Isolation of anaerobic respiratory mutants of Shewannella putrefaciens and genetic analysis of mutants deficient in anaerobic growth on Fe3+.

A genetic approach was used to study (dissimilatory) ferric iron (Fe3+) reduction in Shewanella putrefaciens 200. Chemical mutagenesis procedures and two rapid plate assays were developed to facilitate the screening of Fe3+ reduction-deficient mutants. Sixty-two putative Fe3+ reduction-deficient mutants were identified, and each was subsequently tested for its ability to grow anaerobically on various compounds as sole terminal electron acceptors, including Fe3+, nitrate (NO3-), nitrite (NO2-), manganese oxide (Mn4+), sulfite (SO3(2-)), thiosulfate (S2O3(2-)), trimethylamine N-oxide, and fumarate. A broad spectrum of mutants deficient in anaerobic growth on one or more electron acceptors was identified. Nine of the 62 mutants (designated Fer mutants) were deficient only in anaerobic growth on Fe3+ and retained the ability to grow on all other electron acceptors. These results suggest that S. putrefaciens expresses at least one terminal Fe3+ reductase that is distinct from other terminal reductases coupled to anaerobic growth. The nine Fer mutants were conjugally mated with an S. putrefaciens genomic library harbored in Escherichia coli S17-1. Complemented S. putrefaciens transconjugants were identified by the acquired ability to grow anaerobically on Fe3+ as the sole terminal electron acceptor. All recombinant cosmids that conferred the Fer+ phenotype appeared to carry a common internal region.     

Isolation and characterization of a transposon mutant of Shewanella putrefaciens MR-1 deficient in fumarate reductase

A transposon mutant, designated CMTn-3, of Shewanella putrefaciens MR-1 that was deficient in fumarate reduction was isolated and characterized. In contrast to the wild-type, CMTn-3 could not grow anaerobically with fumarate as the electron acceptor, and it lacked benzyl viologen-linked fumarate reductase activity. Consistent with this, CMTn-3 lacked a 65 kDa c -type cytochrome, which is the same size as the fumarate reductase enzyme. CMTn-3 retained the wild-type ability to use nitrate, iron(III), manganese(IV) and trimethylamine N -oxide (TMAO) as terminal electron acceptors. The results indicate that the loss of the fumarate reductase enzyme does not affect other anaerobic electron transport systems in this bacterium.     

The tetraheme cytochrome CymA is required for anaerobic respiration with dimethyl sulfoxide and nitrite in Shewanella oneidensis

The tetraheme c-type cytochrome, CymA, from Shewanella oneidensis MR-1 has previously been shown to be required for respiration with Fe(III), nitrate, and fumarate [Myers, C. R., and Myers, J. M. (1997) J. Bacteriol. 179, 1143-1152]. It is located in the cytoplasmic membrane where the bulk of the protein is exposed to the periplasm, enabling it to transfer electrons to a series of redox partners. We have expressed and purified a soluble derivative of CymA (CymA(sol)) that lacks the N-terminal membrane anchor. We show here, by direct measurements of electron transfer between the purified proteins, that CymA(sol) efficiently reduces S. oneidensis fumarate reductase. This indicates that no further proteins are required for electron transfer between the quinone pool and fumarate if we assume direct reduction of CymA by quinols. By expressing CymA(sol) in a mutant lacking CymA, we have shown that this soluble form of the protein can complement the defect in fumarate respiration. We also demonstrate that CymA is essential for growth with DMSO (dimethyl sulfoxide) and for reduction of nitrite, implicating CymA in at least five different electron transfer pathways in Shewanella.     

Anaerobic respiratory growth of Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi with trimethylamine N-oxide, nitrate and fumarate: ecological implications

Two symbiotic species, Photobacterium leiognathi and Vibrio fischeri, and one non-symbiotic species, Vibrio harveyi, of the Vibrionaceae were tested for their ability to grow by anaerobic respiration on various electron acceptors, including trimethylamine N-oxide (TMAO) and dimethylsulphoxide (DMSO), compounds common in the marine environment. Each species was able to grow anaerobically with TMAO, nitrate or fumarate, but not with DMSO, as an electron acceptor. Cell growth under microaerophilic growth conditions resulted in elevated levels of TMAO reductase, nitrate reductase and fumarate reductase activity in each strain, whereas growth in the presence of the respective substrate for each enzyme further elevated enzyme activity. TMAO reductase specific activity was the highest of all the reductases. Interestingly, the bacteria-colonized light organs from the two squids, Euprymna scolopes and Euprymna morsei, and the light organ of the ponyfish, Leiognathus equus, also had high levels of TMAO reductase enzyme activity, in contrast to non-symbiotic tissues. The ability of these bacterial symbionts to support cell growth by respiration with TMAO may conceivably eliminate the competition for oxygen needed for both bioluminescence and metabolism.     

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

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.     

Characterization of Tn5 mutants deficient in dissimilatory nitrite reduction in Pseudomonas sp. strain G-179, which contains a copper nitrite reductase.

Tn5 was used to generate mutants that were deficient in the dissimilatory reduction of nitrite for Pseudomonas sp. strain G-179, which contains a copper nitrite reductase. Three types of mutants were isolated. The first type showed a lack of growth on nitrate, nitrite, and nitrous oxide. The second type grew on nitrate and nitrous oxide but not on nitrite (Nir-). The two mutants of this type accumulated nitrite, showed no nitrite reductase activity, and had no detectable nitrite reductase protein bands in a Western blot (immunoblot). Tn5 insertions in these two mutants were clustered in the same region and were within the structural gene for nitrite reductase. The third type of mutant grew on nitrate but not on nitrite or nitrous oxide (N2O). The mutant of this type accumulated significant amounts of nitrite, NO, and N2O during anaerobic growth on nitrate and showed a slower growth rate than the wild type. Diethyldithiocarbamic acid, which inhibited nitrite reductase activity in the wild type, did not affect NO reductase activity, indicating that nitrite reductase did not participate in NO reduction. NO reductase activity in Nir- mutants was lower than that in the wild type when the strains were grown on nitrate but was the same as that in the wild type when the strains were grown on nitrous oxide. These results suggest that the reduction of NO and N2O was carried out by two distinct processes and that mutations affecting nitrite reduction resulted in reduced NO reductase activity following anaerobic growth with nitrate.     

Shewanella oneidensis MR‐1 mutants selected for their inability to produce soluble organic‐Fe(III) complexes are unable to respire Fe(III) as anaerobic electron acceptor

Summary Recent voltammetric analyses indicate that Shewanella putrefaciens strain 200 produces soluble organic‐Fe(III) complexes during anaerobic respiration of sparingly soluble Fe(III) oxides. Results of the present study expand the range of Shewanella species capable of producing soluble organic‐Fe(III) complexes to include Shewanella oneidensis MR‐1. Soluble organic‐Fe(III) was produced by S. oneidensis cultures incubated anaerobically with Fe(III) oxides, or with Fe(III) oxides and the alternate electron acceptor fumarate, but not in the presence of O₂, nitrate or trimethylamine‐N‐oxide. Chemical mutagenesis procedures were combined with a novel MicroElectrode Screening Array (MESA) to identify four (designated Sol) mutants with impaired ability to produce soluble organic‐Fe(III) during anaerobic respiration of Fe(III) oxides. Two of the Sol mutants were deficient in anaerobic growth on both soluble Fe(III)‐citrate and Fe(III) oxide, yet retained the ability to grow on a suite of seven alternate electron acceptors. The rates of soluble organic‐Fe(III) production were proportional to the rates of iron reduction by the S. oneidensis wild‐type and Sol mutant strains, and all four Sol mutants retained wild‐type siderophore production capability. Results of this study indicate that the production of soluble organic‐Fe(III) may be an important intermediate step in the anaerobic respiration of both soluble and sparingly soluble forms of Fe(III) by S. oneidensis.     

Chromate/nitrite interactions in Shewanella oneidensis MR-1: evidence for multiple hexavalent chromium [Cr(VI)] reduction mechanisms dependent on physiological growth conditions

Inhibition of hexavalent chromium [Cr(VI)] reduction due to nitrate and nitrite was observed during tests with Shewanella oneidensis MR-1 (previously named Shewanella putrefaciens MR-1 and henceforth referred to as MR-1). Initial Cr(VI) reduction rates were measured at various nitrite concentrations, and a mixed inhibition kinetic model was used to determine the kinetic parameters-maximum Cr(VI) reduction rate and inhibition constant [V(max,Cr(VI)) and K(i,Cr(VI))]. Values of V(max,Cr(VI)) and K(i,Cr(VI)) obtained with MR-1 cultures grown under denitrifying conditions were observed to be significantly different from the values obtained when the cultures were grown with fumarate as the terminal electron acceptor. It was also observed that a single V(max,Cr(VI)) and K(i,Cr(VI)) did not adequately describe the inhibition kinetics of either nitrate-grown or fumarate-grown cultures. The inhibition patterns indicate that Cr(VI) reduction in MR-1 is likely not limited to a single pathway, but occurs via different mechanisms some of which are dependent on growth conditions. Inhibition of nitrite reduction due to the presence of Cr(VI) was also studied, and the kinetic parameters V(max,NO2) and K(i,NO2) were determined. It was observed that these coefficients also differed significantly between MR-1 grown under denitrifying conditions and fumarate reducing conditions. The inhibition studies suggest the involvement of nitrite reductase in Cr(VI) reduction. Because nitrite reduction is part of the anaerobic respiration process, inhibition due to Cr(VI) might be a result of interaction with the components of the anaerobic respiration pathway such as nitrite reductase. Also, differences in the degree of inhibition of nitrite reduction activity by chromate at different growth conditions suggest that the toxicity mechanism of Cr(VI) might also be dependent on the conditions of growth. Cr(VI) reduction has been shown to occur via different pathways, but to our knowledge, multiple pathways within a single organism leading to Cr(VI) reduction has not been reported previously.     

Effects of ISSo2 insertions in structural and regulatory genes of the trimethylamine oxide reductase of Shewanella oneidensis

We have isolated three Shewanella oneidensis mutants specifically impaired in trimethylamine oxide (TMAO) respiration. The mutations arose from insertions of an ISSo2 element into torA, torR, and torS, encoding, respectively, the TMAO reductase TorA, the response regulator TorR, and the sensor TorS. Although TorA is not the sole enzyme reducing TMAO in S. oneidensis, growth analysis showed that it is the main respiratory TMAO reductase. Use of a plasmid-borne torE'-lacZ fusion confirmed that the TorS-TorR phosphorelay mediates TMAO induction of the torECAD operon.     

A kinetic approach to the dependence of dissimilatory metal reduction by Shewanella oneidensis MR-1 on the outer membrane cytochromes c OmcA and OmcB

The Gram-negative bacterium Shewanella oneidensis MR-1 shows a remarkably versatile anaerobic respiratory metabolism. One of its hallmarks is its ability to grow and survive through the reduction of metallic compounds. Among other proteins, outer membrane decaheme cytochromes c OmcA and OmcB have been identified as key players in metal reduction. In fact, both of these cytochromes have been proposed to be terminal Fe(III) and Mn(IV) reductases, although their role in the reduction of other metals is less well understood. To obtain more insight into this, we constructed and analyzed omcA, omcB and omcA/omcB insertion mutants of S. oneidensis MR-1. Anaerobic growth on Fe(III), V(V), Se(VI) and U(VI) revealed a requirement for both OmcA and OmcB in Fe(III) reduction, a redundant function in V(V) reduction, and no apparent involvement in Se(VI) and U(VI) reduction. Growth of the omcB(-) mutant on Fe(III) was more affected than growth of the omcA(-) mutant, suggesting OmcB to be the principal Fe(III) reductase. This result was corroborated through the examination of whole cell kinetics of OmcA- and OmcB-dependent Fe(III)-nitrilotriacetic acid reduction, showing that OmcB is approximately 11.5 and approximately 6.3 times faster than OmcA at saturating and low nonsaturating concentrations of Fe(III)-nitrilotriacetic acid, respectively, whereas the omcA(-) omcB(-) double mutant was devoid of Fe(III)-nitrilotriacetic acid reduction activity. These experiments reveal, for the first time, that OmcA and OmcB are the sole terminal Fe(III) reductases present in S. oneidensis MR-1. Kinetic inhibition experiments further revealed vanadate (V(2)O(5)) to be a competitive and mixed-type inhibitor of OmcA and OmcB, respectively, showing similar affinities relative to Fe(III)-nitrilotriacetic acid. Neither sodium selenate nor uranyl acetate were found to inhibit OmcA- and OmcB-dependent Fe(III)-nitrilotriacetic acid reduction. Taken together with our growth experiments, this suggests that proteins other than OmcA and OmcB play key roles in anaerobic Se(VI) and U(VI) respiration.     

Reduction of diverse electron acceptors by Aeromonas hydrophila

Aeromonas hydrophila ATCC 7966 grew anaerobically on glycerol with nitrate, fumarate, Fe(III), Co(III), or Se(VI) as the sole terminal electron acceptor, but did not ferment glycerol. Final cell yields were directly proportional to the amount of terminal electron acceptor provided. Twenty-four estuarine mesophilic aeromonads were isolated; all reduced nitrate, Fe(III), or Co(III), and five strains reduced Se(VI). Dissimilatory Fe(III) reduction by A. hydrophila may involve cytochromes. Difference spectra obtained with whole cells showed absorption maxima at wavelengths characteristic of c-type cytochromes (419, 522, and 553 nm). Hydrogen-reduced cytochromes within intact cells were oxidized by the addition of Fe(III) or nitrate. Studies with respiratory inhibitors yielded results consistent with a respiratory chain involving succinate (flavin-containing) dehydrogenase, quinones and cytochromes, and a single Fe(III) reductase. Neither anaerobic respiration nor dissimilatory metal reduction by members of the genus Aeromonas have been reported previously. Received: 24 June 1997 / Accepted: 20 October 1997     

Growth of Campylobacter jejuni supported by respiration of fumarate,nitrate, nitrite,trimethylamine-N-oxide,or dimethyl sulfoxide requires oxygen

The human gastrointestinal pathogen Campylobacter jejuni is a microaerophilic bacterium with a respiratory metabolism. The genome sequence of C. jejuni strain 11168 reveals the presence of genes that encode terminal reductases that are predicted to allow the use of a wide range of alternative electron acceptors to oxygen, including fumarate, nitrate, nitrite, and N- or S-oxides. All of these reductase activities were present in cells of strain 11168, and the molybdoenzyme encoded by Cj0264c was shown by mutagenesis to be responsible for both trimethylamine-N-oxide (TMAO) and dimethyl sulfoxide (DMSO) reduction. Nevertheless, growth of C. jejuni under strictly anaerobic conditions (with hydrogen or formate as electron donor) in the presence of any of the electron acceptors tested was insignificant. However, when fumarate, nitrate, nitrite, TMAO, or DMSO was added to microaerobic cultures in which the rate of oxygen transfer was severely restricted, clear increases in both the growth rate and final cell density compared to what was seen with the control were obtained, indicative of electron acceptor-dependent energy conservation. The C. jejuni genome encodes a single class I-type ribonucleotide reductase (RNR) which requires oxygen to generate a tyrosyl radical for catalysis. Electron microscopy of cells that had been incubated under strictly anaerobic conditions with an electron acceptor showed filamentation due to an inhibition of cell division similar to that induced by the RNR inhibitor hydroxyurea. An oxygen requirement for DNA synthesis can thus explain the lack of anaerobic growth of C. jejuni. The results indicate that strict anaerobiosis is a stress condition for C. jejuni but that alternative respiratory pathways can contribute significantly to energy conservation under oxygen-limited conditions, as might be found in vivo.     

The membrane-bound tetrahaem c-type cytochrome CymA interacts directly with the soluble fumarate reductase in Shewanella

Shewanella spp. demonstrate great variability in the use of terminal electron acceptors in anaerobic respiration; these include nitrate, fumarate, DMSO, trimethylamine oxide, sulphur compounds and metal oxides. These pathways open up possible applications in bioremediation. The wide variety of respiratory substrates for Shewanella is correlated with the evolution of several multi-haem membrane-bound, periplasmic and outer-membrane c-type cytochromes. The 21 kDa c-type cytochrome CymA of the freshwater strain Shewanella oneidensis MR-1 has an N-terminal membrane anchor and a globular tetrahaem periplasmic domain. According to sequence alignments, CymA is a member of the NapC/NirT family. This family of redox proteins is responsible for electron transfer from the quinone pool to periplasmic and outer-membrane-bound reductases. Prior investigations have shown that the absence of CymA results in loss of the ability to respire with Fe(III), fumarate and nitrate, indicating that CymA is involved in electron transfer to several terminal reductases. Here we describe the expression, purification and characterization of a soluble, truncated CymA ('CymA). Potentiometric studies suggest that there are two pairs of haems with potentials of -175 and -261 mV and that 'CymA is an efficient electron donor for the soluble fumarate reductase, flavocytochrome c(3).     

Unexpected chemoreceptors mediate energy taxis towards electron acceptors in Shewanella oneidensis

Shewanella oneidensis uses a wide range of terminal electron acceptors for respiration. In this study, we show that the chemotactic response of S. oneidensis to anaerobic electron acceptors requires functional electron transport systems. Deletion of the genes encoding dimethyl sulphoxide and trimethylamine N -oxide reductases, or inactivation of these molybdoenzymes as well as nitrate reductase by addition of tungstate, abolished electron acceptor taxis. Moreover, addition of nigericin prevented taxis towards trimethylamine N -oxide, dimethyl sulphoxide, nitrite, nitrate and fumarate, showing that this process depends on the ΔpH component of the proton motive force. These data, together with those concerning response to metals ( Bencharit and Ward, 2005 ), support the idea that, in S. oneidensis , taxis towards electron acceptors is governed by an energy taxis mechanism. Surprisingly, energy taxis in S. oneidensis is not mediated by the PAS-containing chemoreceptors but rather by a chemoreceptor (SO2240) containing a Cache domain. Four other chemoreceptors also play a minor role in this process. These results indicate that energy taxis can be mediated by new types of chemoreceptors.