Beliaev and Saffarini, 1998). Membrane fractions for in vitro Fe(III) reductase assays were prepared as follows. Cells were grown anaerobically to mid-log phase in LB supplemented with 10 mM fumarate and 10 mM lactate. The cells were harvested and lysed using a French press. The lysed cells were centrifuged at 14 000 r.p.m., and the supernatant was subjected to ultracentrifugation at 50 000 r.p.m. for 1 h. The pellet (membrane fraction) was resuspended in 10 mM Tris, pH 7.5, by brief sonication. Fe(III) reductase activity in membrane fractions was measured using 10 mM formate, 100 mM Tris-HCl, pH 7.5, 5% glycerol, 1 mM ferrozine and 0.2 mM ferric citrate. The chromophore formed by ferrous iron and ferrozine was measured at 562 nm (Moody and Dailey, 1983).

To detect the presence of c-type cytochromes, cells were centrifuged and the pellets resuspended in SDS loading buffer. The samples were boiled for 3 min and separated by SDS–PAGE using 10% polyacrylamide gels. Haem stains were performed using 3,3′,5,5′-tetramethyl benzidine dihydrochloride as described by Thomas et al. (1976).

Overexpression and purification of recombinant MtrC, MtrA and MtrB proteins

Fragments that corresponded to 1723 bp, 734 bp and 1794 bp of mtrC, mtrA and mtrB were amplified by PCR using the primers 1723C-F/1723C-R, 734 A-F/734A-R and 1749B-F/1749B-R respectively (Table 5). The fragments were cloned into pCR-Script and then subcloned into the expression vector pET 30+. The in frame ligation of the fragments, resulting in N-terminal fusions between the His6-tag and the recombinant MtrC, MtrA and MtrB, was verified by sequencing. The three plasmids, pETA2, pETB6 and pETC4, that contain mtrA, mtrB and mtrC, respectively, were introduced into E. coli BL21 (Novagen). The recombinant E. coli strains were grown at 37°C for 2–3 h (OD600 of 0.6). Expression was induced by the addition of IPTG to a final concentration of 1 mM, followed by incubation for an additional 2 h. The induced cells were then harvested by centrifugation for 10 min at 5000 g at 4°C and stored at −80°C until use. Proteins were purified using the His*Bind kit (Novagen) according to the manufacturer's instructions. The eluted proteins were desalted and concentrated by ultrafiltration using Ultrafree-15 microconcentrators with an exclusion limit of 10 000 molecular weight (Millipore).

Western blotting

Rabbit polyclonal antibodies against the recombinant MtrA, MtrB and MtrC were raised at the University of Massachusetts (Amherst) Animal Care Facility. Cell fractionation and separation of inner and outer membrane fractions were performed as described previously (Beliaev and Saffarini, 1998). Proteins were resolved on SDS polyacrylamide gels and transferred to Immun-Blot polyvinylidene difluoride (PVDF) membranes using a Trans-Blot electrophoretic transfer cell (Bio-Rad). The membranes were incubated with a 1:1000 to 1:5000 dilution of affinity-purified polyclonal rabbit antisera raised against recombinant MtrC, MtrA or MtrB, and the reactive bands were visualized with an Opti-4CN substrate kit (Bio-Rad).

Acknowledgements

This work was supported by National Science Foundation grant MCB 9604298 and a University of Wisconsin–Milwaukee Faculty award. We thank M. McBride for helpful comments and critical reading of the manuscript.

Footnotes

  1. Present addresses: Oak Ridge National Laboratory, Oak Ridge, TN, USA. Immonogen, Inc., Cambridge, MA, USA.

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