Bd Oxidase Research Articles

Pyrrolo[3,4-c]pyridine-1,3(2H)-diones: A Novel Antimycobacterial Class Targeting Mycobacterial Respiration.

High-throughput screening of a library of small polar molecules against Mycobacterium tuberculosis led to the identification of a phthalimide-containing ester hit compound (1), which was optimized for metabolic stability by replacing the ester moiety with a methyl oxadiazole bioisostere. A route utilizing polymer-supported reagents was designed and executed to explore structure-activity relationships with respect to the N-benzyl substituent, leading to compounds with nanomolar activity. The frontrunner compound (5h) from these studies was well tolerated in mice. A M. tuberculosis cytochrome bd oxidase deletion mutant (ΔcydKO) was hyper-susceptible to compounds from this series, and a strain carrying a single point mutation in qcrB, the gene encoding a subunit of the menaquinol cytochrome c oxidoreductase, was resistant to compounds in this series. In combination, these observations indicate that this novel class of antimycobacterial compounds inhibits the cytochrome bc1 complex, a validated drug target in M. tuberculosis.

Molecular evolution of cytochrome bd oxidases across proteobacterial genomes.

This work is aimed to resolve the complex molecular evolution of cytochrome bd ubiquinol oxidase, a nearly ubiquitous bacterial enzyme that is involved in redox balance and bioenergetics. Previous studies have created an unclear picture of bd oxidases phylogenesis without considering the existence of diverse types of bd oxidases. Integrated approaches of genomic and protein analysis focused on proteobacteria have generated a molecular classification of diverse types of bd oxidases, which produces a new scenario for interpreting their evolution. A duplication of the original gene cluster of bd oxidase might have occurred in the ancestors of extant α-proteobacteria of the Rhodospirillales order, such as Acidocella, from which the bd-I type of the oxidase might have diffused to other proteobacterial lineages. In contrast, the Cyanide-Insensitive Oxidase type may have differentiated into recognizable subtypes after another gene cluster duplication. These subtypes are widespread in the genomes of α-, β-, and γ-proteobacteria, with occasional instances of lateral gene transfer. In resolving the evolutionary pattern of proteobacterial bd oxidases, this work sheds new light on the basal taxa of α-proteobacteria from which the γ-proteobacterial lineage probably emerged.

Conservation analysis of the CydX protein yields insights into small protein identification and evolution

BackgroundThe reliable identification of proteins containing 50 or fewer amino acids is difficult due to the limited information content in short sequences. The 37 amino acid CydX protein in Escherichia coli is a member of the cytochrome bd oxidase complex, an enzyme found throughout Eubacteria. To investigate the extent of CydX conservation and prevalence and evaluate different methods of small protein homologue identification, we surveyed 1095 Eubacteria species for the presence of the small protein.ResultsOver 300 homologues were identified, including 80 unannotated genes. The ability of both closely-related and divergent homologues to complement the E. coli ΔcydX mutant supports our identification techniques, and suggests that CydX homologues retain similar function among divergent species. However, sequence analysis of these proteins shows a great degree of variability, with only a few highly-conserved residues. An analysis of the co-variation between CydX homologues and their corresponding cydA and cydB genes shows a close synteny of the small protein with the CydA long Q-loop. Phylogenetic analysis suggests that the cydABX operon has undergone horizontal gene transfer, although the cydX gene likely evolved in a progenitor of the Alpha, Beta, and Gammaproteobacteria. Further investigation of cydAB operons identified two additional conserved hypothetical small proteins: CydY encoded in CydAQlong operons that lack cydX, and CydZ encoded in more than 150 CydAQshort operons.ConclusionsThis study provides a systematic analysis of bioinformatics techniques required for the unique challenges present in small protein identification and phylogenetic analyses. These results elucidate the prevalence of CydX throughout the Proteobacteria, provide insight into the selection pressure and sequence requirements for CydX function, and suggest a potential functional interaction between the small protein and the CydA Q-loop, an enigmatic domain of the cytochrome bd oxidase complex. Finally, these results identify other conserved small proteins encoded in cytochrome bd oxidase operons, suggesting that small protein subunits may be a more common component of these enzymes than previously thought.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-946) contains supplementary material, which is available to authorized users.

Regulation of nitrite resistance of the cytochrome cbb3 oxidase by cytochrome c ScyA in Shewanella oneidensis.

Cytochrome c proteins, as enzymes to exchange electrons with substrates or as pure electron carriers to shuttle electrons, play vital roles in bacterial respiration and photosynthesis. In Shewanella oneidensis, a research model for the respiratory diversity, at least 42 c-type cytochromes are predicted to be encoded in the genome and are regarded to be the foundation of its highly branched electron transport pathways. However, only a small number of c-type cytochromes have been extensively studied. In this study, we identify soluble cytochrome c ScyA as an important factor influencing the nitrite resistance of a strain devoid of the bd oxidase by utilizing a newly developed transposon mutagenesis vector, which enables overexpression of the gene(s) downstream of the insertion site. We show that when in overabundance ScyA facilitates growth against nitrite inhibition by enhancing nitrite resistance of the cbb3 oxidase. Based on the data presented in this study, we suggest two possible mechanisms underlying the observed effect of ScyA: (1) ScyA increases electron flow to the cbb3 oxidase; (2) ScyA promotes nitrite resistance of the cbb3 oxidase, possibly by direct interaction.

Evidence for the requirement of CydX in function but not assembly of the cytochrome bd oxidase in Shewanella oneidensis

BackgroundCytochrome bd oxidase, existing widely in bacteria, produces a proton motive force by the vectorial charge transfer of protons and more importantly, endows bacteria with a number of vitally important physiological functions, such as enhancing tolerance to various stresses. Although extensively studied as a CydA–CydB two-subunit complex for decades, the complex in certain groups of bacteria is recently found to in fact consist of an additional subunit, which is functionally essential. MethodsWe investigated the assembly of the CydA–CydB complex using BiFC. We investigated the function of CydX using mutational analysis. ResultsCydX, a 38-amino-acid inner-membrane protein, is associated with the CydA–CydB complex in Shewanella oneidensis, a facultative anaerobe renowned for its respiratory versatility. It is clear that CydX is neither required for the in vivo assembly of the CydA–CydB complex nor relies on the complex for its translocation and integration into the membrane. The N-terminal segment (1–25 amino acid residues) and short periplasmic overhang of CydX, with respect to functionality, are important whereas the remaining C-terminal segment is rather flexible. ConclusionBased on these findings, we postulate that CydX may function by positioning and stabilizing the prosthetic hemes, especially heme d in the CydA–CydB complex although a role of participating in catalytic reaction is not excluded. General significanceThe work provides novel insights into our understanding of the small subunit of the cytochrome bd oxidase.

HrtBA and menaquinones control haem homeostasis in Lactococcus lactis.

Lactococcus lactis is a fermenting Gram-positive bacterium widely used for production of dairy products. Lacking haem biosynthesis genes, L. lactis can still shift to an energetically favourable respiratory metabolism by activating a terminal cytochrome bd oxidase when haem is added to an aerated culture. Haem intracellular homeostasis is mediated by the hrtRBA operon encoding the conserved membrane HrtBA haem efflux permease and the unique intracellular haem sensor and regulator, HrtR. Here we report that membrane-associated menaquinones (MK) favour the accumulation of reduced haem in membranes. An oxidative environment, provided by oxygen, prevents and reverses haemin reduction by MK and thus limits haem accumulation in membranes. HrtBA counteracts MK-dependent membrane retention of excess haem in membrane, suggesting direct efflux from this compartment. Moreover, both HrtBA and MK-mediated reduction have a strong impact on haem intracellular pools, as determined via HrtR haem sensor induction, suggesting that intracellular haem acquisition is controlled at the membrane level without the need for dedicated import systems. Our conclusions lead to a new hypothesis of haem acquisition and regulation in which HrtBA and the bacterial membrane have central roles in L. lactis.

Fast electron transfer between hemes and binding of internal ligand to heme d upon photodissociation of CO from cytochrome bd oxidase

Fast electron transfer between hemes and binding of internal ligand to heme d upon photodissociation of CO from cytochrome bd oxidase

Cytochrome bd oxidase from Escherichia coli is a quinol peroxidase mechanistically adjusted to protecting the cell from hydrogen peroxide

Cytochrome bd oxidase from Escherichia coli is a quinol peroxidase mechanistically adjusted to protecting the cell from hydrogen peroxide

Cytochrome bd oxidase and bacterial tolerance to oxidative and nitrosative stress

Cytochrome bd oxidase and bacterial tolerance to oxidative and nitrosative stress

The small RNA RyhB homologs from Salmonella typhimurium participate in the response to S-nitrosoglutathione-induced stress

Typically, the expression of sRNAs is activated in response to environmental stimuli in order to regulate gene expression through post-transcriptional mechanisms. In the present work we show that the Salmonellatyphimurium paralogous sRNAs RyhB-1 and RyhB-2 are induced in response to the nitrosating agent S-nitrosoglutathione (GSNO). Inactivation of these sRNAs decreased S. typhimurium resistance to GSNO and increased the levels of nitrosylated proteins. These results prompted us to evaluate a possible role of these sRNAs in nitrosative stress resistance. RNA profiling was used as a screen to identify novel RyhB-1 and RyhB-2 regulated targets. A subset of genes was filtered based on their potential role in the response to nitrosative stress and their expression was analyzed by quantitative RT-PCR in wild type, single and double mutant strains (ΔryhB1, ΔryhB2 and ΔryhB1 ΔryhB2) treated with GSNO. In response to GSNO RyhB-1 and RyhB-2 negatively regulate the expression of the genes cyoABC (cytochrome bo oxidase), cydB (cytochrome bd oxidase), cybC (cytochrome b-562), and positively regulate the nirBCD operon (nitrite reductase system). Together, these results suggest that RyhB-1 and RyhB-2 finely tune the expression of genes coding for cytochrome oxidases and the nitrate reductase system, allowing the cell to cope with GSNO-induced stress.

The multiple roles of hypothetical gene BPSS1356 in Burkholderia pseudomallei.

Burkholderia pseudomallei is an opportunistic pathogen and the causative agent of melioidosis. It is able to adapt to harsh environments and can live intracellularly in its infected hosts. In this study, identification of transcriptional factors that associate with the β′ subunit (RpoC) of RNA polymerase was performed. The N-terminal region of this subunit is known to trigger promoter melting when associated with a sigma factor. A pull-down assay using histidine-tagged B. pseudomallei RpoC N-terminal region as bait showed that a hypothetical protein BPSS1356 was one of the proteins bound. This hypothetical protein is conserved in all B. pseudomallei strains and present only in the Burkholderia genus. A BPSS1356 deletion mutant was generated to investigate its biological function. The mutant strain exhibited reduced biofilm formation and a lower cell density during the stationary phase of growth in LB medium. Electron microscopic analysis revealed that the ΔBPSS1356 mutant cells had a shrunken cytoplasm indicative of cell plasmolysis and a rougher surface when compared to the wild type. An RNA microarray result showed that a total of 63 genes were transcriptionally affected by the BPSS1356 deletion with fold change values of higher than 4. The expression of a group of genes encoding membrane located transporters was concurrently down-regulated in ΔBPSS1356 mutant. Amongst the affected genes, the putative ion transportation genes were the most severely suppressed. Deprivation of BPSS1356 also down-regulated the transcriptions of genes for the arginine deiminase system, glycerol metabolism, type III secretion system cluster 2, cytochrome bd oxidase and arsenic resistance. It is therefore obvious that BPSS1356 plays a multiple regulatory roles on many genes.

Subunit CydX of Escherichia coli cytochrome bd ubiquinol oxidase is essential for assembly and stability of the di‐heme active site

Cytochrome bd ubiquinol oxidase uses the electron transport from ubiquinol to oxygen to establish a proton gradient across the membrane. The enzyme complex consists of subunits CydA and B and contains two b- and one d-type hemes as cofactors. Recently, it was proposed that a third subunit named CydX is essential for the function of the complex. Here, we show that CydX is indeed a subunit of purified Escherichia coli cytochrome bd oxidase and that the small protein is needed either for the assembly or the stability of the active site di-heme center and, thus, is essential for oxidase activity.

Energetics of pathogenic bacteria and opportunities for drug development.

The emergence and spread of drug-resistant pathogens and our inability to develop new antimicrobials to overcome resistance has inspired scientists to consider new targets for drug development. Cellular bioenergetics is an area showing promise for the development of new antimicrobials, particularly in the discovery of new anti-tuberculosis drugs where several new compounds have entered clinical trials. In this review, we have examined the bioenergetics of various bacterial pathogens, highlighting the versatility of electron donor and acceptor utilisation and the modularity of electron transport chain components in bacteria. In addition to re-examining classical concepts, we explore new literature that reveals the intricacies of pathogen energetics, for example, how Salmonella enterica and Campylobacter jejuni exploit host and microbiota to derive powerful electron donors and sinks; the strategies Mycobacterium tuberculosis and Pseudomonas aeruginosa use to persist in lung tissues; and the importance of sodium energetics and electron bifurcation in the chemiosmotic anaerobe Fusobacterium nucleatum. A combination of physiological, biochemical, and pharmacological data suggests that, in addition to the clinically-approved target F1Fo-ATP synthase, NADH dehydrogenase type II, succinate dehydrogenase, hydrogenase, cytochrome bd oxidase, and menaquinone biosynthesis pathways are particularly promising next-generation drug targets. The realisation of cellular energetics as a rich target space for the development of new antimicrobials will be dependent upon gaining increased understanding of the energetic processes utilised by pathogens in host environments and the ability to design bacterial-specific inhibitors of these processes.

Cytochrome bd Oxidase and Hydrogen Peroxide Resistance in Mycobacterium tuberculosis

Cytochrome bd oxidase is a prokaryotic respiratory oxidase, phylogenetically unrelated to the well-known heme-copper oxidases, which catalyzes the reduction of O2 to 2H2O. Apart from having a role in energy metabolism, this enzyme plays other key functions relevant to physiology and, particularly, to adaptation to stress conditions (reference 1 and references therein). The enzyme is encoded in several pathogens, and its expression was found to correlate positively with virulence in some bacteria. In Mycobacterium tuberculosis, a transient upregulation of cytochrome bd was observed in vivo during the transition from acute to chronic infection of mouse lungs, as was reduced virulence of a mutant strain defective in the cytochrome bd -associated transporter CydC (2). Stimulation …

Membrane-bound oxygen reductases of the anaerobic sulfate-reducing Desulfovibrio vulgaris Hildenborough: roles in oxygen defence and electron link with periplasmic hydrogen oxidation

Cytoplasmic membranes of the strictly anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough contain two terminal oxygen reductases, a bd quinol oxidase and a cc(b/o)o3 cytochrome oxidase (Cox). Viability assays pointed out that single Δbd, Δcox and double ΔbdΔcox deletion mutant strains were more sensitive to oxygen exposure than the WT strain, showing the involvement of these oxygen reductases in the detoxification of oxygen. The Δcox strain was slightly more sensitive than the Δbd strain, pointing to the importance of the cc(b/o)o3 cytochrome oxidase in oxygen protection. Decreased O2 reduction rates were measured in mutant cells and membranes using lactate, NADH, ubiquinol and menadiol as substrates. The affinity for oxygen measured with the bd quinol oxidase (Km, 300 nM) was higher than that of the cc(b/o)o3 cytochrome oxidase (Km, 620 nM). The total membrane activity of the bd quinol oxidase was higher than that of the cytochrome oxidase activity in line with the higher expression of the bd oxidase genes. In addition, analysis of the ΔbdΔcox mutant strain indicated the presence of at least one O2-scavenging membrane-bound system able to reduce O2 with menaquinol as electron donor with an O2 affinity that was two orders of magnitude lower than that of the bd quinol oxidase. The lower O2 reductase activity in mutant cells with hydrogen as electron donor and the use of specific inhibitors indicated an electron transfer link between periplasmic H2 oxidation and membrane-bound oxygen reduction via the menaquinol pool. This linkage is crucial in defence of the strictly anaerobic bacterium Desulfovibrio against oxygen stress.

Evidence for a Key Role of Cytochrome bo3 Oxidase in Respiratory Energy Metabolism of Gluconobacter oxydans

The obligatory aerobic acetic acid bacterium Gluconobacter oxydans oxidizes a variety of substrates in the periplasm by membrane-bound dehydrogenases, which transfer the reducing equivalents to ubiquinone. Two quinol oxidases, cytochrome bo3 and cytochrome bd, then catalyze transfer of the electrons from ubiquinol to molecular oxygen. In this study, mutants lacking either of these terminal oxidases were characterized. Deletion of the cydAB genes for cytochrome bd had no obvious influence on growth, whereas the lack of the cyoBACD genes for cytochrome bo3 severely reduced the growth rate and the cell yield. Using a respiration activity monitoring system and adjusting different levels of oxygen availability, hints of a low-oxygen affinity of cytochrome bd oxidase were obtained, which were supported by measurements of oxygen consumption in a respirometer. The H(+)/O ratio of the ΔcyoBACD mutant with mannitol as the substrate was 0.56 ± 0.11 and more than 50% lower than that of the reference strain (1.26 ± 0.06) and the ΔcydAB mutant (1.31 ± 0.16), indicating that cytochrome bo3 oxidase is the main component for proton extrusion via the respiratory chain. Plasmid-based overexpression of cyoBACD led to increased growth rates and growth yields, both in the wild type and the ΔcyoBACD mutant, suggesting that cytochrome bo3 might be a rate-limiting factor of the respiratory chain.

OxyR acts as a transcriptional repressor of hydrogen peroxide‐inducible antioxidant genes in Corynebacterium glutamicum R

OxyR, a LysR-type transcriptional regulator, has been established as a redox-responsive activator of antioxidant genes in bacteria. This study shows that OxyR acts as a transcriptional repressor of katA, dps, ftn and cydA in Corynebacterium glutamicum R. katA encodes H2O2-detoxifing enzyme catalase, dps and ftn are implicated in iron homeostasis and cydA encodes a subunit of cytochrome bd oxidase. Quantitative RT-PCR analyses revealed that expression of katA and dps, but not of ftn and cydA, was induced by H2O2. Disruption of the oxyR gene encoding OxyR resulted in a marked increase in katA and dps mRNAs to a level higher than that induced by H2O2, and the oxyR-deficient mutant showed a H2O2-resistant phenotype. This is in contrast to the conventional OxyR-dependent regulatory model. ftn and cydA were also upregulated by oxyR disruption but to a smaller extent. Electrophoretic mobility shift assays revealed that the OxyR protein specifically binds to all four upstream regions of the respective genes under reducing conditions. We observed that the oxidized form of OxyR similarly bound to not only the target promoter regions, but also nonspecific DNA fragments. Based on these findings, we propose that the transcriptional repression by OxyR is alleviated under oxidative stress conditions in a titration mechanism due to the decreased specificity of its DNA-binding activity. DNase I footprinting analyses revealed that the OxyR-binding site in the four target promoters is ~ 50 bp in length and has multiple T-N11-A motifs, a feature of LysR-type transcriptional regulators, but no significant overall sequence conservation.

Cytochrome bd oxidase from Escherichia coli displays high catalase activity: An additional defense against oxidative stress

Cytochrome bd oxygen reductase from Escherichia coli has three hemes, b558, b595 and d. We found that the enzyme, as-prepared or in turnover with O2, rapidly decomposes H2O2 with formation of approximately half a mole of O2 per mole of H2O2. Such catalase activity vanishes upon cytochrome bd reduction, does not compete with the oxygen-reductase activity, is insensitive to NO, CO, antimycin-A and N-ethylmaleimide (NEM), but is inhibited by cyanide (Ki ∼2.5μM) and azide. The activity, possibly associated with heme-b595, was also observed in catalase-deficient E. coli cells following cytochrome bd over-expression suggesting a protective role against oxidative stress in vivo.

The Brucella abortus General Stress Response System Regulates Chronic Mammalian Infection and Is Controlled by Phosphorylation and Proteolysis

Virulence of pathogenic bacteria is often determined by their ability to adapt to stress. The Brucella abortus general stress response (GSR) system is required for chronic mammalian infection and is regulated by phosphorylation and proteolysis. The B. abortus GSR signaling pathway has multiple layers of post-translational control and is a determinant of chronic infection. This study provides new, molecular level insight into chronic Brucella infection. Brucella spp. are adept at establishing a chronic infection in mammals. We demonstrate that core components of the α-proteobacterial general stress response (GSR) system, PhyR and σ(E1), are required for Brucella abortus stress survival in vitro and maintenance of chronic murine infection in vivo. ΔphyR and ΔrpoE1 null mutants exhibit decreased survival under acute oxidative and acid stress but are not defective in infection of primary murine macrophages or in initial colonization of BALB/c mouse spleens. However, ΔphyR and ΔrpoE1 mutants are attenuated in spleens beginning 1 month postinfection. Thus, the B. abortus GSR system is dispensable for colonization but is required to maintain chronic infection. A genome-scale analysis of the B. abortus GSR regulon identified stress response genes previously linked to virulence and genes that affect immunomodulatory components of the cell envelope. These data support a model in which the GSR system affects both stress survival and the interface between B. abortus and the host immune system. We further demonstrate that PhyR proteolysis is a unique feature of GSR control in B. abortus. Proteolysis of PhyR provides a mechanism to avoid spurious PhyR protein interactions that inappropriately activate GSR-dependent transcription. We conclude that the B. abortus GSR system regulates acute stress adaptation and long term survival within a mammalian host and that PhyR proteolysis is a novel regulatory feature in B. abortus that ensures proper control of GSR transcription.

Impacts of nitrate and nitrite on physiology of Shewanella oneidensis.

Shewanella oneidensis exhibits a remarkable versatility in anaerobic respiration, which largely relies on its diverse respiratory pathways. Some of these are expressed in response to the existence of their corresponding electron acceptors (EAs) under aerobic conditions. However, little is known about respiration and the impact of non-oxygen EAs on the physiology of the microorganism when oxygen is present. Here we undertook a study to elucidate the basis for nitrate and nitrite inhibition of growth under aerobic conditions. We discovered that nitrate in the form of NaNO3 exerts its inhibitory effects as a precursor to nitrite at low concentrations and as an osmotic-stress provider (Na+) at high concentrations. In contrast, nitrite is extremely toxic, with 25 mM abolishing growth completely. We subsequently found that oxygen represses utilization of all EAs but nitrate. To order to utilize EAs with less positive redox potential, such as nitrite and fumarate, S. oneidensis must enter the stationary phase, when oxygen respiration becomes unfavorable. In addition, we demonstrated that during aerobic respiration the cytochrome bd oxidase confers S. oneidensis resistance to nitrite, which likely functions via nitric oxide (NO).