Cytochrome bd-I Research Articles

Improved poly(3-hydroxybutyrate) production in Escherichia coli by inactivation of cytochrome bd-II oxidase or/and NDH-II dehydrogenase in low efficient respiratory chains

In order to redirect more carbon flux from TCA cycle into poly(3-hydroxybutyrate) (PHB) biosynthesis pathway via increasing respiratory efficiency, appB and ndh genes encoding cytochrome bd-II oxidase and NDH-II dehydrogenase were inactivated in Escherichia coli JM109/pBHR68. All appB or/and ndh knockout strains exhibited significantly increased PHB accumulation accompanying with increased NAD(P)H/NAD(P)+ ratio and intracellular acetyl-CoA pool. Among them, the Δndh strain could accumulate up to 6.16g/L PHB from 20g/L glucose and 3.5g/L PHB from 20g/L xylose, respectively, a 1.76-fold and 3.43-fold increase compared to the wild-type control. The PHB production of this strain reached 28.23g/L in a 5-L fermentor study, which was 2.70-fold as much as that of the wild-type control. These results indicated that inactivating the cytochrome bd-II oxidase or/and NDH-II dehydrogenase of the aerobic respiratory chain is a simple and effective strategy to improve PHB biosynthesis in E. coli. To date, this is the first time to improve PHB production by inactivation of cytochrome bd-II oxidase or/and NDH-II dehydrogenase in low efficient respiratory chains.

Delocalised electron transport and chemiosmosis in Escherichia coli

The membrane organisation of electron transport and chemiosmosis remains a topic of intense debate, with current models in many bioenergetic membranes favouring the assembly of multiple chemiosmotic components into supercomplexes that could control the pathways of electron flow and utilisation of the proton-motive force. We set out to investigate the distribution and dynamics of OXPHOS components in the plasma membrane of Escherichia coli using a combination of fluorescent protein tagging and fluorescence microscopy with dynamic tracking and single-particle analysis. Complexes investigated included NDH-1, SDH, Cytochrome bd-I and the proton-translocating ATPase, which could all be tagged with a variety of fluorescent proteins, with minimal loss of function [1,2]. Fluorescence microscopy in vivo showed that all complexes tested are concentrated in mobile domains in the membrane, with dimensions of about 100–200 nm and containing 10 s to 100 s of the tagged complex. Simultaneous visualisation of pairs of tagged complexes showed that different complexes are concentrated in separate domains, with no significant co-localisation and therefore no supercomplexes [2]. Since the pairs of complexes tested include the two complexes involved in one of the major respiratory electron transport pathways, and a major source and sink for the proton-motive force, it follows that both electron transport and the proton motive force are largely delocalised over the entire membrane area in E. coli. Consistent with this model, we observed rapid long-range diffusion of a fluorescent quinone analogue. We suggest that long-range quinone diffusion serves to carry electrons between islands of distinct electron transport complexes in the membrane.

Organization of the Escherichia coli aerobic enzyme complexes of oxidative phosphorylation in dynamic domains within the cytoplasmic membrane.

The Escherichia coli cytoplasmic membrane contains the enzyme complexes of oxidative phosphorylation (OXPHOS). Not much is known about their supramolecular organization and their dynamics within the membrane in this model organism. In mitochondria and other bacteria, it was demonstrated by nondenaturing electrophoretic methods and electron microscopy that the OXPHOS complexes are organized in so-called supercomplexes, stable assemblies with a defined number of the individual enzyme complexes. To investigate the organization of the E. coli enzyme complexes of aerobic OXPHOS in vivo, we established fluorescent protein fusions of the NADH:ubiquinone oxidoreductase, the succinate:ubiquinone oxidoreductase, the cytochrome bd-I, and the cytochrome bo3 terminal oxidases, and the FoF1 ATP-synthase. The fusions were integrated in the chromosome to prevent artifacts caused by protein overproduction. Biochemical analysis revealed that all modified complexes were fully assembled, active, and stable. The distribution of the OXPHOS complexes in living cells was determined using total internal reflection fluorescence microscopy. The dynamics within the membrane were detected by fluorescence recovery after photobleaching. All aerobic OXPHOS complexes showed an uneven distribution in large mobile patches within the E. coli cytoplasmic membrane. It is discussed whether the individual OXPHOS complexes are organized as clustered individual complexes, here called “segrazones.”

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 formate:oxygen oxidoreductase supercomplex of Escherichia coli aerobic respiratory chain

The Escherichia coli formate:oxygen oxidoreductase supercomplex (FdOx) was investigated with respect to function and composition. Formate oxidoreductase activity was detected in blue native polyacrylamide gel electrophoresis (BN-PAGE) resolved membranes of E. coli, which were also capable of cyanide sensitive formate:oxygen oxidoreductase activity. The latter was compromised in strains devoid of specific oxygen reductases, particularly, in those devoid of cytochrome bo3 or bdI. A principal component analysis (PCA) integrating E. coli aerobic respiratory chain gene transcription, enzyme activity and growth dynamics was performed, correlating formate:oxygen oxidoreductase activity and the transcription of the genes encoding cytochromes bo3 and bdI, and corroborating previous evidence that associated these complexes in FdOx.

Cytochrome bd-I in Escherichia coli is less sensitive than cytochromes bd-II or bo′' to inhibition by the carbon monoxide-releasing molecule, CORM-3: N-acetylcysteine reduces CO-RM uptake and inhibition of respiration

Background: CO-releasing molecules (CO-RMs) are potential therapeutic agents, able to deliver CO – a critical gasotransmitter – in biological environments. CO-RMs are also effective antimicrobial agents; although the mechanisms of action are poorly defined, haem-containing terminal oxidases are primary targets. Nevertheless, it is clear from several studies that the effects of CO-RMs on biological systems are frequently not adequately explained by the release of CO: CO-RMs are generally more potent inhibitors than is CO gas and other effects of the molecules are evident. Methods: Because sensitivity to CO-RMs cannot be predicted by sensitivity to CO gas, we assess the differential susceptibilities of strains, each expressing only one of the three terminal oxidases of E. coli — cytochrome bd-I, cytochrome bd-II and cytochrome bo′, to inhibition by CORM-3. We present the first sensitive measurement of the oxygen affinity of cytochrome bd-II (Km 0.24μM) employing globin deoxygenation. Finally, we investigate the way(s) in which thiol compounds abolish the inhibitory effects of CORM-2 and CORM-3 on respiration, growth and viability, a phenomenon that is well documented, but poorly understood. Results: We show that a strain expressing cytochrome bd-I as the sole oxidase is least susceptible to inhibition by CORM-3 in its growth and respiration of both intact cells and membranes. Growth studies show that cytochrome bd-II has similar CORM-3 sensitivity to cytochrome bo′. Cytochromes bo′ and bd-II also have considerably lower affinities for oxygen than bd-I. We show that the ability of N-acetylcysteine to abrogate the toxic effects of CO-RMs is not attributable to its antioxidant effects, or prevention of CO targeting to the oxidases, but may be largely due to the inhibition of CO-RM uptake by bacterial cells. Conclusions: A strain expressing cytochrome bd-I as the sole terminal oxidase is least susceptible to inhibition by CORM-3. N-acetylcysteine is a potent inhibitor of CO-RM uptake by E. coli. General significance: Rational design and exploitation of CO-RMs require a fundamental understanding of their activity. CO and CO-RMs have multifaceted effects on mammalian and microbial cells; here we show that the quinol oxidases of E. coli are differentially sensitive to CORM-3. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Cytochrome bd-II from aerobic respiratory chain of Escherichia coli is a proton-motive force generator

Cytochrome bd-II from aerobic respiratory chain of Escherichia coli is a proton-motive force generator

The aerobic respiratory chain of Escherichia coli: from genes to supercomplexes

In spite of the large number of reports on the aerobic respiratory chain of Escherichia coli, from gene transcription regulation to enzyme kinetics and structural studies, an integrative perspective of this pathway is yet to be produced. Here, a multi-level analysis of the aerobic respiratory chain of E. coli was performed to find correlations between gene transcription, enzyme activity, growth dynamics, and supercomplex formation and composition. The transcription level of all genes encoding the aerobic respiratory chain of E. coli varied significantly in response to bacterial growth. Coordinated expression patterns were observed between the genes encoding NADH : quinone oxidoreductase and complex I (NDH-1), alternative NADH : quinone oxidoreductase (NDH-2) and cytochrome bdI, and also between sdhA and appC, encoding succinate dehydrogenase and cytochrome bdII, respectively. In general, the rates of the respiratory chain activities increased from mid-exponential to late-stationary phase, with no significant further variation occurring until the mid-stationary phase. Multi-level correlations between gene transcription, enzyme activity and growth dynamics were also found in this study. The previously reported NADH dehydrogenase and formate : oxygen oxidoreductase supercomplexes of E. coli were already assembled at mid-exponential phase and remained throughout growth. A new succinate oxidase supercomplex composed of succinate dehydrogenase and cytochrome bdII was identified, in agreement with the suggestion provided by the coordinated transcription of sdhA and appC.

Oxoferryl-Porphyrin Radical Catalytic Intermediate in Cytochrome bd Oxidases Protects Cells from Formation of Reactive Oxygen Species

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b(558) that donates electrons to a binuclear heme b(595)/heme d center. The reaction with O(2) and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O(2), the O-O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b(595). Compound I accumulates to 0.75-0.85 per enzyme in agreement with its much higher rate of formation (~20,000 s(-1)) compared with its rate of decay (~1,900 s(-1)). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b(558) before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O-O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O-O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species.

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.

Clustering and dynamics of cytochrome bd‐I complexes in the Escherichia coli plasma membrane in vivo

The cytochrome bd-I complex of Escherichia coli is a respiratory terminal oxidase and an integral component of the cytoplasmic membrane. As with other respiratory components, the organization and dynamics of this complex in living membranes is unknown. We set out to visualize the distribution and dynamics of this complex in vivo. By exchanging cydB for cydB-gfpgcn4 on the E. coli chromosome, we produced a strain (YTL01) that expresses functional GFP-tagged cytochrome bd-I terminal oxidase complexes under wild-type genetic control. We imaged live YTL01 cells using video-rate epifluorescence and total internal reflection fluorescence (TIRF) microscopy in combination with fluorescence recovery after photobleaching (FRAP) and saw mobile spots of GFP fluorescence in plasma membranes. Numbers of GFP molecules per spot were quantified by step-wise photobleaching giving a broad distribution with a mean of approximately 76, indicating that cytochrome bd-I is concentrated in mobile patches in the E. coli plasma membrane. We hypothesize that respiration occurs in mobile membrane patches which we call 'respirazones'.

Sequence analysis of cytochrome bd oxidase suggests a revised topology for subunit I

Numerous sequences of the cytochrome bd quinol oxidase (cytochrome bd) have recently become available for analysis. The analysis has revealed a small number of conserved residues, a new topology for subunit I and a phylogenetic tree involving extensive horizontal gene transfer. There are 20 conserved residues in subunit I and two in subunit II. Algorithms utilizing multiple sequence alignments predicted a revised topology for cytochrome bd, adding two transmembrane helices to subunit I to the seven that were previously indicated by the analysis of the sequence of the oxidase from E. coli. This revised topology has the effect of relocating the N-terminus and C-terminus to the periplasmic and cytoplasmic sides of the membrane, respectively. The new topology repositions I-H19, the putative ligand for heme b 595, close to the periplasmic edge of the membrane, which suggests that the heme b 595/heme d active site of the oxidase is located near the outer (periplasmic) surface of the membrane. The most highly conserved region of the sequence of subunit I contains the sequence GRQPW and is located in a predicted periplasmic loop connecting the eighth and ninth transmembrane helices. The potential importance of this region of the protein was previously unsuspected, and it may participate in the binding of either quinol or heme d. There are two very highly conserved glutamates in subunit I, E99 and E107, within the third transmembrane helix ( E. coli cytochrome bd-I numbering). It is speculated that these glutamates may be part of a proton channel leading from the cytoplasmic side of the membrane to the heme d oxygen-reactive site, now placed near the periplasmic surface. The revised topology and newly revealed conserved residues provide a clear basis for further experimental tests of these hypotheses. Phylogenetic analysis of the new sequences of cytochrome bd reveals considerable deviation from the 16sRNA tree, suggesting that a large amount of horizontal gene transfer has occurred in the evolution of cytochrome bd.

Effects of sigmaS and the transcriptional activator AppY on induction of the Escherichia coli hya and cbdAB-appA operons in response to carbon and phosphate starvation.

The transcriptional regulation of two energy metabolism operons, hya and cbdAB-appA, has been investigated during carbon and phosphate starvation. The hya operon encodes hydrogenase 1, and the cbdAB-appA operon encodes cytochrome bd-II oxidase and acid phosphatase, pH 2.5. Both operons are targets for the transcriptional activator AppY. In exponential growth, expression of the hya and cbd operons was reduced in an rpoS mutant lacking the RNA polymerase sigmaS factor, and the induction of the two operons by entry into stationary phase in rich medium was strongly dependent on sigmaS. Both operons were induced by carbon starvation, but only induction of the hya operon was dependent on sigmaS, whereas that of the cbd promoter was dependent on AppY. The appY gene also showed sigmaS-dependent induction by carbon starvation. The cbd and hya operons were also found to exhibit a sigmaS-dependent transient twofold induction by osmotic upshift. Like the cbd operon, the hya operon was highly induced by phosphate starvation. For both operons the induction was strongly dependent on AppY. The induction ratio of the two operons was the same in rpoS+ and rpoS mutant strains, indicating that the phosphate starvation-induced increase in sigmaS concentration is not involved in the phosphate regulation of these operons.

Purification of a cytochrome bd terminal oxidase encoded by the Escherichia coli app locus from a delta cyo delta cyd strain complemented by genes from Bacillus firmus OF4.

Escherichia coli GK100, with deletions in the operons encoding its two terminal oxidases, cytochrome bo and ctyochrome bd, was complemented for growth on succinate by a recombinant plasmid (pMS100) containing a 3.4-kb region of DNA from alkaliphilic Bacillus firmus OF4. The complementing DNA was predicted to encode five proteins, but neither sequence analysis nor complementation experiments with subclones provided insight into the basis for the complementation. Cytochrome difference spectra of everted membrane vesicles from the transformed strain had characteristics of a cytochrome bd spectrum but with features different from those observed for alkaliphile membranes. To determine the bacterial source and identity of the structural genes for the cytochrome bd in the transformed mutant, the complex was extracted and partially purified. On sodium dodecyl sulfate-polyacrylamide gels, two polypeptides were resolved from the preparation, 43 (subunit I) and 27 (subunit II) kDa. An internal peptide from subunit I was sequenced, and it yielded the same primary sequence as is found in positions 496 to 510 of E. coli appC. Consistent with the microsequencing results pMS100 failed to complement a triple mutant of E. coli carrying a deletion in appB as well as in the cyo and cyd loci. The deduced sequence of AppBC had been predicted to be very similar to the sequence of CydAB (J. Dassa et al., Mol. Gen. Genet. 229:341-352, 1991) but this is the first demonstration that the former is indeed a cytochrome bd terminal oxidase. The enzyme catalyzed oxygen uptake coupled to quinol or N,N,N',N'-tetramethyl-p-phenylenediamine oxidation, and the activity was sensitive to cyanide. No cross-reactivity to subunit-specific polyclonal antibodies directed against the two individual subunits of cyd-encoded cytochrome bd was detected. Since this is the second cytochrome bd discovered in E. coli, it is proposed that the two complexes be designated cytochrome bd-I (cydAB-encoded enzyme) and cytochrome bd-II (appBC-encoded enzyme). In addition, cbdAB is suggested as a more appropriate gene designation for cytochrome bd than either appBC or cyxAB.