Discovery of 1-hydroxy-2-methylquinolin-4(1H)-one derivatives as new cytochrome bd oxidase inhibitors for tuberculosis therapy.

The cytochrome bcc-aa3 oxidase (Cyt-bcc) of Mycobacterium tuberculosis (Mtb) is a promising anti-tuberculosis target. However, when Cyt-bcc is inhibited, cytochrome bd terminal oxidase (Cyt-bd) can still maintain the activity of the respiratory chain and drive ATP synthesis. Through virtual screening and biological validation, we discovered two FDA-approved drugs, ivacaftor and roquinimex, exhibited moderate binding affinity to Cyt-bd. Structural modifications of them led to 1-hydroxy-2-methylquinolin-4(1H)-one derivatives as potent new Cyt-bd inhibitors. Compound 8d binds to Cyt-bd with a Kd value of 4.17 µM and inhibits the growth of the Cyt-bcc knock-out strain (ΔqcrCAB, Cyt-bd+) with a MIC value of 6.25 µM. The combination of 8d with the Cyt-bcc inhibitor Q203 completely inhibited oxygen consumption of the wild-type strain and the inverted-membrane vesicles expressing M. tuberculosis Cyt-bd (ΔcydAB::MtbCydAB+). Our study provides a promising starting point for the development of novel dual chemotherapies for tuberculosis.

The cryo-EM structure of the bd oxidase from M. tuberculosis reveals a unique structural framework and enables rational drug design to combat TB.

New drugs are urgently needed to combat the global TB epidemic. Targeting simultaneously multiple respiratory enzyme complexes of Mycobacterium tuberculosis is regarded as one of the most effective treatment options to shorten drug administration regimes, and reduce the opportunity for the emergence of drug resistance. During infection and proliferation, the cytochrome bd oxidase plays a crucial role for mycobacterial pathophysiology by maintaining aerobic respiration at limited oxygen concentrations. Here, we present the cryo-EM structure of the cytochrome bd oxidase from M. tuberculosis at 2.5 Å. In conjunction with atomistic molecular dynamics (MD) simulation studies we discovered a previously unknown MK-9-binding site, as well as a unique disulfide bond within the Q-loop domain that defines an inactive conformation of the canonical quinol oxidation site in Actinobacteria. Our detailed insights into the long-sought atomic framework of the cytochrome bd oxidase from M. tuberculosis will form the basis for the design of highly specific drugs to act on this enzyme.

Bacterial Oxidases of the Cytochrome bd Family: Redox Enzymes of Unique Structure, Function, and Utility As Drug Targets.

Significance: Cytochrome bd is a ubiquinol:oxygen oxidoreductase of many prokaryotic respiratory chains with a unique structure and functional characteristics. Its primary role is to couple the reduction of molecular oxygen, even at submicromolar concentrations, to water with the generation of a proton motive force used for adenosine triphosphate production. Cytochrome bd is found in many bacterial pathogens and, surprisingly, in bacteria formally denoted as anaerobes. It endows bacteria with resistance to various stressors and is a potential drug target. Recent Advances: We summarize recent advances in the biochemistry, structure, and physiological functions of cytochrome bd in the light of exciting new three-dimensional structures of the oxidase. The newly discovered roles of cytochrome bd in contributing to bacterial protection against hydrogen peroxide, nitric oxide, peroxynitrite, and hydrogen sulfide are assessed. Critical Issues: Fundamental questions remain regarding the precise delineation of electron flow within this multihaem oxidase and how the extraordinarily high affinity for oxygen is accomplished, while endowing bacteria with resistance to other small ligands. Future Directions: It is clear that cytochrome bd is unique in its ability to confer resistance to toxic small molecules, a property that is significant for understanding the propensity of pathogens to possess this oxidase. Since cytochrome bd is a uniquely bacterial enzyme, future research should focus on harnessing fundamental knowledge of its structure and function to the development of novel and effective antibacterial agents.

Formate dehydrogenase, ubiquinone, and cytochrome bd-I are required for peptidoglycan recognition protein-induced oxidative stress and killing in Escherichia coli.

Mammalian Peptidoglycan Recognition Proteins (PGRPs) kill bacteria through induction of synergistic oxidative, thiol, and metal stress. PGRPs induce oxidative stress in bacteria through a block in the respiratory chain, which results in decreased respiration and incomplete reduction of oxygen (O2) to hydrogen peroxide (H2O2). In this study we identify the site of PGRP-induced generation of H2O2 in Escherichia coli. Tn-seq screening of E. coli Tn10 insertion library revealed that mutants in formate dehydrogenase (FDH) genes had the highest survival following PGRP treatment. Mutants lacking functional FDH-O had abolished PGRP-induced H2O2 production and the highest resistance to PGRP-induced killing, and formate enhanced PGRP-induced killing and H2O2 production in an FDH-dependent manner. Mutants in ubiquinone synthesis (but not menaquinone and demethylmenaquinone) and cytochrome bd-I (but not cytochromes bo3 and bd-II) also had completely abolished PGRP-induced H2O2 production and high resistance to PGRP-induced killing. Because electrons in the respiratory chain flow from dehydrogenases' substrates through quinones and then cytochromes to O2, these results imply that the site of PGRP-induced incomplete reduction of O2 to H2O2 is downstream from dehydrogenases and ubiquinone at the level of cytochrome bd-I, which results in oxidative stress. These results reveal several essential steps in PGRP-induced bacterial killing.

The cytochrome d oxidase complex regulated by fexA is an Achilles' heel in the in vivo survival of vibrio vulnificus.

Vibrio vulnificus is a halophilic estuarine bacterium causing severe opportunistic infections. To successfully establish an infection, V. vulnificus must adapt to redox fluctuations in vivo. In the present study, we show that deletion of V. vulnificus fexA gene caused hypersensitivity to acid and reactive oxygen species. The ΔfexA mutant exhibited severe in vivo survival defects. For deeper understanding the role of fexA gene on the successful V. vulnificus infection, we analyzed differentially expressed genes in ΔfexA mutant in comparison with wild type under aerobic, anaerobic or in vivo culture conditions by genome-scale DNA microarray analyses. Twenty-two genes were downregulated in the ΔfexA mutant under all three culture conditions. Among them, cydAB appeared to dominantly contribute to the defective phenotypes of the ΔfexA mutant. The fexA deletion induced compensatory point mutations in the cydAB promoter region over subcultures, suggesting essentiality. Those point mutations (PcydSMs) restored bacterial growth, motility, cytotoxicity ATP production and mouse lethality in the ΔfexA mutant. These results indicate that the cydAB operon, being regulated by FexA, plays a crucial role in V. vulnificus survival under redox-fluctuating in vivo conditions. The FexA-CydAB axis should serve an Achilles heel in the development of therapeutic regimens against V. vulnificus infection.

Cytochrome bd-Dependent Bioenergetics and Antinitrosative Defenses in Salmonella Pathogenesis.

In the course of an infection, Salmonella enterica occupies diverse anatomical sites with various concentrations of oxygen (O2) and nitric oxide (NO). These diatomic gases compete for binding to catalytic metal groups of quinol oxidases. Enterobacteriaceae express two evolutionarily distinct classes of quinol oxidases that differ in affinity for O2 and NO as well as stoichiometry of H+ translocated across the cytoplasmic membrane. The investigations presented here show that the dual function of bacterial cytochrome bd in bioenergetics and antinitrosative defense enhances Salmonella virulence. The high affinity of cytochrome bd for O2 optimizes respiratory rates in hypoxic cultures, and thus, this quinol oxidase maximizes bacterial growth under O2-limiting conditions. Our investigations also indicate that cytochrome bd, rather than cytochrome bo, is an intrinsic component of the adaptive antinitrosative toolbox of Salmonella Accordingly, induction of cytochrome bd helps Salmonella grow and respire in the presence of inhibitory NO. The combined antinitrosative defenses of cytochrome bd and the flavohemoglobin Hmp account for a great part of the adaptations that help Salmonella recover from the antimicrobial activity of NO. Moreover, the antinitrosative defenses of cytochrome bd and flavohemoglobin Hmp synergize to promote Salmonella growth in systemic tissues. Collectively, our investigations indicate that cytochrome bd is a critical means by which Salmonella resists the nitrosative stress that is engendered in the innate response of mammalian hosts while it concomitantly allows for proper O2 utilization in tissue hypoxia. IMPORTANCE: It is becoming quite apparent that metabolism is critically important to the virulence potential of pathogenic microorganisms. Bacterial cells use a variety of terminal electron acceptors to power electron transport chains and metabolic processes. Of all the electron acceptors available to bacteria, utilization of O2 yields the most energy while diversifying the type of substrates that a pathogen can use. Recent investigations have demonstrated important roles for bd-type quinol oxidases with high affinity for O2 in bacterial pathogenesis. The investigations presented here have revealed that cytochrome bd potentiates virulence of a clinically relevant bacterial pathogen by fueling bioenergetics of prokaryotic cells while protecting the respiratory chain against NO toxicity. The adaptive antinitrosative defenses afforded by cytochrome bd synergize with other NO-detoxifying systems to preserve cellular bioenergetics, thereby promoting bacterial virulence in tissue hypoxia.

Structure of a bd oxidase indicates similar mechanisms for membrane-integrated oxygen reductases.

The cytochrome bd oxidases are terminal oxidases that are present in bacteria and archaea. They reduce molecular oxygen (dioxygen) to water, avoiding the production of reactive oxygen species. In addition to their contribution to the proton motive force, they mediate viability under oxygen-related stress conditions and confer tolerance to nitric oxide, thus contributing to the virulence of pathogenic bacteria. Here we present the atomic structure of the bd oxidase from Geobacillus thermodenitrificans, revealing a pseudosymmetrical subunit fold. The arrangement and order of the heme cofactors support the conclusions from spectroscopic measurements that the cleavage of the dioxygen bond may be mechanistically similar to that in the heme-copper-containing oxidases, even though the structures are completely different.

Component identification of electron transport chains in curdlan-producing Agrobacterium sp. ATCC 31749 and its genome-specific prediction using comparative genome and phylogenetic trees analysis.

Agrobacterium sp. ATCC 31749 (formerly named Alcaligenes faecalis var. myxogenes) is a non-pathogenic aerobic soil bacterium used in large scale biotechnological production of curdlan. However, little is known about its genomic information. DNA partial sequence of electron transport chains (ETCs) protein genes were obtained in order to understand the components of ETC and genomic-specificity in Agrobacterium sp. ATCC 31749. Degenerate primers were designed according to ETC conserved sequences in other reported species. DNA partial sequences of ETC genes in Agrobacterium sp. ATCC 31749 were cloned by the PCR method using degenerate primers. Based on comparative genomic analysis, nine electron transport elements were ascertained, including NADH ubiquinone oxidoreductase, succinate dehydrogenase complex II, complex III, cytochrome c, ubiquinone biosynthesis protein ubiB, cytochrome d terminal oxidase, cytochrome bo terminal oxidase, cytochrome cbb (3)-type terminal oxidase and cytochrome caa (3)-type terminal oxidase. Similarity and phylogenetic analyses of these genes revealed that among fully sequenced Agrobacterium species, Agrobacterium sp. ATCC 31749 is closest to Agrobacterium tumefaciens C58. Based on these results a comprehensive ETC model for Agrobacterium sp. ATCC 31749 is proposed.

The cytochrome bcc-aa3-type respiratory chain of Rhodococcus rhodochrous.

Rhodococcus rhodochrous is an active soil bacterium belonging to the Nocardia group of high GC gram-positive bacteria. It is rich in various enzymes and thus important in the industrial production of chemicals and bioremediation. In this work, the respiratory chain of this aerobic organism was investigated and characterized. Grown under highly aerobic conditions, the membrane fraction of R. rhodochrous cells only contained a-, b- and c-type cytochromes, suggesting that it is the cytochrome bcc-aa(3)-type pathway that mainly operates under these conditions. In contrast, the d-type cytochrome was also present under microaerobic conditions, indicating that the alternative pathway of the bd-type oxidase works in these circumstances. In addition, the results of H(+)/O ratio measurements indicate that these two pathways have different energy efficiencies.

Compensations for diminished terminal oxidase activity in Escherichia coli: cytochrome bd-II-mediated respiration and glutamate metabolism.

Escherichia coli possesses cytochrome bo' (CyoABCDE), cytochrome bd-I (CydAB), and cytochrome bd-II (AppBC) quinol oxidases, all of which can catalyze the terminal step in the aerobic respiratory chain, the reduction of oxygen by ubiquinol. Although CydAB has a role in the generation of DeltapH, AppBC has been proposed to alleviate the accumulation of electrons in the quinone pool during respiratory stress via electroneutral ubiquinol oxidation. A cydB mutant strain exhibited lower respiration rates while maintaining a wild type growth rate. Transcriptomic analysis revealed a dramatic up-regulation of AppBC in the cydB strain, accompanied by the induction of genes involved in glutamate/gamma-aminobutyric acid (GABA) antiport, the GABA shunt, the glyoxylate shunt, respiration (including appBC), motility, and osmotic stress. Transcription factor modeling suggests that the underpinning regulation is largely controlled by H-NS, GadX, FlhDC, and AppY. The transcriptional adaptations imply that cydB cells contribute to the proton motive force via consumption of intracellular protons and glutamate/GABA antiport. Indeed, supplementation of culture medium with l-glutamate stimulates growth in a cydB strain. Phenotype analyses of the cydB strain confirm decreased motility and elevated acid resistance and also an elevated cytochrome d spectroscopic signal in cells grown at low pH. We propose a mechanism via which E. coli can compensate for the loss of cytochrome bd-I activity; cytochrome bd-II-mediated quinol oxidation prevents the accumulation of NADH, whereas GABA synthesis/antiport maintains the proton motive force for ATP production.

Cytochrome d but not cytochrome o rescues the toluidine blue growth sensitivity of arc mutants of Escherichia coli.

The Arc (anoxic redox control) two-component signal transduction system, consisting of the ArcB sensor kinase and the ArcA response regulator, allows adaptive responses of Escherichia coli to changes of O(2) availability. The arcA gene was previously known as the dye gene because null mutants were growth sensitive to the photosensitizer redox dyes toluidine blue and methylene blue, a phenotype whose molecular basis still remains elusive. In this study we report that the toluidine blue O (TBO) effect on the arc mutants is light independent and observed only during aerobic growth conditions. Moreover, 16 suppressor mutants with restored growth were generated and analyzed. Thirteen of those possessed insertion elements upstream of the cydAB operon, rendering its expression ArcA independent. Also, it was found that, in contrast to cythocrome d, cythocrome o was not able to confer toluidine blue resistance to arc mutants, thereby representing an intriguing difference between the two terminal oxidases. Finally, a mechanism for TBO sensitivity and resistance is discussed.

Presence and expression of terminal oxygen reductases in strictly anaerobic sulfate-reducing bacteria isolated from salt-marsh sediments.

In the anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough genes were found encoding membrane terminal oxygen reductases of two types: a cytochrome c oxidase and a cytochrome bd oxidase, both enzymes are terminal oxidases typical of facultative or aerobic microorganisms (Heidelberg JF, et al., The genome sequence of the anaerobic, sulfate-reducing bacterium D. vulgaris Hildenborough. Nat Biotechnol 2004; 22: 554-9). To apprehend the presence of both oxidases in other sulfate-reducing bacteria (SRB), several assays were performed on isolates recovered from salt-marsh sediments in Portugal, representative of the different phylogenetic groups identified. Hybridization and PCR experiments for DNA sequencing were performed on the chosen isolates. Primers were selected to amplify conserved regions of cytochrome c oxidases and cytochrome bd oxidases taking into consideration alignment of corresponding subunit I sequences. The results showed that both oxidase genes are present on the chromosome of several isolates characterized as Desulfovibrio. These genes were shown to be transcribed, as demonstrated by Reverse Transcriptase-PCR experiments on total RNA. In order to assess the relative contribution of each oxidase to oxygen consumption, oxygen uptake was measured for each isolate and further characterized by the effect of cyanide on oxygen consumption. It was concluded that cytochrome bd oxidase was the terminal membrane oxygen reductase allowing oxygen consumption. In addition, it was observed that isolates containing cytochrome bd oxidase had higher resistance to air exposure, suggesting an important role of this enzyme in survival to air exposure. The pattern for the presence of oxygen reductase genes was compared to the physiological pattern of substrate use, which was determined for each isolate. Salinity tolerance, pH and temperature growth of each isolate were also analyzed.

Characterization of photosystem II in salt-stressed cyanobacterial Spirulina platensis cells.

PSII activity was inhibited after Spirulina platensis cells were incubated with different salt concentrations (0-0.8 M NaCl) for 12 h. Flash-induced fluorescence kinetics showed that in the absence of DCMU, the half time of the fast and slow components decreased while that of the middle component increased considerably with increasing salt concentration. In the presence of DCMU, fluorescence relaxation was dominated by a 0.6s component in control cells. After salt stress, this was partially replaced by a faster new component with half time of 20-50 ms. Thermoluminescence measurements revealed that S(2)Q(A)(-) and S(2)Q(B)(-) recombinations were shifted to higher temperatures in parallel and the intensities of the thermoluminescence emissions were significantly reduced in salt-stressed cells. The period-four oscillation of the thermoluminescence B band was highly damped. There were no significant changes in contents of CP47, CP43, cytochrome c550, and D1 proteins. However, content of the PsbO protein in thylakoid fraction decreased but increased significantly in soluble fraction. The results suggest that salt stress leads to a modification of the Q(B) niche at the acceptor side and an increase in the stability of the S(2) state at the donor side, which is associated with a dissociation of the PsbO protein.

Cytochrome bd from Azotobacter vinelandii: evidence for high-affinity oxygen binding.

Cytochrome bd from Azotobacter vinelandii is a respiratory quinol oxidase that is highly efficient in reducing intracellular oxygen concentration, thus enabling nitrogen fixation under ambient aerobic conditions. Equilibrium measurements of O2 binding to ferrous heme d in the one-electron-reduced form of the A. vinelandii enzyme give Kd(O2) = 0.5 microM, close to the value for the Escherichia coli cytochrome bd (ca. 0.3 microM); thus, both enzymes have similar, high affinity for oxygen. The reaction of the A. vinelandii cytochrome bd in the one-electron-reduced and fully reduced states with O2 is extremely fast approaching the diffusion-controlled limit in water. In the fully reduced state, the rate of O2 binding depends linearly on the oxygen concentration consistently with a simple, single-step process. In contrast, in the one-electron-reduced state the rate of oxygen binding is hyperbolic, implying a more complex binding pattern. Two possible explanations for the saturation kinetics are considered: (A) There is a spectroscopically silent prebinding of oxygen to an unidentified low-affinity saturatable site followed by the oxygen transfer to heme d. (B) Oxygen binding to heme d requires an "activated" state of the enzyme in which an oxygen channel connecting heme d to the bulk is open. This channel is permanently open in the fully reduced enzyme (hence no saturation behavior) but flickers between the open and closed states in the one-electron-reduced enzyme.

[Use of antidepressant drugs in schizophrenic patients with depression]. / Intérêt des antidépresseurs chez le patient schizophrène présentant un syndrome dépressif.

INTRODUCTION: Depression is common in people with schizophrenia and is associated with substantial morbidity explaining also the considerable attention and recognition of this entity as suggested by the inclusion of the post-psychotic depression in DSM IV and ICD 10. The prevalence of this disorder varies according to the type of approach used (range between 7% to 75%). Prescription of antidepressants plus antipsychotic treatment is frequent in clinical practice (11 to 43%). BACKGROUND: Pharmacokinetic and metabolic interactions have been identified. The cytochrome P450 has been identified as being implicated in the metabolism of most psychotropics, mainly through the CYP1A2, CYP2C19, CYP2D6, CYP3A4 isoenzymes. Tricyclic antidepressants are likely to increase chlorpromazine plasma levels. Similarly, antipsychotics such as perphenazine, chlorpromazine or haloperidol can increase antidepressant plasma levels, through the inhibition of CYP 450 isoenzymes (CYP2D6). Most of the Specific Serotonin Recapture Inhibitors (SSRIs) are likely to inhibit one or several CYP450 isoenzymes. The inhibition is moderate to marked for CYP1A2 (fluvoxamine and fluoxetine), CYP2C19 (fluoxetine, fluvoxamine and sertraline), CYP2D6 (paroxetine, fluoxetine and sertraline), and CYP3A4 (fluvoxamine, fluoxetine and sertraline). In the US, one-fourth of psychiatrists report the use of depression-rating scales in schizophrenic patients. Non specific scales (Hamilton Depression Rating Scale or Beck Depression Inventory) are the most commonly used in spite of the fact that these scales do not allow the distinction of depressive from negative symptoms in schizophrenic patients. LITERATURE FINDINGS: Due to these limitations, more specific assessment tools for depressive symptoms in schizophrenia are required. Two specific scales for assessing depressive symptoms in schizophrenic patients have been constructed and validated. The Calgary Depression Scale (CDS) is a nine item scale, each item scored from 0 to 3. This scale was derived from the HDRS and the Present State Examination. Factor analysis showed that the CDS is unidimensional, has high internal consistency, and significant strong correlation with scores on the HDRS, Beck and BPRS depression scales. The CDS has been validated in different languages (Brazilian, Danish, French...). It has been shown that there is no overlap between negative or extrapyramidal and depressive symptoms assessed by the PDS in schizophrenic patients. The Psychotic Depression Scale (PDS) is a 32 item scale derived from the HDRS, PANSS, CPRS and AMDP, each item being rated from 0 to 7. A principal component analysis of the PDS items using a Varimax rotation disclosed 8 orthogonal components that account for 71% of the variance. These components involved the following dimensions : depressive mood, inhibition, vegetative signs, paranoid signs, strangeness of thought, inverse vegetative signs, guilt feelings and cognitive signs. The analysis revealed that the 'depressive mood' factor of the PDS was correlated with the 'depressive' factor and was slightly correlated with the cognitive factor of the PANSS. This first factor was not correlated with either the "negative" factor of the PANSS, or the Positive or Excitement factor of the PANSS. Hence, this PDS, factor distinguished depressive signs from negative symptoms. Due to their metrologic properties, specific scales should be preferred. However, only one open trial (of an antipsychotic) and two double blind controlled trials (one comparison of 2 antipsychotics and one comparison of an cholinesterase inhibitor versus placebo) have been published using the CDS. Likewise, only one double blind controlled trial using the PDS (comparison of 2 antipsychotics) has been published. No study of the effect of antidepressants in depressed schizophrenic patients has been published, using either the CDS or the PDS assessment criteria. DISCUSSION: These specific scales are rarely used in clinical practice. Only about 1% of the US psychiatrists reported the use of the Calgary Depression Scale. Several open clinical trials have assessed the efficacy of antidepressant agents added to antipsychotic in patients with schizophrenia. They have produced inconsistent results but have suffered from methodological limits (short duration of the studies, non homogeneous inclusion criteria, heterogeneous assessment methods...). Due to the lack of a reference drug, double blind placebo-controlled trial are necessary. A recent meta-analysis has been performed on results of trials that have investigated the clinical efficacy of antidepressant medication (either tricyclics, SSRIs or others) in the treatment of depression in schizophrenic patients. In a subset of 5 trials (209 patients), the proportion improved in the antidepressant group was 26% (95% CI 10 to 42) higher than in the placebo group. The estimated number needed to treat was 4. In a subgroup of 6 studies (267 patients), the standardized mean difference on the HDRS score was -0.27 (95% CI - 0.7 to -0.2). There was no evidence that antidepressant treatment induced a deterioration of psychotic symptoms in these trials. CONCLUSION: The results provide weak evidence for the efficacy of antidepressants in patients with schizophrenia and depression. Today, the only SSRI tested in the treatment of depression in schizophrenic patients is sertraline. One study led to positive results. Since the meta-analysis, one additional study has been performed comparing sertraline to placebo. No difference between the 2 treatment groups was demonstrated but the power of the trial was rather low. Further research is required to determine the best approach towards treating depression in patients with schizophrenia, with clinical trials performed for longer periods, using appropriate assessment criteria such as depressive symptoms and quality of life.

Characterization and expression analysis of the cytochrome bd oxidase operon from Desulfovibrio gigas.

Although classified as anaerobic, Desulfovibrio gigas contains a functional canonical membrane respiratory chain, including a cytochrome bd quinol oxidase as its terminal element. In the present study, we report the identification of the operon cydAB encoding the two subunits of cytochrome bd from this bacterium. Two hypothetical promoter regions and sequences resembling transcriptional regulators-binding sites have been identified. Amino acid sequence analysis revealed a high similarity to cytochrome bd from other organisms, presenting the conserved residues typical from these proteins. Reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analysis confirmed the operon transcription. Gene expression was assessed by real-time RT-PCR in cells grown in different media and under exposure to oxygen and nitric oxide. mRNA levels were slightly enhanced in the presence of 150 microM: NO. However, in the presence of 10 microM: NO, a decrease was observed of the steady-state population of cydAB mRNA. No considerable effect was observed in the presence of fumarate/sulfate medium, 60 microM: O2 or 10 microM: NO.

Pressure-regulated biosynthesis of cytochrome bd in piezo- and psychrophilic deep-sea bacterium Shewanella violacea DSS12.

The genes of cytochrome bd-encoding cydAB were identified from a deep-sea bacterium Shewanella violacea DSS12. These showed significant homologies with known cydAB gene sequences from various organisms. Additionally, highly conserved regions that are important for the enzymatic function were also conserved in cydA of S. violacea. Based on the results, transcriptional analysis of cydAB operon and cydDC operon (required for assembly of cytochrome bd) of S. violacea in microaerobic condition was performed under the growth condition of various pressures. The gene of cydA was expressed even under the condition of atmospheric pressure and its expression was enhanced with pressurization. On the other hand, the expression of cydC was strongly depressed under the condition of atmospheric pressure compared with the case under high pressure. It appeared spectrophotometrically that loss of cytochrome bd in S. violacea under atmospheric pressure shown in previous study is caused mainly by the loss of cydDC. Further, under the growth condition of atmospheric pressure, either less amount or no d-type cytochrome was expressed compared with the case of high-pressure condition even if the organism was grown under alkaline condition or in the presence of uncoupler, which are the inducible condition of d-type cytochrome in Escherichia coli. These results suggested that the significant amount of d-type cytochrome expression is specific event under the growth condition of high pressure.

Intracellular expression of Vitreoscilla hemoglobin modifies microaerobic Escherichia coli metabolism through elevated concentration and specific activity of cytochrome o.

The function of the reversible oxygen-binding hemoprotein from Vitreoscilla (VHb), which enhances oxygen-limited cell growth and recombinant protein production when functionally expressed in Escherichia coli, was investigated in wild-type E. coli and in E. coli mutants lacking one of the two terminal oxidases, cytochrome o complex (aerobic terminal oxidase, Cyo) or cytochrome d complex (microaerobic terminal oxidase, Cyd). Deconvolution of VHb, cytochrome o, and cytochrome d bands from in vivo absorption spectra revealed a 5-fold enhancement in cytochrome o content and a 1.5-fold increment in cytochrome d by VHb under microaerobic environments (dissolved oxygen less than 2% air saturation). Based upon oxygen uptake kinetics measurements of these mutants, the apparent oxygen affinity of the Cyo(+), Cyd(-) E. coli was increased in the presence of VHb, but no difference in the apparent K(m) was observed for the Cyo(-), Cyd(+) strain. Results suggest that the expression of VHb in E. coli increases the level and activity of terminal oxidases and thereby improves the efficiency of microaerobic respiration and growth.