INTRODUCTION

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Shewanella putrefaciens MR-1 is a gram-negative facultative anaerobe with remarkable respiratory plasticity that can couple its anaerobic growth, and link respiratory proton translocation, to the reduction of a variety of compounds including manganese(IV) oxides, iron(III) oxides, fumarate, nitrate, trimethylamine N-oxide (TMAO), and many others (30, 32-34, 38, 40). Previous studies suggested that this bacterium contains a potentially complex multi-component branched electron transport system that includes cytochromes, quinones, dehydrogenases, and iron-sulfur proteins (30-36, 38, 40, 41). Some electron transport components are common to the use of several electron acceptors (32, 34), while others are unique to specific electron acceptors (35).

The mutant CMTn-1, which was previously generated by transposon mutagenesis of the rifampin-resistant derivative MR-1A (32), cannot use nitrate, Fe(III), or fumarate as electron acceptors, and its ability to reduce Mn(IV) is markedly compromised; it retains the ability to use TMAO and O₂ as electron acceptors (32, 34). This is the same electron acceptor phenotype as that of the menaquinone-deficient mutant CMA-1 (32). In fact, compared to MR-1, CMTn-1 is markedly deficient in menaquinones, although unlike CMA-1 it cannot be restored to a wild-type phenotype by supplementation with menaquinone or menaquinone precursors (32). Subsequent characterization of CMTn-1 demonstrated that the transposon interrupted the cymA gene, which encodes a 21-kDa tetraheme cytochrome c (34). The wild-type cymA gene complements CMTn-1, restoring the CymA cytochrome and the cells' ability to utilize nitrate, Fe(III), Mn(IV), and fumarate (34); the complemented mutant, however, still has very low levels of menaquinone. The phenotype of CMTn-1 is therefore apparently due to the loss of the CymA cytochrome and not to a deficiency in menaquinones. However, two questions regarding the mutant CMTn-1 remain: (i) what is the reason for the menaquinone deficiency in CMTn-1? and (ii) would the phenotype of a cymA knockout be the same if menaquinone levels were normal? The latter is particularly important because the putative role of CymA orthologs in other bacteria (e.g., NapC of Thiosphaera pantotropha) is to accept electrons from membrane-bound quinones and transfer them to downstream electron transport components (e.g., nitrate reductase) (5).

This report demonstrates that menaquinone levels are markedly depressed in the spontaneous rifampin-resistant mutant MR-1A, which served as the parent of CMTn-1. This suggests that the rifampin resistance, and not the cymA knockout, is responsible for the depressed menaquinone levels in CMTn-1. This report also describes the isolation of a cymA knockout directly from MR-1 by a site-directed gene replacement approach. This cymA knockout (MR1-CYMA) has the same electron transport phenotype as CMTn-1 but has menaquinone levels comparable to those of the wild-type MR-1. The electron transport phenotype of MR1-CYMA can be restored to that of the wild-type by complementation with the cymA gene. The requirement for CymA in anaerobic electron transport to Fe(III), fumarate, nitrate, and Mn(IV) is therefore not dependent on the levels of menaquinone in these cells.

	MATERIALS AND METHODS

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Materials. All materials were from sources previously described (34) with the following exceptions. The Expand High Fidelity PCR system and Expand Long Template PCR system were from Boehringer-Mannheim (Indianapolis, Ind.). Restriction enzymes and biotinylated RNA markers were from New England BioLabs (Beverly, Mass.). Custom oligonucleotide primers were synthesized by Operon Technologies (Alameda, Calif.) or by Genemed Biotechnologies (South San Francisco, Calif.). Vitamin K₂ (menaquinone-4, MK-4) and coenzyme Q₆ (ubiquinone-6) and Q₁₀ (ubiquinone-10) standards were obtained from Sigma Chemical Co. (St. Louis, Mo.), and 1,4-dihydroxy-2-naphthoic acid (DHNA) was obtained from Aldrich Chemical (Milwaukee, Wis.).

Bacterial strains, plasmids, media, and growth conditions. A list of the bacteria and plasmids used in this study is presented in Table 1. For molecular biology purposes, S. putrefaciens and Escherichia coli were grown aerobically on Luria-Bertani (LB) medium (49) supplemented, when required, with antibiotics at the following concentrations: ampicillin, 50 µg ml¹; kanamycin, 50 µg ml¹; chloramphenicol, 34 µg ml¹; rifampin, 50 µg ml¹. E. coli was grown at 37°C unless indicated otherwise. S. putrefaciens was grown at room temperature (23 to 25°C), except where indicated otherwise, under either aerobic or anaerobic conditions as previously described (30) in defined medium (40) supplemented with 15 mM lactate. For anaerobic growth or for testing electron acceptor use, the medium was also supplemented with vitamin-free Casamino Acids (0.1 g liter¹) and with one of the following electron acceptors: 20 mM TMAO, 20 mM fumarate, 10 mM ferric citrate, 2 mM nitrate, or 5 mM MnO₂. For growth on TMAO, the medium was also supplemented with 30 mM HEPES to buffer against alkalinization by the product trimethylamine. Antibiotics were included when needed at the concentrations listed above.

DNA manipulations. Restriction digests and mapping, cloning, subcloning, and DNA electrophoresis were done according to standard techniques (49) following manufacturers' recommendations as appropriate. DNA ligations were done with Fast-Link DNA ligase (Epicentre Technologies, Madison, Wis.) or T4 DNA ligase (Life Technologies, Rockville, Md.). Isolation of plasmid and cosmid DNA was accomplished with the QIAprep Spin Plasmid kit (Qiagen, Chatsworth, Calif.). The sizes of DNA fragments, RNA, and proteins were estimated based on their relative electrophoretic mobilities to known standards by using a computer program kindly provided by G. Raghava (46). Colony PCR (51) with primers specific to cymA was utilized to screen transconjugants for gene replacement events.

Northern blotting and RT-PCR. Total RNA was purified from anaerobically grown cells by a hot phenol method, followed by treatment with RNase-free DNase as previously described (34). All standard precautions to prevent RNase contamination were followed (49). Electrophoresis of RNA and Northern blot analysis were done as previously described (34) with the following exceptions. Immediately after electrophoresis, the gel was sequentially soaked (20 min each) in 50 mM NaOH-150 mM NaCl and 150 mM NaCl-100 mM Tris-HCl (pH 7.5). The RNA was transferred to a Magna (MSI, Westboro, Mass.) nylon membrane (0.45 µm pore size) by using 10× SSC (1× SSC is 0.15 M NaCl plus 15 mM sodium citrate) and the Posiblot pressure blotter (Stratagene, La Jolla, Calif.) at 70 to 75 mm Hg pressure for 1 h. The RNA was fixed to the membrane by UV cross-linking (33 mJ cm²). The membrane was prehybridized, hybridized, and washed as previously described (34) prior to development with the Phototope Star detection system (New England BioLabs). Biotinylated DNA probes were generated with a PCR-based labeling system (25).

Quinone analysis. Quinones were extracted from the cells essentially as described previously (21). The cells were harvested by centrifugation, and the cell pellets were washed in cold 20 mM MgCl₂-0.1 M triethanolamine-HCl (pH 7.2); the washed pellets were resuspended in this buffer (1.0 ml of buffer per 0.6 g wet cell weight). Per 0.6 g of cells, 6.0 ml of methanol-acetone (1/1 [vol/vol]) was added to the cell suspension, followed by vigorous agitation and incubation at room temperature for 30 min. Petroleum ether (2.0 ml) was added, followed by vigorous agitation. Following a 1-min centrifugation at 2,800 × g at 15°C to break the phases, the upper layer was removed; the lower layer was reextracted with another 2.0 ml of petroleum ether, followed by centrifugation and recovery of the upper layer. The upper layers were pooled and evaporated under a stream of nitrogen at 37°C. The dried residue was dissolved in chloroform-methanol (2/1 [vol/vol]; 50 µl per 0.6 g original wet cell weight).

1

Miscellaneous procedures. Growth was assessed by measuring culture turbidity at 500 nm in a Beckman DU-64 spectrophotometer. Nitrate (9) and nitrite (52) levels were determined colorimetrically in cell-free filtrates. The Fe(II) level was determined by a ferrozine extraction procedure (23, 39). The Mn(II) level was determined in filtrates by a formaldoxime method (3, 7), and MnO₂ was prepared as described previously (38).

	RESULTS

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Construction of cymA insertion mutation. While an early report suggested that pACYC184 (which contains the p15A origin of replication) could be used as a suicide vector in MR-1 (48), this was subsequently shown not to be the case (37). For these studies we utilized the mobilizable vector pEP185.2; it contains the R6K origin of replication, which requires the pir gene product for replication (20); pir is carried on the chromosome of the E. coli donor strain S17-1 pir. In several matings of E. coli S17-1 pir/pEP185.2 with MR-1, we were unable to obtain any MR-1 cells which survived chloramphenicol selection because of resistance encoded by the cat gene of pEP185.2. These results suggested that pEP185.2 is not maintained in MR-1 and could therefore be useful as a suicide vector for gene replacement strategies.

Characterization and complementation of the cymA insertion mutant. A cymA probe detected RNA bands of approximately 900 and 1,200 bases in MR-1 cells grown anaerobically with TMAO as the electron acceptor (Fig. 1A). These two bands were previously detected in Northern blot analysis of total RNA from MR-1 cells grown anaerobically with fumarate or TMAO as the electron acceptor (34); the smaller band was predominant in fumarate-grown cells, and the larger band could represent a cymA transcript generated from a different transcriptional start site, or it could represent a larger mRNA precursor (34). Even though the cymA transcript is more prominent in fumarate-grown cells (34), we used TMAO-grown cells to provide for direct comparisons because MR1-CYMA will not grow on fumarate (see below). These two cymA RNA bands were also detected in MR-1 cells containing the control vector pVK100, but they were absent in the gene replacement mutant MR1-CYMA (Fig. 1A). Plasmid pCMTN1-VK (34) is pVK100 containing an insert consisting of the 561-bp cymA gene and 262 and 166 bp of the associated 5' and 3' DNA from MR-1, respectively (i.e., the insert spans bases 604 to 1592 of the published sequence [34]). Plasmid pCMTN1-VK restored the 900-base cymA transcript to MR1-CYMA (Fig. 1A). Since the levels of the cymA transcript are low in TMAO-grown cells, RT-PCR analysis of total RNA from these strains was done with primers bracketing bases 950 to 1290 of the published sequence (34). RT-PCR analysis confirmed the presence of the expected 341-bp RT-PCR product in MR-1 and MR-1/pVK100, as well as its absence in MR1-CYMA (Fig. 1B); plasmid pCMTN1-VK restored this 341-bp RT-PCR product, derived from the cymA transcript, to MR1-CYMA (Fig. 1B).

View larger version (45K): [in this window] [in a new window]   FIG. 1.   Expression of cymA in S. putrefaciens cells grown anaerobically with TMAO as the electron acceptor as determined by Northern blotting (A) and RT-PCR (B). The lanes contain samples from the following strains: lanes 1, MR1-CYMA(pCMTN1-VK); lanes 2, MR1-CYMA(pVK100); lanes 3, MR-1(pVK100); lanes 4, MR-1. (A) Northern blot of 100 µg of total RNA from each strain hybridized with a biotin-labelled cymA probe (representing bases 604 to 1588 of the published sequence [34]). No bands were seen in lane 2 with extended exposure times. Lane M, 3 µl of biotinylated RNA markers (New England BioLabs), with their sizes in bases indicated at the right; the position of each marker was assessed from a short exposure. The major cymA RNA is indicated by the arrow at the left. (B) RT-PCR of 2 µg of total RNA from each strain with primers specific for cymA. The sizes of the DNA markers (lane M) in kilobases are indicated at the right. The cymA product from RT-PCR is indicated by the arrow at the left.

View larger version (37K): [in this window] [in a new window]   FIG. 2.   Heme-stained SDS-PAGE profiles of subcellular fractions prepared from S. putrefaciens cells grown anaerobically with TMAO as the electron acceptor. The lanes were loaded with 50 µg of protein of each of the following subcellular fractions: cytoplasmic membrane (lanes 1 to 3), soluble fraction (lanes 4 to 6), and outer membrane (lanes 7 to 9). Fractions were prepared from MR-1/pVK100 (lanes 1, 4, and 7), MR1-CYMA/pVK100 (lanes 2, 5, and 8), and MR1-CYMA/pCMTN1-VK (lanes 3, 6, and 9). The bars and numbers at the right indicate the migration and masses of the protein standards.

Quinone analysis of various strains. TLC analysis of quinones from the various strains (MR-1, MR-1/pVK100, MR1-CYMA/pVK100, MR1-CYMA/pCMTN1-VK) demonstrated that there were no apparent significant differences in ubiquinone or menaquinone content (Fig. 3, lanes 3 to 6). Based on HPLC analysis of extracts pooled from two or more cultures, levels of menaquinone (21 to 23 nmol per g of wet cells), methylmenaquinone (4.3 to 5.0 nmol per g of wet cells), and total ubiquinones (5.9 to 7.2 nmol per g of wet cells) were similar for all four strains. The spectra of the quinones in spots I and II were typical for menaquinones, whereas those in spots III to V were typical for ubiquinones (Fig. 4). Therefore the knockout of cymA in MR1-CYMA is solely responsible for the electron transport phenotype, as this mutant retains wild-type levels of menaquinone. This is in contrast to the marked deficiency of menaquinones in the previous mutant, CMTn-1, and its parent, MR-1A (Fig. 3, lanes 7 and 8). Based on HPLC analysis, MR-1A and its derived strains contained similar levels of total ubiquinones (4.0 to 7.2 nmol per g of wet cells), whereas menaquinone and methylmenaquinone were not detected. This suggests that the spontaneous rifampin resistance in MR-1A is somehow responsible for the menaquinone deficiency in CMTn-1 and that it is not related to the knockout of cymA in CMTn-1. Even though the introduction of the wild-type cymA gene into CMTn-1 did restore its ability to reduce Mn(IV), Fe(III), nitrate, and fumarate (34), the levels of menaquinones remained markedly depressed in CMTn-1/pCMTN1-VK (Fig. 3, lane 9). It is clear that the presence or absence of CymA does not influence the levels of menaquinones. Conversely, the levels of menaquinones do not affect the overall electron acceptor phenotype of a cymA knockout.

View larger version (98K): [in this window] [in a new window]   FIG. 3.   Thin-layer chromatogram of quinone standards and of quinones isolated from S. putrefaciens cells grown anaerobically with TMAO as the electron acceptor. Lanes 1 and 2 were loaded with ubiquinone-6 and menaquinone-4 standards, respectively. The other lanes were loaded with quinone extracts (equivalent to 0.10 g of wet cells) isolated from cells of the following strains: lane 3, MR-1; lane 4, MR-1/pVK100; lane 5, MR1-CYMA/pVK100; lane 6, MR1-CYMA/pCMTN1-VK; lane 7, MR-1A; lane 8, CMTn-1/pVK100; lane 9, CMTn-1/pCMTN1-VK. The samples were loaded at the bottom, and migration was upward. The quinone standards did not exactly migrate with the quinones from S. putrefaciens because the length and composition of the isoprenyl side chain affect the migration. In a previous report (32), spots I and II were identified as methylmenaquinone and menaquinone, respectively, because of their migration relative to the identified quinones in S. putrefaciens ATCC 8071 (14). Spots III, IV, and V were identified as ubiquinones by a similar comparison.

View larger version (18K): [in this window] [in a new window]   FIG. 4.   UV absorption spectra for quinone peaks. The quinone spots were recovered from the TLC plate (Fig. 3) and were subjected to HPLC analysis with diode array detection. Each quinone eluted as a single peak. The spectra for the quinones in spots I and II are typical menaquinone spectra, with max at ~245, 270, and 330 to 340 nm (42), while those of spots III, IV, and V are consistent with ubiquinones with a max of ~275 nm.

View larger version (20K): [in this window] [in a new window]   FIG. 5.   (A) Thin-layer chromatogram of quinone standards and of quinones isolated from S. putrefaciens cells grown anaerobically with fumarate as the electron acceptor. Lanes 1 and 2 were loaded with ubiquinone-6 and menaquinone-4 standards, respectively. The other lanes were loaded with quinone extracts isolated from cells of the following strains: lane 3, MR-1 (equivalent to 0.18 g of wet cells); lane 4, MR-1A (equivalent to 0.72 g of wet cells). The samples were loaded at the bottom, and migration was upward. The numbered quinone spots were recovered from the TLC plate and subjected to HPLC analysis using diode array detection. Each quinone eluted as a single predominant peak, and the UV absorption spectra of the quinone peaks from MR-1A are shown (B). The spectra for the quinones in spots I and II are typical menaquinone spectra, with max at ~245, 270, and 330 to 340 nm (42). The spectrum for spot VI (not shown) was consistent with ubiquinone with a single max of 275 nm (42).

View larger version (20K): [in this window] [in a new window]   FIG. 6.   Anaerobic growth of MR-1 versus MR-1A (A) or MR-1B (B) cells on fumarate. The medium was supplemented with vehicle (control; dimethylformamide only), MK-4 (10 µM), or DHNA (10 µM). Growth was measured by increases in optical density (OD) measured at 500 nm with a Beckman DU-64 spectrophotometer. Points represent the means for n = 2, and bars represent the range of high and low values; for points lacking apparent range bars, the bars were smaller than the points as shown.

View larger version (69K): [in this window] [in a new window]   FIG. 7.   Thin-layer chromatogram of quinone standards and of quinones isolated from S. putrefaciens cells grown anaerobically with fumarate as the electron acceptor and supplemented with either DHNA or MK-4. Standards were loaded in lanes 1 to 3 as follows: lane 1, ubiquinone-10; lane 2, DHNA; lane 3, menaquinone-4. The other lanes were loaded with quinone extracts (equivalent to 0.18 g of wet cells) isolated from cells of the following strains: lanes 4 to 6, MR-1; lanes 7 to 9, MR-1A; lanes 10 to 12, MR-1B. During cell growth, the medium was supplemented with 10 µM DHNA (lanes 5, 8, and 11), 10 µM MK-4 (lanes 6, 9, and 12), or the solvent for these quinones as a control (lanes 4, 7, and 10). The samples were loaded at the bottom, and migration was upward. As described above for MR-1, spots I and II are methylmenaquinone and menaquinone, respectively, and spots III, IV, and V are ubiquinones. The prominent presence of MK-4 in cells grown in the presence of MK-4 is indicated at the right. The DHNA standard (15 nmol; lane 2) was not visually detectable under these conditions. The spectra for the spots indicated by VII (not shown) were consistent with ubiquinone.

	DISCUSSION

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The interpretation of previous studies with the cymA mutant CMTn-1 (34) were complicated by its low levels of menaquinones (32), which could have contributed to its phenotype. This issue required clarification because the putative role of CymA orthologs in other bacteria is to accept electrons from membrane-bound quinones and transfer them to downstream electron transport components (5, 17, 24). The results in this study clarify the role of CymA in anaerobic electron transport in MR-1. The menaquinone levels in the mutant MR1-CYMA are the same as those in the wild type, and yet the knockout of cymA renders the cells unable to reduce nitrate, Fe(III), and Mn(IV) and unable to grow anaerobically with fumarate as the electron acceptor. Complementing MR1-CYMA with wild-type cymA restored its phenotype to that of the wild type. It seems likely that CymA is a common component in the electron transport chains for at least these four anaerobic electron acceptors and that these chains diverge to separate components somewhere after CymA. CymA is not required for aerobic growth or for anaerobic growth coupled to TMAO reduction. Since the CymA-deficient mutant of MR-1 bears the same electron transport deficiencies as the menaquinone-deficient mutant CMA-1 (which is restored to a wild-type phenotype by DHNA, a precursor in the menaquinone synthesis pathway) (32), it is possible that CymA is somehow involved in the transfer of electrons from menaquinone to other electron transport components. One possible role for CymA is to transfer electrons to components which are localized in the periplasm and/or outer membrane. For example, the majority of Fe(III) reductase activity in MR-1 is localized in the outer membrane (31). MR-1 localizes a majority of its membrane-bound cytochromes to the outer membrane when grown under anaerobic conditions (30), and one or more of these outer membrane cytochromes could be part of the electron transport chain for the reduction of Fe(III) and/or Mn(IV) (36). CymA is also necessary for fumarate reduction, which is catalyzed by a periplasmic enzyme in S. putrefaciens (26, 29, 44). However, CymA does not likely serve as a terminal reductase for these various oxidants, e.g., a mutant lacking the 65-kDa fumarate reductase is only deficient in anaerobic growth on fumarate (35).

Prior to this report, a successful suicide vector approach had not been described for MR-1. One report indicated that pACYC184 could not replicate in MR-1 (48), but this was subsequently disproven (37). Another report described the use of pJQ200KS to generate a gene replacement knockout of the fumarate reductase of S. putrefaciens (now S. frigidimarina [47]) NCIMB 400 (13). This use cannot be extrapolated to MR-1 because MR-1 maintains pJQ200KS as a free replicon (C. Myers, unpublished data); this is not surprising because it contains the p15A origin of replication from pACYC184 (45). The use of pEP185.2 was successful in the present study, and the plasmid should prove useful as a suicide vector for the directed replacement of other genes in MR-1.

Quinone analyses of several other strains of S. putrefaciens, including the type strain, ATCC 8071, have been reported. Most strains synthesize two or three ubiquinones (Q-6, Q-7, Q-8) (1, 27, 42), consistent with the three ubiquinones seen in our TLC analysis of MR-1 (Fig. 3). In our previous studies (32), these same three ubiquinones were present and were identified based on relative migration in TLC; the HPLC spectral analysis (_max at 275 nm) is consistent with their identity as ubiquinones (42). Other strains of S. putrefaciens synthesize two predominant menaquinones, MK-7 and methylmenaquinone-7 (MMK-7), while a few strains also produce small amounts of MK-8 (1, 27, 42). Our TLC analysis demonstrated two prominent menaquinones in MR-1, which were previously designated MK and MMK based on relative migration in TLC (32); the HPLC spectral analysis (_max at 245, 270, and 330 to 340 nm) is consistent with their identity as MK and MMK (42).

The exact reason for the depressed menaquinone levels in the spontaneous Rf^r strain MR-1A is not known. However, they are not due to the presence of rifampin itself as MR-1A cells grown in the absence of rifampin also have markedly depressed menaquinone levels (not shown). The decreased menaquinone levels could be due to changes in the RNA polymerase. Rifampin binds the  (RpoB) subunit of RNA polymerase, and several mutations in rpoB are associated with rifampin resistance (6, 15). In other species, mutations in rpoB leading to rifampin resistance can have pleiotropic effects, including effects on promoter clearance, RNA elongation, transcriptional termination, paused transcription complexes, and DNA supercoiling (11, 12, 16). While the core RNA polymerase (₂') carries out elongation and termination of RNA synthesis, the addition of a  subunit to make the holopolymerase (₂') is required for initiation of RNA synthesis (54). The  subunit of the holoenzyme plays a prominent role in the recognition of promoters (54), and most eubacteria synthesize several different  factors which direct RNA polymerase to distinct promoters (54). The effect of rifampin on gene expression can vary significantly depending on the  factor which initiates transcription from that gene (53). Some mutations in rpoB can lead to decreased transcription of certain  factors, thereby affecting the expression of several genes (56). While the exact rpoB mutation responsible for the Rf^r phenotype of MR-1A is unknown at this time, it is plausible that it could be responsible for decreased transcription of one or more of the genes required for menaquinone synthesis. The addition of MK-4 or DHNA to the medium significantly improved the rate of growth of MR-1A on fumarate as well as the menaquinone content of these cells (Fig. 6 and 7).

To the best of our knowledge, this is the first report of a spontaneous rifampin-resistant mutant that leads to markedly depressed levels of menaquinones. This marked effect on menaquinone levels is of concern because spontaneous Rf^r mutants are frequently used as recipients in mating protocols designed to generate mutants. Even though the reduced levels of menaquinones in the Rf^r strains (MR-1A and CMTn-1) did not affect the ultimate electron acceptor phenotype of a cymA knockout, effects related to the Rf^r phenotype must be considered possible contributing factors to the phenotypes of other Shewanella mutants, e.g., those lacking fumarate reductase (13, 35) or MtrB (a putative outer membrane protein required for Fe(III) and Mn(IV) reduction) (4). Even if menaquinone levels are not affected by other rpoB mutations, other as yet uncharacterized effects must be considered. Caution in interpreting the phenotypes of Shewanella mutants derived from Rf^r strains is therefore warranted. Given the potential for rpoB mutations to affect paused transcriptional complexes and transcriptional termination, particular caution is advised in interpreting the phenotypes resulting from mutations of genes arranged in operons, e.g., mtrAB of MR-1 (4).

The interpretation of results for the previously described mutant CMA-1 are not complicated by Rf^r effects, as it was isolated directly from MR-1 by acridine orange mutagenesis (32). CMA-1 is MK deficient and cannot reduce Fe(III), Mn(IV), or nitrate or grow anaerobically with fumarate. Supplementation of the medium with either MK-4 or DHNA restores the presence of MK in CMA-1 and its ability to utilize nitrate, Fe(III), Mn(IV), and fumarate (32). A recent menC mutant isolated directly from MR-1 confirmed this role for MK (D. K. Newman and R. G. Kolter, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. K-17, p. 404, 1999), i.e., it is deficient in o-succinylbenzoic acid synthase, an enzyme involved in menaquinone synthesis; similar to CMA-1, it could not reduce fumarate, Fe(III), or Mn(IV) and was restored to the wild-type phenotype by supplementation of the medium with DHNA (Newman and Kolter, Abstr. 99th Gen. Meet. Am. Soc. Microbiol.).

In summary, by using a site-directed gene replacement approach, a cymA knockout strain (MR1-CYMA), which was derived directly from MR-1, lost the ability to reduce Fe(III), Mn(IV), and nitrate and to grow with fumarate as an electron acceptor. All of these functions, were restored to wild-type efficacy, and the presence of the cymA transcript and CymA protein was also restored, by complementation with the cymA gene. However, unlike the previous mutant CMTn-1, MR1-CYMA retained menaquinone levels comparable to those of the wild type, thereby providing a definitive demonstration of the requirement for CymA in anaerobic electron transport associated with at least four different electron acceptors. This also represents the first successful use of a suicide vector for directed gene replacement in MR-1. Furthermore, the depressed menaquinone levels in CMTn-1 were the result of the Rf^r phenotype of the parent strain, MR-1A, and were unrelated to the interruption of the cymA gene. Rifampin-resistant strains of Shewanella should be used with caution, particularly when analyzing electron transport processes.