Branched Electron Transport Chain Research Articles

Genetically dissecting the electron transport chain of a soil bacterium reveals a generalizable mechanism for biological phenazine-1-carboxylic acid oxidation.

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa.

Bactericidal and sterilizing activity of sudapyridine-clofazimine-TB47 combined with linezolid or pyrazinamide in a murine model of tuberculosis.

As an obligate aerobe, Mycobacterium tuberculosis relies on its branched electron transport chain (ETC) for energy production through oxidative phosphorylation. Regimens targeting ETC exhibit promising potential to enhance bactericidal activity against M. tuberculosis and hold the prospect of shortening treatment duration. Our previous research demonstrated that the bacteriostatic drug candidate TB47 (T) inhibited the growth of M. tuberculosis by targeting the cytochrome bc1 complex and exhibited synergistic activity with clofazimine (C). Here, we found synergistic activities between C and sudapyridine (S), a structural analog of bedaquiline (B). S has shown similar anti-tuberculosis efficacy and may share a mechanism of action with B, which inhibits ATP synthesis and the energy metabolism of bacteria. We evaluated the efficacy of SCT in combination with linezolid (L) or pyrazinamide (Z) using a well-established murine model of tuberculosis. Compared to the BPa(pretomanid)L regimen, SCT and SCTL demonstrated similar bactericidal and sterilizing activities. There was no significant difference in activity between SCT and SCTL. In contrast, SCZ and SCTZ showed much higher activities, with none of the 15 mice experiencing relapse after 2 months of treatment with either SCZ or SCTZ. However, T did not contribute to the activity of the SCZ. Our findings emphasize the efficacy and the potential clinical significance of combination therapy with ETC inhibitors. Additionally, cross-resistance exists not only between S and B but also between S/B and C. This is supported by our findings, as spontaneous S-resistant mutants exhibited mutations in Rv0678, which are associated with cross-resistance to B and C.

Copper Metabolism in Naegleria gruberi and Its Deadly Relative Naegleria fowleri.

Although copper is an essential nutrient crucial for many biological processes, an excessive concentration can be toxic and lead to cell death. The metabolism of this two-faced metal must be strictly regulated at the cell level. In this study, we investigated copper homeostasis in two related unicellular organisms: nonpathogenic Naegleria gruberi and the “brain-eating amoeba” Naegleria fowleri. We identified and confirmed the function of their specific copper transporters securing the main pathway of copper acquisition. Adjusting to different environments with varying copper levels during the life cycle of these organisms requires various metabolic adaptations. Using comparative proteomic analyses, measuring oxygen consumption, and enzymatic determination of NADH dehydrogenase, we showed that both amoebas respond to copper deprivation by upregulating the components of the branched electron transport chain: the alternative oxidase and alternative NADH dehydrogenase. Interestingly, analysis of iron acquisition indicated that this system is copper-dependent in N. gruberi but not in its pathogenic relative. Importantly, we identified a potential key protein of copper metabolism of N. gruberi, the homolog of human DJ-1 protein, which is known to be linked to Parkinson’s disease. Altogether, our study reveals the mechanisms underlying copper metabolism in the model amoeba N. gruberi and the fatal pathogen N. fowleri and highlights the differences between the two amoebas.

Nitric Oxide-Dependent Electron Transport Chain Inhibition by the Cytochrome bc1 Inhibitor and Pretomanid Combination Kills Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of human tuberculosis, harbors a branched electron transport chain, preventing the bactericidal action of cytochrome bc1 inhibitors (e.g., TB47). Here, we investigated, using luminescent mycobacterial strains, the in vitro combination activity of cytochrome bc1 inhibitors and nitric oxide (NO) donors including pretomanid (PMD) and explored the mechanisms of combination activity. The TB47 and PMD combination quickly abolished the light emission of luminescent bacilli, as was the case for the combination of TB47 and aurachin D, a putative cytochrome bd inhibitor. The TB47 and PMD combination inhibited M. tuberculosis oxygen consumption, decreased ATP levels, and had a delayed bactericidal effect. The NO scavenger carboxy-PTIO prevented the bactericidal activity of the drug combination, suggesting the requirement for NO. In addition, cytochrome bc1 inhibitors were largely bactericidal when administered with DETA NONOate, another NO donor. Proteomic analysis revealed that the cotreated bacilli had a compromised expression of the dormancy regulon proteins, PE/PPE proteins, and proteins required for the biosynthesis of several cofactors, including mycofactocin. Some of these proteomic changes, e.g., the impaired dormancy regulon induction, were attributed to PMD. In conclusion, combination of cytochrome bc1 inhibitors with PMD inhibited M. tuberculosis respiration and killed the bacilli. The activity of cytochrome bc1 inhibitors can be greatly enhanced by NO donors. Monitoring of luminescence may be further exploited to screen cytochrome bd inhibitors.

Involvement of the cbb3-Type Terminal Oxidase in Growth Competition of Bacteria, Biofilm Formation, and in Switching between Denitrification and Aerobic Respiration.

Paracoccus denitrificans has a branched electron transport chain with three terminal oxidases transferring electrons to molecular oxygen, namely aa3-type and cbb3-type cytochrome c oxidases and ba3-type ubiquinol oxidase. In the present study, we focused on strains expressing only one of these enzymes. The competition experiments showed that possession of cbb3-type oxidase confers significant fitness advantage during oxygen-limited growth and supports the biofilm lifestyle. The aa3-type oxidase was shown to allow rapid aerobic growth at a high oxygen supply. Activity of the denitrification pathway that had been expressed in cells grown anaerobically with nitrate was fully inhibitable by oxygen only in wild-type and cbb3 strains, while in strains aa3 and ba3 dinitrogen production from nitrate and oxygen consumption occurred simultaneously. Together, the results highlight the importance of the cbb3-type oxidase for the denitrification phenotype and suggest a way of obtaining novel bacterial strains capable of aerobic denitrification.

Reductive Stress: New Insights in Physiology and Drug Tolerance of Mycobacterium.

Significance:Mycobacterium tuberculosis (Mtb) encounters reductive stress during its infection cycle. Notably, host-generated protective responses, such as acidic pH inside phagosomes and lysosomes, exposure to glutathione in alveolar hypophase (i.e., a thin liquid lining consisting of surfactant and proteins in the alveolus), and hypoxic environments inside granulomas are associated with the accumulation of reduced cofactors, such as nicotinamide adenine dinucleotide (reduced form), nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide (reduced form), and nonprotein thiols (e.g., mycothiol), leading to reductive stress in Mtb cells. Dissipation of this reductive stress is important for survival of the bacterium. If reductive stress is not dissipated, it leads to generation of reactive oxygen species, which may be fatal for the cells. Recent Advances: This review focuses on mechanisms utilized by mycobacteria to sense and respond to reductive stress. Importantly, exposure of Mtb cells to reductive stress leads to growth inhibition, altered metabolism, modulation of virulence, and drug tolerance. Mtb is equipped with thiol buffering systems of mycothiol and ergothioneine to protect itself from various redox stresses. These systems are complemented by thioredoxin and thioredoxin reductase (TR) systems for maintaining cellular redox homeostasis. A diverse array of sensors is used by Mycobacterium for monitoring its intracellular redox status. Upon sensing reductive stress, Mtb uses a flexible and robust metabolic system for its dissipation. Branched electron transport chain allows Mycobacterium to function with different terminal electron acceptors and modulate proton motive force to fulfill energy requirements under diverse scenarios. Interestingly, Mtb utilizes variations in the tricarboxylic cycle and a number of dehydrogenases to dissipate reductive stress. Upon prolonged exposure to reductive stress, Mtb utilizes biosynthesis of storage and virulence lipids as a dissipative mechanism. Critical Issues: The mechanisms utilized by Mycobacterium for sensing and tackling reductive stress are not well characterized. Future Directions: The precise role of thiol buffering and TR systems in neutralizing reductive stress is not well defined. Genetic systems that respond to metabolic reductive stress and thiol reductive stress need to be mapped. Genetic screens could aid in identification of such systems. Given that management of reductive stress is critical for both actively replicating and persister mycobacteria, an improved understanding of the mechanisms used by mycobacteria for dissipation of reductive stress may lead to identification of vulnerable choke points that could be targeted for killing Mtb in vivo.

Insights into the Fundamental Physiology of the Uncultured Fe-Oxidizing Bacterium Leptothrix ochracea.

Leptothrix ochracea is known for producing large volumes of iron oxyhydroxide sheaths that alter wetland biogeochemistry. For over a century, these delicate structures have fascinated microbiologists and geoscientists. Because L. ochracea still resists long-term in vitro culture, the debate regarding its metabolic classification dates back to 1885. We developed a novel culturing technique for L. ochracea using in situ natural waters and coupled this with single-cell genomics and nanoscale secondary-ion mass spectrophotometry (nanoSIMS) to probe L. ochracea's physiology. In microslide cultures L. ochracea doubled every 5.7 h and had an absolute growth requirement for ferrous iron, the genomic capacity for iron oxidation, and a branched electron transport chain with cytochromes putatively involved in lithotrophic iron oxidation. Additionally, its genome encoded several electron transport chain proteins, including a molybdopterin alternative complex III (ACIII), a cytochrome bd oxidase reductase, and several terminal oxidase genes. L. ochracea contained two key autotrophic proteins in the Calvin-Benson-Bassham cycle, a form II ribulose bisphosphate carboxylase, and a phosphoribulose kinase. L. ochracea also assimilated bicarbonate, although calculations suggest that bicarbonate assimilation is a small fraction of its total carbon assimilation. Finally, L. ochracea's fundamental physiology is a hybrid of those of the chemolithotrophic Gallionella-type iron-oxidizing bacteria and the sheathed, heterotrophic filamentous metal-oxidizing bacteria of the Leptothrix-Sphaerotilus genera. This allows L. ochracea to inhabit a unique niche within the neutrophilic iron seeps.IMPORTANCELeptothrix ochracea was one of three groups of organisms that Sergei Winogradsky used in the 1880s to develop his hypothesis on chemolithotrophy. L. ochracea continues to resist cultivation and appears to have an absolute requirement for organic-rich waters, suggesting that its true physiology remains unknown. Further, L. ochracea is an ecological engineer; a few L. ochracea cells can generate prodigious volumes of iron oxyhydroxides, changing the ecosystem's geochemistry and ecology. Therefore, to determine L. ochracea's basic physiology, we employed new single-cell techniques to demonstrate that L. ochracea oxidizes iron to generate energy and, despite having predicted genes for autotrophic growth, assimilates a fraction of the total CO2 that autotrophs do. Although not a true chemolithoautotroph, L. ochracea's physiological strategy allows it to be flexible and to extensively colonize iron-rich wetlands.

Generation of the membrane potential and its impact on the motility, ATP production and growth in Campylobacter jejuni.

The generation of a membrane potential (Δψ), the major constituent of the proton motive force (pmf), is crucial for ATP synthesis, transport of nutrients and flagellar rotation. Campylobacter jejuni harbors a branched electron transport chain, enabling respiration with different electron donors and acceptors. Here, we demonstrate that a relatively high Δψ is only generated in the presence of either formate as electron donor or oxygen as electron acceptor, in combination with an acceptor/donor respectively. We show the necessity of the pmf for motility and growth of C. jejuni. ATP generation is not only accomplished by oxidative phosphorylation via the pmf, but also by substrate-level phosphorylation via the enzyme AckA. In response to a low oxygen tension, C. jejuni increases the transcription and activity of the donor complexes formate dehydrogenase (FdhABC) and hydrogenase (HydABCD) as well as the transcription of the alternative respiratory acceptor complexes. Our findings suggest that in the gut of warm-blooded animals, C. jejuni depends on at least formate or hydrogen as donor (in the anaerobic lumen) or oxygen as acceptor (near the epithelial cells) to generate a pmf that sustains efficient motility and growth for colonization and pathogenesis.

An Investigation into Membrane Bound Redox Carriers Involved in Energy Transduction Mechanism in Brevibacterium linens DSM 20158 with Unsequenced Genome

Brevibacterium linens (B. linens) DSM 20158 with an unsequenced genome can be used as a non-pathogenic model to study features it has in common with other unsequenced pathogens of the same genus on the basis of comparative proteome analysis. The most efficient way to kill a pathogen is to target its energy transduction mechanism. In the present study, we have identified the redox protein complexes involved in the electron transport chain of B. linens DSM 20158 from their clear homology with the shot-gun genome sequenced strain BL2 of B. linens by using the SDS-Polyacrylamide gel electrophoresis coupled with nano LC-MS/MS mass spectrometry. B. linens is found to have a branched electron transport chain (Respiratory chain), in which electrons can enter the respiratory chain either at NADH (Complex I) or at Complex II level or at the cytochrome level. Moreover, we are able to isolate, purify, and characterize the membrane bound Complex II (succinate dehydrogenase), Complex III (menaquinone cytochrome c reductase cytochrome c subunit, Complex IV (cytochrome c oxidase), and Complex V (ATP synthase) of B. linens strain DSM 20158.

Analysis of Escherichia coli mutants with a linear respiratory chain.

The respiratory chain of E. coli is branched to allow the cells' flexibility to deal with changing environmental conditions. It consists of the NADH:ubiquinone oxidoreductases NADH dehydrogenase I and II, as well as of three terminal oxidases. They differ with respect to energetic efficiency (proton translocation) and their affinity to the different quinone/quinol species and oxygen. In order to analyze the advantages of the branched electron transport chain over a linear one and to assess how usage of the different terminal oxidases determines growth behavior at varying oxygen concentrations, a set of isogenic mutant strains was created, which lack NADH dehydrogenase I as well as two of the terminal oxidases, resulting in strains with a linear respiratory chain. These strains were analyzed in glucose-limited chemostat experiments with defined oxygen supply, adjusting aerobic, anaerobic and different microaerobic conditions. In contrast to the wild-type strain MG1655, the mutant strains produced acetate even under aerobic conditions. Strain TBE032, lacking NADH dehydrogenase I and expressing cytochrome bd-II as sole terminal oxidase, showed the highest acetate formation rate under aerobic conditions. This supports the idea that cytochrome bd-II terminal oxidase is not able to catalyze the efficient oxidation of the quinol pool at higher oxygen conditions, but is functioning mainly under limiting oxygen conditions. Phosphorylation of ArcA, the regulator of the two-component system ArcBA, besides Fnr the main transcription factor for the response towards different oxygen concentrations, was studied. Its phosphorylation pattern was changed in the mutant strains. Dephosphorylation and therefore inactivation of ArcA started at lower aerobiosis levels than in the wild-type strain. Notably, not only the micro- and aerobic metabolism was affected by the mutations, but also the anaerobic metabolism, where the respiratory chain should not be important.

The Twin-Arginine Translocation System: Contributions to the Pathobiology of Campylobacter Jejuni

The twin-arginine translocation (Tat) system is specialized in the transport of folded proteins across the cytoplasmic membrane. Although the mechanisms that govern the Tat transport and its scope are not well understood, this system and its cognate substrates are involved in important functions that facilitate the adaptation and survival of bacteria. Evidence also exists that connects the Tat system to virulent traits of clinically relevant pathogens. Of interest is Campylobacter jejuni, an important foodborne pathogen that is capable of surviving in different hosts and environmental niches. Recent studies have shown that the Tat system in this bacterium mediates key metabolic and stress resistance traits. Furthermore, the majority of the identified Tat substrates in C. jejuni are cofactor-containing redox proteins that contribute to the bacterium?s branched electron transport chain, a component essential for survival under differing conditions. These studies as well as the absence of Tat homologs in the sequenced genomes of animals suggest that the Tat system might pose an attractive target for therapeutics against C. jejuni.

Dynamics and Co-Localization of the Electron Transport Chain of Escherichia Coli: Investigations Through Fluorescence Microscopy

Total Internal Reflection Fluorescence Microscopy (TIRFM) is a key tool in probing the dynamics and distribution of fluorescently tagged fusion proteins. This non-invasive in vivo technique is suited to the investigation of membrane-bound proteins due to the low cytoplasmic response, sometimes allowing for ∼millisecond trajectory sampling of membrane bound fusion proteins. In addition, multi-wavelength TIRFM set-ups allow for investigations into co-localization of multiple fluorescently labeled protein species.Presented here are simultaneous dual-color TIRFM investigations of key proteins involved in the branched Electron Transport Chain (ETC) in Escherichia coli, using a series of single and dual-labeled mutant strains. Of interest was the comparative degree to which these proteins reflected a ‘free-diffusion-collision’ model of protein interactions, to that of a ‘solid state’ model in which reacting proteins are confined and co-localized.This builds upon previous work that identified a key protein in the ETC as having a highly non-random heterogeneous distribution, being located into functional mobile membrane patches.We investigated the spatial distribution and the degree of co-localization of these protein pairs at the cellular level. We quantified the diffusion behavior of the observed trajectories comparing the relative populations which displayed freely diffusing characteristics (through standard Brownian motion), with the populations that exhibited confined or anomalous modes. We discuss the implications of the observed dynamics at the systems level, and how this relates to the overall functioning of the branched ETC.

The role of respiratory donor enzymes in Campylobacter jejuni host colonization and physiology

The human pathogen Campylobacter jejuni utilizes oxidative phosphorylation to meet all of its energy demands. The genome sequence of this bacterium encodes a number of respiratory enzymes in a branched electron transport chain that predicts the utilization of a number of electron transport chain donor and acceptor molecules. Three of these electron donor enzymes: hydrogenase, formate dehydrogenase, and 2-oxoglutarate:acceptor oxidoreductase (OOR), oxidize hydrogen, formate and α-ketoglutarate as electron donors, respectively. Mutations were created in these donor enzymes to isolate mutants in hydrogenase (HydB::CM), formate dehydrogenase (Fdh::CM), and OOR (OorB::CM), as well as a strain with insertions in both hydrogenase and formate dehydrogenase (Hyd::Fdh). These mutants are deficient in their respective enzyme activities and do not reduce the components of the electron transport chain when provided with their respective substrates. The presence of either hydrogen or formate in the media stimulated the growth of wild type (WT) C. jejuni (but not the associated mutant strains) and at least one of these alternative substrates is required for growth of the OOR mutant strain OorB::CM. Finally, the importance of hydrogenase, formate dehydrogenase and OOR as well as the complex I of C. jejuni are elucidated by chicken colonization assays, where the double mutant Hyd::Fdh, OorB::CM and nuo mutants are severely impaired in host colonization.

Lactobacillus plantarum WCFS1 Electron Transport Chains

Lactobacillus plantarum WCFS1 requires both heme and menaquinone to induce respiration-like behavior under aerobic conditions. The addition of these compounds enhanced both biomass production, without progressive acidification, and the oxygen consumption rate. When both heme and menaquinone were present, L. plantarum WCFS1 was also able to reduce nitrate. The ability to reduce nitrate was severely inhibited by the glucose levels that are typically found in L. plantarum growth media (1 to 2% [vol/vol] glucose). In contrast, comparable mannitol levels did not inhibit the reduction of nitrate. L. plantarum reduced nitrate with concomitant formation of nitrite and ammonia. Genes that encode a bd-type cytochrome (cydABCD) and a nitrate reductase (narGHJI) were identified in the genome of L. plantarum. The narGHJI operon is part of a cluster of genes that includes the molybdopterin cofactor biosynthesis genes and narK. Besides a menaquinone source, isogenic mutants revealed that cydA and ndh1 are required for the aerobic-respiration-like response and narG for nitrate reduction. The ndh1 mutant was still able to reduce nitrate. The existence of a nonredundant branched electron transport chain in L. plantarum WCFS1 that is capable of using oxygen or nitrate as a terminal electron acceptor is proposed.

Role of Campylobacter jejuni Respiratory Oxidases and Reductases in Host Colonization

Campylobacter jejuni is the leading cause of human food-borne bacterial gastroenteritis. The C. jejuni genome sequence predicts a branched electron transport chain capable of utilizing multiple electron acceptors. Mutants were constructed by disrupting the coding regions of the respiratory enzymes nitrate reductase (napA::Cm), nitrite reductase (nrfA::Cm), dimethyl sulfoxide, and trimethylamine N-oxide reductase (termed Cj0264::Cm) and the two terminal oxidases, a cyanide-insensitive oxidase (cydA::Cm) and cbb3-type oxidase (ccoN::Cm). Each strain was characterized for the loss of the associated enzymatic function in vitro. The strains were then inoculated into 1-week-old chicks, and the cecal contents were assayed for the presence of C. jejuni 2 weeks postinoculation. cydA::Cm and Cj0264c::Cm strains colonized as well as the wild type; napA::Cm and nrfA::Cm strains colonized at levels significantly lower than the wild type. The ccoN::Cm strain was unable to colonize the chicken; no colonies were recovered at the end of the experiment. While there appears to be a role for anaerobic respiration in host colonization, oxygen is the most important respiratory acceptor for C. jejuni in the chicken cecum.

ALTERNATIVE NAD(P)H DEHYDROGENASES OF PLANT MITOCHONDRIA

Plant mitochondria have a highly branched electron transport chain that provides great flexibility for oxidation of cytosolic and matrix NAD(P)H. In addition to the universal electron transport chain found in many organisms, plants have alternative NAD(P)H dehydrogenases in the first part of the chain and a second oxidase, the alternative oxidase, in the latter part. The alternative activities are nonproton pumping and allow for NAD(P)H oxidation with varying levels of energy conservation. This provides a mechanism for plants to, for example, remove excess reducing power and balance the redox poise of the cell. This review presents our current understanding of the alternative NAD(P)H dehydrogenases present in plant mitochondria.

The respiratory chain of Corynebacterium glutamicum

Corynebacterium glutamicum is an aerobic bacterium that requires oxygen as exogenous electron acceptor for respiration. Recent molecular and biochemical analyses together with information obtained from the genome sequence showed that C. glutamicum possesses a branched electron transport chain to oxygen with some remarkable features. Reducing equivalents obtained by the oxidation of various substrates are transferred to menaquinone via at least eight different dehydrogenases, i.e. NADH dehydrogenase, succinate dehydrogenase, malate:quinone oxidoreductase, pyruvate:quinone oxidoreductase, d-lactate dehydrogenase, l-lactate dehydrogenase, glycerol-3-phosphate dehydrogenase and l-proline dehydrogenase. All these enzymes contain a flavin cofactor and, except succinate dehydrogenase, are single subunit peripheral membrane proteins located inside the cell. From menaquinol, the electrons are passed either via the cytochrome bc 1 complex to the aa 3-type cytochrome c oxidase with low oxygen affinity, or to the cytochrome bd-type menaquinol oxidase with high oxygen affinity. The former branch is exceptional, in that it does not involve a separate cytochrome c for electron transfer from cytochrome c 1 to the Cu A center in subunit II of cytochrome aa 3. Rather, cytochrome c 1 contains two covalently bound heme groups, one of which presumably takes over the function of a separate cytochrome c. The bc 1 complex and cytochrome aa 3 oxidase form a supercomplex in C. glutamicum. The phenotype of defined mutants revealed that the bc 1– aa 3 branch, but not the bd branch, is of major importance for aerobic growth in minimal medium. Changes of the efficiency of oxidative phosphorylation caused by qualitative changes of the respiratory chain or by a defective F 1F 0-ATP synthase were found to have strong effects on metabolism and amino acid production. Therefore, the system of oxidative phosphorylation represents an attractive target for improving amino acid productivity of C. glutamicum by metabolic engineering.

Roles for the two 1-butanol dehydrogenases of Pseudomonas butanovora in butane and 1-butanol metabolism.

Pseudomonas butanovora grown on butane or 1-butanol expresses two 1-butanol dehydrogenases, a quinoprotein (BOH) and a quinohemoprotein (BDH). BOH exhibited high affinity towards 1-butanol (K(m) = 1.7 +/- 0.2 microM). BOH also oxidized butyraldehyde and 2-butanol (K(m) = 369 +/- 85 microM and K(m) = 662 +/- 98 microM, respectively). The mRNA induction profiles of BOH and BDH at three different levels of 1-butanol, a nontoxic level (0.1 mM), a growth-supporting level (2 mM), and a toxic level (40 mM), were similar. When cells were grown in citrate-containing medium in the presence of different levels of 1-butanol, wild-type P. butanovora could tolerate higher levels of 1-butanol than the P. butanovora boh::tet strain and the P. butanovora bdh::kan strain. A model is proposed in which the electrons from 1-butanol oxidation follow a branched electron transport chain. BOH may be coupled to ubiquinone, with the electrons being transported to a cyanide-sensitive terminal oxidase. In contrast, electrons from BDH may be transferred to a terminal oxidase that is less sensitive to cyanide. The former pathway may function primarily in energy generation, while the latter may be more important in the detoxification of 1-butanol.

Cytochrome bd biosynthesis in Bacillus subtilis: characterization of the cydABCD operon.

Under aerobic conditions Bacillus subtilis utilizes a branched electron transport chain comprising various cytochromes and terminal oxidases. At present there is evidence for three types of terminal oxidases in B. subtilis: a caa3-, an aa3-, and a bd-type oxidase. We report here the cloning of the structural genes (cydA and cydB) encoding the cytochrome bd complex. Downstream of the structural genes, cydC and cydD are located. These genes encode proteins showing similarity to bacterial ATP-binding cassette (ABC)-type transporters. Analysis of isolated cell membranes showed that inactivation of cydA or deletion of cydABCD resulted in the loss of spectral features associated with cytochrome bd. Gene disruption experiments and complementation analysis showed that the cydC and cydD gene products are required for the expression of a functional cytochrome bd complex. Disruption of the cyd genes had no apparent effect on the growth of cells in broth or defined media. The expression of the cydABCD operon was investigated by Northern blot analysis and by transcriptional and translational cyd-lacZ fusions. Northern blot analysis confirmed that cydABCD is transcribed as a polycistronic message. The operon was found to be expressed maximally under conditions of low oxygen tension.

A critique of the use of inhibitors to estimate partitioning of electrons between mitochondrial respiratory pathways in plants

The contribution of individual plant mitochondrial respiratory pathways to total respiration is commonly assessed by titration with specific inhibitors of different components in the branched electron transport chain. A pathway's contribution is equal to the activity when the other branch is blocked by an inhibitor multiplied by the degree (0‐1.0) to which this activity is engaged when both pathways are operating. According to Bahr and Bonner (1973. J. Biol. Chem. 218: 3441–3445) the plot of the activities of identical titrations, one performed in the absence and the other in the presence of a specific inhibitor of the other branch of the respiratory chain, yields a straight line whose slope indicates the engagement of the titrated pathway during uninhibited respiration. An initial slope of zero may occur if electron flux is diverted between pathways during titrations. However, beyond the breakpoint (representing the point of pathway saturation), a straight line is obtained with a slope representing engagement. This technique assumes that the kinetics of inhibiting a specific component of the respiratory chain are independent of the absolute rate of electron flux through the total pathway. To test this assumption, the activity of respiratory pathways in isolated soybean (Glycine max [L]. Merr. cv. Stevens) mitochondria was titrated with specific inhibitors of the cytochrome and alternative oxidases. Under these conditions, the electron flux through a given pathway was manipulated by poising the rate of succinate oxidation with the succinate dehydrogenase inhibitor malonate. Construction of activity plots in the presence versus absence of malonate failed to result in straight lines for either KCN (when titrating the cytochrome pathway) or salicylhydroxamic acid (when titrating the alternative pathway). Rather, the resultant plots were always curvilinear whenever the activity in the presence of malonate divided by the activity in the absence of malonate was less than 1.0. In no case could the real engagement of the pathway be precisely estimated from the titration data. Titrations of cytochrome pathway activity in isolated potato tuber (Solanum tuberosum L. cv. Sabago and Canabex) mitochondria (which lack the alternative oxidase) showed that as the inhibitor concentration was increased, so did the reduction status of the ubiquinone pool, to a new steady state. The dependence of inhibition kinetics on the rate of flux through the pathway, and the increase in ubiquinone pool reduction upon KCN addition, are explained in terms of the elasticity of component enzymes as outlined in the theory of metabolic control analysis. The implications of this finding for the use of titrations to estimate engagement of plant respiratory pathways are discussed.