An Escherichia coli mutant containing only demethylmenaquinone,but no menaquinone: effects on fumarate,dimethylsulfoxide, trimethylamine N-oxide and nitrate respiration

The mutant strain AN70 (ubiE) of Escherichia coli which is known to lack ubiquinone (Young IG et al. 1971), was analyzed for menaquinone (MK) and demethylmenaquinone (DMK) contents. In contrast to the wild-type, strain AN70 contained only DMK, but no MK. The mutant strain was able to grow with fumarate, trimethylamine N-oxide (TMAO) and dimethylsulfoxide (DMSO), but not with nitrate as electron acceptor. The membranes catalyzed anaerobic respiration with fumarate and TMAO at 69 and 74% of wild-type rates. DMSO respiration was reduced to 38% of wild-type activities and nitrate respiration was missing (8% of wild-type), although the respective enzymes were present in wild-type rates. The results complement earlier findings which demonstrated a role for DMK only in TMAO respiration (Wissenbach et al. 1990). It is concluded, that DMK (in addition to MK) can serve as a redox mediator in fumarate, TMAO and to some extent in DMSO respiration, but not in nitrate respiration. In strain AN70 (ubiE) the lack of ubiquinone (Q) is due to a defect in a specific methylation step of Q biosynthesis. Synthesis of MK from DMK appears to depend on the same gene (ubiE).Abbreviations DMSO = dimethylsulfoxide - DMS = dimethylsulfide - TMAO = trimethylamine N-oxide - TMA = trimethylamine - BV = benzylviologen - BV_red = reduced benzylyiologen - Q = ubiquinone - MK = menaquinone - DMK = demethylmenaquinone - NQ = naphthoquinone     

The specific functions of menaquinone and demethylmenaquinone in anaerobic respiration with fumarate,dimethylsulfoxide, trimethylamine N-oxide and nitrate by Escherichia coli

The respiratory activities of E. coli with H₂ as donor and with nitrate, fumarate, dimethylsulfoxide (DMSO) or trimethylamine N-oxide (TMAO) as acceptor were measured using the membrane fraction of quinone deficient strains. The specific activities of the membrane fraction lacking naphthoquinones with fumarate, DMSO or TMAO amounted to 2% of those measured with the membrane fraction of the wild-type strain. After incorporation of vitamin K₁ [instead of menaquinone (MK)] into the membrane fraction deficient of naphthoquinones, the activities with fumarate or DMSO were 92% or 17%, respectively, of the activities which could be theoretically achieved. Incorporation of demethylmenaquinone (DMK) did not lead to a stimulation of the activities of the mutant. In contrast, the electron transport activity with TMAO was stimulated by the incorporation of either vitamin K₁ or DMK. Nitrate respiration was fully active in membrane fractions lacking either naphthoquinones or Q, but was 3% of the wild-type activity, when all quinones were missing. Nitrate respiration was stimulated on the incorporation of either vitamin K₁ or Q into the membrane fraction lacking quinones, while the incorporation of DMK was without effect. These results suggest that MK is specifically involved in the electron transport chains catalyzing the reduction of fumarate or DMSO, while either MK or DMK serve as mediators in TMAO reduction. Nitrate respiration requires either Q or MK.Abbreviations DMK demethylmenaquinone - MK menaquinone - Q ubiquinone - DMSO dimethylsulfoxide - TMAO trimethylamine N-oxide - DMS dimethylsulfide - TMA trimethylamine - BV benzylviologen     

Role of menaquinone in the reduction of fumarate, nitrate, iron(III) and manganese(IV) by Shewanella putrefaciens MR-1

Abstract Mutants of Shewanella putrefaciens MR-1 deficient in menaquinone and methylmenaquinone, but which have wild-type levels of ubiquinone, retain the ability to use trimethylamine N -oxide as an electron acceptor, but they lose the ability to use nitrate, iron(III), and fumarate as electron acceptors. These mutants also show a reduced rate of manganese(IV) reduction. One of these mutants could be restored to essentially wild-type phenotype by supplementing the medium with 1,4-dihydroxy-2-naphthoic acid. A requirement for naphthoquinones in iron(III) reduction and a preference for naphthoquinones in manganese(IV) reduction provide further support that the metal reducing systems in MR-1 are linked to anaerobic respiration.     

The dependence on quinone specificity of terminal electron transport of bacteria

Bacterial quinones were extracted with pentane, and homologues or other quinones were reincorporated. In spite of the redox potential difference of 110 mV, menaquinone and demethylmenaquinone could replace each other in aerobic electron transport and fumarate respiration ofHaemophilus influenzae RAMC 18 Bensted andProteus mirabilis Harding & Nicholson. The enzymes involved may recognize the naphthoquinone structure and are not specific for menaquinone or demethylmenaquinone. Ubiquinone was not replaced in aerobic electron transport by naphthoquinones withPseudomonas fluorescens 28/5 Rhodes orAcinetobacter sp. 661/60 Mannheim, probably owing to the specificity for benzoquinones of the enzymes involved, since the redox potential difference between demethylmenaquinone and ubiquinone is only 76 mV.Haemophilus parainfluenzae 429 Pittman, which resembles aerobic bacteria with respect to the terminal electron transport system, could incorporate demethylmenaquinone or menaquinone. This organism seems to be defective in the synthesis of naphthoquinones but possesses the enzyme system for fumarate respiration.Haemophilus influenzae RAMC 18 Bensted, which produces only demethylmenaquinone, seems to be defective in synthesizing ubiquinone, but it also possesses the enzymes for a ubiquinonemediated aerobic respiration.     

Anaerobic fumarate transport in Escherichia coli by an fnr-dependent dicarboxylate uptake system which is different from the aerobic dicarboxylate uptake system.

Escherichia coli grown anaerobically with fumarate as electron acceptor is able to take up C4-dicarboxylates by a specific transport system. The system differs in all tested parameters from the known aerobic C4-dicarboxylate transporter. The anaerobic transport system shows higher transport rates (95 mumol/g [dry weight] per min versus 30 mumol/g/min) and higher Kms (400 versus 30 microM) for fumarate than for the aerobic system. Mutants lacking the aerobic dicarboxylate uptake system are able to grow anaerobically at the expense of fumarate respiration and transport dicarboxylates with wild-type rates after anaerobic but not after aerobic growth. Transport by the anaerobic system is stimulated by preloading the bacteria with dicarboxylates. The anaerobic transport system catalyzes homologous and heterologous antiport of dicarboxylates, whereas the aerobic system operates only in the unidirectional mode. The anaerobic antiport is measurable only in anaerobically grown bacteria with fnr+ backgrounds. Additionally, the system is inhibited by incubation of resting bacteria with physiological electron acceptors such as O2, nitrate, dimethyl sulfoxide, and fumarate. The inhibition is reversed by the presence of reducing agents. It is suggested that the physiological role of the system is a fumarate/succinate antiport under conditions of fumarate respiration.     

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.     

Involvement of cyclic AMP (cAMP) and cAMP receptor protein in anaerobic respiration of Shewanella oneidensis

Shewanella oneidensis is a metal reducer that can use several terminal electron acceptors for anaerobic respiration, including fumarate, nitrate, dimethyl sulfoxide (DMSO), trimethylamine N-oxide (TMAO), nitrite, and insoluble iron and manganese oxides. Two S. oneidensis mutants, SR-558 and SR-559, with Tn5 insertions in crp, were isolated and analyzed. Both mutants were deficient in Fe(III) and Mn(IV) reduction. They were also deficient in anaerobic growth with, and reduction of, nitrate, fumarate, and DMSO. Although nitrite reductase activity was not affected by the crp mutation, the mutants failed to grow with nitrite as a terminal electron acceptor. This growth deficiency may be due to the observed loss of cytochromes c in the mutants. In contrast, TMAO reduction and growth were not affected by loss of cyclic AMP (cAMP) receptor protein (CRP). Fumarate and Fe(III) reductase activities were induced in rich medium by the addition of cAMP to aerobically growing wild-type S. oneidensis. These results indicate that CRP and cAMP play a role in the regulation of anaerobic respiration, in addition to their known roles in catabolite repression and carbon source utilization in other bacteria.     

Metabolic characterization of lactic acid bacterium Lactococcus garvieae sk11, capable of reducing ferric iron, nitrate, and fumarate

A lactic acid bacterium capable of anaerobic respiration was isolated from soil with ferric iron-containing glucose basal medium and identified as L. garvieae by using 16S rDNA sequence homology. The isolate reduced ferric iron, nitrate, and fumarate to ferrous iron, nitrite, and succinate, respectively, under anaerobic N2 atmosphere. Growth of the isolate was increased about 30-39% in glucose basal medium containing nitrate and fumarate, but not in the medium containing ferric iron. Specifically, metabolic reduction of nitrate and fumarate is thought to be controlled by the specific genes fnr, encoding FNR-like protein, and nir, regulating fumarate-nitrate reductase. Reduction activity of ferric iron by the isolate was estimated physiologically, enzymologically, and electrochemically. The results obtained led us to propose that the isolate metabolized nitrate and fumarate as an electron acceptor and has specific enzymes capable of reducing ferric iron in coupling with anaerobic respiration.     

On the function of the various quinone species in Escherichia coli

The respiratory chain of Escherichia?coli contains three quinones. Menaquinone and demethylmenaquinone have low midpoint potentials and are involved in anaerobic respiration, while ubiquinone, which has a high midpoint potential, is involved in aerobic and nitrate respiration. Here, we report that demethylmenaquinone plays a role not only in trimethylaminooxide-, dimethylsulfoxide- and fumarate-dependent respiration, but also in aerobic respiration. Furthermore, we demonstrate that demethylmenaquinone serves as an electron acceptor for oxidation of succinate to fumarate, and that all three quinol oxidases of E.?coli accept electrons from this naphtoquinone derivative.     

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.     

A membrane-bound nitrate reductase encoded by the narGHJI operon is responsible for anaerobic respiration in Halomonas maura

The halophilic bacterium Halomonas maura is capable of anaerobic respiration on nitrates. By insertional mutagenesis with the minitransposon Tn-5 we obtained the mutant Tc62, which was incapable of anaerobic respiration on nitrates. An analysis of the regions adjacent to the transposon allowed us to characterize the membrane-bound anaerobic-respiratory nitrate reductase narGHJI gene cluster in H. maura. We identified consensus sequences for fumarate and nitrate reductase regulator (FNR)-like protein-binding sites in the promoter regions of the nar genes and consensus sequences corresponding to the NarL binding sites upstream of the nar genes. RT-PCR analysis showed that the narGHJI operon was expressed in response to anaerobic conditions when nitrate was available as electron acceptor. This membrane-bound nitrate reductase is the only enzyme responsible for anaerobic respiration on nitrate in H. maura. In this article we discuss the possible relationship between this enzyme and a dissimilatory nitrate-reduction-to-ammonia process (DNRA) in H. maura and its role in the colonization of the rhizosphere.     

Analysis of the fnrL gene and its function in Rhodobacter capsulatus.

The fnr gene encodes a regulatory protein involved in the response to oxygen in a variety of bacterial genera. For example, it was previously shown that the anoxygenic, photosynthetic bacterium Rhodobacter sphaeroides requires the fnrL gene for growth under anaerobic, photosynthetic conditions. Additionally, the FnrL protein in R. sphaeroides is required for anaerobic growth in the dark with an alternative electron acceptor, but it is not essential for aerobic growth. In this study, the fnrL locus from Rhodobacter capsulatus was cloned and sequenced. Surprisingly, an R. capsulatus strain with the fnrL gene deleted grows like the wild type under either photosynthetic or aerobic conditions but does not grow anaerobically with alternative electron acceptors such as dimethyl sulfoxide (DMSO) or trimethylamine oxide. It is demonstrated that the c-type cytochrome induced upon anaerobic growth on DMSO is not synthesized in the R. capsulatus fnrL mutant. In contrast to wild-type strains, R. sphaeroides and R. capsulatus fnrL mutants do not synthesize the anaerobically, DMSO-induced reductase. Mechanisms that explain the basis for FnrL function in both organisms are discussed.