-
[show abstract]
[hide abstract]
ABSTRACT: 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 0.24 μM) 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 requires 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.
Biochimica et Biophysica Acta 04/2013; · 4.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Aims: Carbon monoxide-releasing molecules are being developed with the ultimate goal of safely utilizing the therapeutic potential of CO clinically. One such application is antimicrobial activity; therefore we aimed to characterize and compare the effects of the CO-releasing molecule, CORM-3, and its inactivated counterpart, where all labile CO has been removed, at the transcriptomic and cellular level. Results: We found that both compounds are able to penetrate the cell, but the inactive form is not inhibitory to bacterial growth under conditions where CORM-3 is. Transcriptomic analyses revealed that the bacterial response to iCORM-3 is much lower than to the active compound and that a wide range of processes appear to be affected by CORM-3 and to a lesser extent iCORM-3, including energy metabolism, membrane transport, motility and the metabolism of many sulfur-containing species including cysteine and methionine. Innovation: This work has demonstrated that both CORM-3 and its inactivated counterpart react with cellular functions to yield a complex response at the transcriptomic level. A full understanding of the actions of both compounds is vital in order to understand the toxic effects of CO-releasing molecules. Conclusion: This work has furthered our understanding of how CORM-3 behaves at the cellular level and identifies the responses that occur when the host is exposed to the Ru-compound as well as those that result from the released CO. This is a vital step in laying the groundwork for future development of optimized CO-RMs for eventual use in antimicrobial therapy.
Antioxidants & Redox Signaling 03/2013; · 8.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Aims: Carbon monoxide (CO) delivered to cells and tissues by CO-releasing molecules (CO-RMs) has beneficial and toxic effects not mimicked by CO gas. The metal carbonyl Ru(CO)3Cl(glycinate) (CORM-3) is a novel, potent antimicrobial agent. Here we established its mode of action. Results: CORM-3 inhibits respiration in several bacterial and yeast pathogens. In anoxic Escherichia coli suspensions, CORM-3 first stimulates, then inhibits respiration, but much higher concentrations of CORM-3 than of a classic protonophore are required for stimulation. Proton translocation measurements (H+/O quotients, i.e. H+ extrusion on pulsing anaerobic cells with O2) show that respiratory stimulation cannot be attributed to true 'uncoupling', i.e. dissipation of the protonmotive force, or to direct stimulation of oxidase activity. Our data are consistent with CORM-3 facilitating the electrogenic transmembrane movement of K+ (or Na+) causing a stimulation of respiration and H+ pumping to compensate for the transient drop in membrane potential (ΔΨ). The effects on respiration are not mimicked by CO gas or control Ru compounds that do not release CO. Inhibition of respiration and loss of bacterial viability elicited by CORM-3 are reversible by white light, unambiguously identifying heme-containing oxidase(s) as target(s). Innovation: This is the most complete study to date of the antimicrobial action of a CO-RM. Noteworthy are the demonstration of respiratory stimulation, electrogenic ion transport and photosensitive activity, establishing terminal oxidases and ion transport as primary targets. Conclusion: CORM-3 has multifaceted effects: increased membrane permeability, inhibition of terminal oxidases and perhaps other unidentified mechanisms underlie its effectiveness in tackling microbial pathogenesis.
Antioxidants & Redox Signaling 11/2012; · 8.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Carbon monoxide (CO) is a colourless and odourless gas that has long been considered as a potent respiratory poison. Recent advances have demonstrated its production by haem oxygenases in both mammals and microbes, and it has roles as a gasotransmitter in higher organisms. This review concentrates on the application of CO, via carbon monoxide-releasing molecules (CO-RMs), as an anti-bacterial agent. Currently, the scope of literature on the effects of CO on bacteria is small, and we have included discussions on the production of CO by bacteria via haem oxygenase enzymes, the use of CO as an energy source, and existing knowledge on CO sensors in bacteria. CO is known to target haem proteins and is an effective inhibitor of respiration, even when provided at concentrations much higher than prevailing oxygen. We review here data suggesting that CO-RMs are more effective inhibitors of respiration than is CO gas, perhaps due to the ability of CO-RMs to deliver CO selectively to intracellular targets. We also consider the recently reported transcriptomic consequences of CO-RM treatment of Escherichia coli, revealing a myriad of unexpected targets for CO and potential CO sensors. Finally, we consider the use of CO and CO-RMs as anti-bacterial agents in vivo, and the future prospects for this gaseous molecule.
Current pharmaceutical biotechnology 09/2012; 13(6):760-8. · 3.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Abstract The microaerophilic pathogen Campylobacter jejuni possesses inducible systems for resisting NO. Two globins-Cgb (a single-domain globin) and Ctb (a truncated globin)-are up-regulated in response to NO via the positively acting transcription factor NssR. Our aims were to determine whether these oxygen-binding globins also function in severely oxygen-limited environments, as in the host. At growth-limiting oxygen transfer rates, bacteria were more S-nitrosoglutathione (GSNO) sensitive, irrespective of the presence of Cgb, Ctb, or NssR. Pregrowth of cells with GSNO enhanced GSNO resistance, even in nssR and cgb mutants, but transcriptomic profiling of oxygen-limited, NO-exposed cells failed to reveal the NssR regulon. Nevertheless, globin expression in an Escherichia coli mutant lacking the NO-detoxifying flavohemoglobin Hmp showed that Cgb and Ctb consume NO aerobically or anoxically and offer some protection to respiratory inhibition by NO. The constitutively expressed nitrite reductase NrfA does not provide resistance under oxygen-limited conditions. We, therefore, hypothesize that, although Cgb and NrfA can detoxify NO, even anoxically, they are neither up-regulated nor functional under physiologically relevant oxygen-limited conditions and, second, responses to NO do not stem from trancriptional regulation. Antioxid. Redox Signal. 00, 000-000.
Antioxidants & Redox Signaling 07/2012; · 8.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Abstract Significance: The formation and degradation of S-nitrosothiols (SNOs) are important mechanisms of post-translational protein modification and appear to be ubiquitous in biology. These processes play well-characterized roles in eukaryotic cells, including a variety of pathologies and in relation to chronic conditions. We know little of the roles of these processes in pathogenic and other bacteria. Recent Advances: It is clear, mostly from growth and transcriptional studies, that bacteria sense and respond to exogenous SNOs. These responses are phenotypically and mechanistically distinct from the responses of bacteria to nitric oxide (NO) and NO-releasing agents, as well as peroxynitrite. Small SNOs, such as S-nitrosoglutathione (GSNO), are accumulated by bacteria with the result that intracellular S-nitrosoproteins (the 'S-nitrosoproteome') are detectable. Recently, conditions for endogenous SNO formation in enterobacteria have been described. Critical Issues: The propensity of intracellular proteins to form SNOs is presumably constrained by the same rules of selectivity that have been discovered in eukaryotic systems, but is also influenced by uniquely bacterial NO detoxification systems, exemplified by the flavohemoglobin Hmp in enterobacteria and NO reductase of meningococci. Furthermore, the bacterial expression of such proteins impacts upon the formation of SNOs in mammalian hosts. Future Directions: The impairment during bacterial infections of specific SNO events in the mammalian host is of considerable interest in the context of proteins involved in innate immunity and intracellular signalling. In bacteria, numerous mechanisms of S-nitrosothiol degradation have been reported (e.g., GSNO reductase); others are thought to operate, based on consideration of their mammalian counterparts. The nitrosothiols of bacteria and particularly of pathogens warrant more intensive investigation. Antioxid. Redox Signal. 00, 000-000.
Antioxidants & Redox Signaling 07/2012; · 8.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Understanding gene regulation requires knowledge of changes in transcription factor (TF) activities. Simultaneous direct measurement of numerous TF activities is currently impossible. Nevertheless, statistical approaches to infer TF activities have yielded non-trivial and verifiable predictions for individual TFs. Here, global statistical modelling identifies changes in TF activities from transcript profiles of Escherichia coli growing in stable (fixed oxygen availabilities) and dynamic (changing oxygen availability) environments. A core oxygen-responsive TF network, supplemented by additional TFs acting under specific conditions, was identified. The activities of the cytoplasmic oxygen-responsive TF, FNR, and the membrane-bound terminal oxidases implied that, even on the scale of the bacterial cell, spatial effects significantly influence oxygen-sensing. Several transcripts exhibited asymmetrical patterns of abundance in aerobic to anaerobic and anaerobic to aerobic transitions. One of these transcripts, ndh, encodes a major component of the aerobic respiratory chain and is regulated by oxygen-responsive TFs ArcA and FNR. Kinetic modelling indicated that ArcA and FNR behaviour could not explain the ndh transcript profile, leading to the identification of another TF, PdhR, as the source of the asymmetry. Thus, this approach illustrates how systematic examination of regulatory responses in stable and dynamic environments yields new mechanistic insights into adaptive processes.
Open biology. 07/2012; 2(7):120091.
-
[show abstract]
[hide abstract]
ABSTRACT: Carbon monoxide-releasing molecules (CO-RMs) emulate the beneficial (e.g., anti-inflammatory) effects of CO in biology. CO release from CO-RMs is routinely determined in the presence of reduced deoxy-myoglobin by measuring the formation of carboxy-myoglobin (Mb-CO). Previous studies have highlighted discrepancies between the apparent CO release rates of some CO-RMs established using this assay versus other experimental data where a slower or more complex mechanism of release is suggested. It has been hypothesized that some CO-RMs require a CO acceptor, believed to be reduced myoglobin in Mb-CO assays, in order to facilitate the release of CO. Here, we show, for the first time, that CO is not liberated from the ruthenium (Ru)-based [Ru(CO)(3)Cl(2)](2) (CORM-2) and [Ru(CO)(3)Cl(glycinate)] (CORM-3) at an appreciable rate in the presence of reduced myoglobin alone. Rather, we confirm that it is the reducing agent sodium dithionite that facilitates release of CO from these CO-RMs. Other sulfite compounds, namely sodium sulfite and potassium metabisulfite, also promote the liberation of CO from CORM-3. We describe an alternative oxy-hemoglobin assay that eliminates dithionite and suggest that the efficacy of CO-RMs results from intracellular interactions with anions that facilitate CO delivery to therapeutic targets.
Analytical Biochemistry 05/2012; 427(1):36-40. · 3.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The ethanol-producing bacterium Zymomonas mobilis is of great interest from a bioenergetic perspective because, although it has a very high respiratory capacity, the respiratory system does not appear to be primarily required for energy conservation. To investigate the regulation of respiratory genes and function of electron transport branches in Z. mobilis, several mutants of the common wild-type strain Zm6 (ATCC 29191) were constructed and analyzed. Mutant strains with a chloramphenicol-resistance determinant inserted in the genes encoding the cytochrome b subunit of the bc (1) complex (Zm6-cytB), subunit II of the cytochrome bd terminal oxidase (Zm6-cydB), and in the catalase gene (Zm6-kat) were constructed. The cytB and cydB mutants had low respiration capacity when cultivated anaerobically. Zm6-cydB lacked the cytochrome d absorbance at 630 nm, while Zm6-cytB had very low spectral signals of all cytochromes and low catalase activity. However, under aerobic growth conditions, the respiration capacity of the mutant cells was comparable to that of the parent strain. The catalase mutation did not affect aerobic growth, but rendered cells sensitive to hydrogen peroxide. Cytochrome c peroxidase activity could not be detected. An upregulation of several thiol-dependent oxidative stress-protective systems was observed in an aerobically growing ndh mutant deficient in type II NADH dehydrogenase (Zm6-ndh). It is concluded that the electron transport chain in Z. mobilis contains at least two electron pathways to oxygen and that one of its functions might be to prevent endogenous oxidative stress.
Archives of Microbiology 01/2012; 194(6):461-71. · 1.43 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We describe a hybrid transcriptomic and modelling analysis of the dynamics of a bacterial response to stress, namely the addition of 200 µM Zn to Escherichia coli growing in severely Zn-depleted medium and of cells growing at different Zn concentrations at steady state. Genes that changed significantly in response to the transition were those reported previously to be associated with zinc deficiency (zinT, znuA, ykgM) or excess (basR, cpxP, cusF). Cellular Zn levels were confirmed by ICP-AES to be 14- to 28-fold greater after Zn addition but there was also 6- to 8-fold more cellular Fe 30 min after Zn addition. Statistical modelling of the transcriptomic data generated from the Zn shift focused on the role of ten key regulators; ArsR, BaeR, CpxR, CusR, Fur, OxyR, SoxS, ZntR, ZraR and Zur. The data and modelling reveal a transient change in the activity of the iron regulator Fur and of the oxidative stress regulator SoxS, neither of which is evident from the steady-state transcriptomic analyses. We hypothesize a competitive binding mechanism that combines these observations and existing data on the physiology of Zn and Fe uptake. Formalizing the mechanism in a differential equation model shows that it can reproduce qualitatively the behaviour seen in the data. This gives new insights into the interplay of these two fundamental metal ions in gene regulation and bacterial physiology, as well as highlighting the importance of dynamic studies to reverse-engineer systems behaviour.
Microbiology 01/2012; 158(Pt 1):284-92. · 3.06 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Oxygen affinity in heme-containing proteins is determined by a number of factors, such as the nature and conformation of the distal residues that stabilize the heme bound-oxygen via hydrogen-bonding interactions. The truncated hemoglobin III from Campylobacter jejuni (Ctb) contains three potential hydrogen-bond donors in the distal site: TyrB10, TrpG8, and HisE7. Previous studies suggested that Ctb exhibits an extremely slow oxygen dissociation rate due to an interlaced hydrogen-bonding network involving the three distal residues. Here we have studied the structural and kinetic properties of the G8(WF) mutant of Ctb and employed state-of-the-art computer simulation methods to investigate the properties of the O(2) adduct of the G8(WF) mutant, with respect to those of the wild-type protein and the previously studied E7(HL) and/or B10(YF) mutants. Our data indicate that the unique oxygen binding properties of Ctb are determined by the interplay of hydrogen-bonding interactions between the heme-bound ligand and the surrounding TyrB10, TrpG8, and HisE7 residues.
Biochemistry 05/2011; 50(19):3946-56. · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Carbon monoxide (CO) is a classical respiratory inhibitor, but CO-releasing molecules (CO-RMs) have therapeutic value, increasing phagocytosis, and reducing sepsis-induced lethality. CORM-3, Ru(CO)(3) Cl(glycinate), a ruthenium-based carbonyl that liberates CO under physiological conditions, has previously been shown to inhibit bacterial growth and respiration, even at high concentrations of oxygen. Here, we report the effects of CORM-3 on the microaerophilic foodborne pathogen Campylobacter jejuni. Even at CO-RM (i.e., CO) concentrations that exceed dissolved oxygen levels, CORM-3 does not inhibit microaerobic growth. This insensitivity is not due to failure of CORM-3 to penetrate cells, as revealed by assay with extracellular myoglobin and by the ability of CO from externally added CORM-3 to bind intracellular membrane-associated respiratory oxidases. Even at almost 200 μ M oxygen, CORM-3 inhibits formate-dependent respiration and leads to generation of hydrogen peroxide. This work shows that CO-RMs have valuable properties as antimicrobial agents; however, growth inhibition does not always accompany inhibition of respiration, even when ambient oxygen concentrations are low.
International Union of Biochemistry and Molecular Biology Life 05/2011; 63(5):363-71. · 3.51 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Oxygen availability is the major determinant of the metabolic modes adopted by Escherichia coli. Although much is known about E. coli gene expression and metabolism under fully aerobic and anaerobic conditions, the intermediate oxygen tensions that are encountered
in natural niches are understudied. Here, for the first time, the transcript profiles of E. coli K-12 across the physiologically significant range of oxygen availabilities are described. These suggested a progressive switch
to aerobic respiratory metabolism and a remodeling of the cell envelope as oxygen availability increased. The transcriptional
responses were consistent with changes in the abundance of cytochrome bd and bo′ and the outer membrane protein OmpW. The observed transcript and protein profiles result from changes in the activities of
regulators that respond to oxygen itself or to metabolic and environmental signals that are sensitive to oxygen availability
(aerobiosis). A probabilistic model (TFInfer) was used to predict the activity of the indirect oxygen-sensing two-component
system ArcBA across the aerobiosis range. The model implied that the activity of the regulator ArcA correlated with aerobiosis
but not with the redox state of the ubiquinone pool, challenging the idea that ArcA activity is inhibited by oxidized ubiquinone.
The amount of phosphorylated ArcA correlated with the predicted ArcA activities and with aerobiosis, suggesting that fermentation
product-mediated inhibition of ArcB phosphatase activity is the dominant mechanism for regulating ArcA activity under the
conditions used here.
Journal of Biological Chemistry 03/2011; 286(12):10147-10154. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Campylobacter jejuni, a major food-borne intestinal pathogen, preferentially utilizes a few specific amino acids and some organic acids such as pyruvate and L- and D-lactate as carbon sources, which may be important for growth in the avian and mammalian gut. Here, we identify the enzymatic basis for C. jejuni growth on L-lactate. Despite the presence of an annotated gene for a fermentative lactate dehydrogenase (cj1167), no evidence for lactate excretion could be obtained in C. jejuni NCTC 11168, and inactivation of the cj1167 gene did not affect growth on lactate as carbon source. Instead, L-lactate utilization in C. jejuni NCTC 11168 was found to proceed via two novel NAD-independent L-LDHs; a non-flavin iron-sulfur containing three subunit membrane-associated enzyme (Cj0075c-73c), and a flavin and iron-sulfur containing membrane-associated oxidoreductase (Cj1585c). Both enzymes contribute to growth on L-lactate, as single mutants in each system grew as well as wild-type on this substrate, while a cj0075c cj1585c double mutant showed no L-lactate oxidase activity and did not utilize or grow on L-lactate; D-lactate-dependent growth was unaffected. Orthologues of Cj0075c-73c (LldEFG/LutABC) and Cj1585c (Dld-II) were recently shown to represent two novel families of L- and D-lactate oxidases; this is the first report of a bacterium where both enzymes are involved in L-lactate utilization only. The cj0075c-73c genes are located directly downstream of a putative lactate transporter gene (cj0076c, lctP), which was also shown to be specific for L-lactate. The avian and mammalian gut environment contains dense populations of obligate anaerobes that excrete lactate; our data indicate that C. jejuni is well equipped to use L- and D-lactate as both electron-donor and carbon source.
Environmental Microbiology 01/2011; 13(1):48-61. · 5.84 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Campylobacter jejuni possesses NO-responsive and -detoxifying mechanisms to survive NO during transmission and pathogenesis. C. jejuni possesses two hemoglobins. The first (Cgb) is a single-domain (non-flavo)hemoglobin encoded by gene Cj1586 (cgb), mutation of which leads to hypersensitivity to S-nitrosoglutathione and NO. Transcription of cgb is induced by nitrosative stress and confers resistance to NO, presumably via a Cgb-catalyzed dioxygenase or denitrosylase reaction that converts NO and oxygen to nitrate. Expression of Cgb in response to NO is mediated via the positively-acting transcription factor NssR, which regulates expression of a small regulon that includes cgb and ctb (Cj0465c), the latter encoding the truncated hemoglobin, Ctb. The role of Ctb is unclear: it is not directly involved in NO detoxification but is implicated in oxygen delivery or metabolism. Here, we describe attempts to define a function for Ctb by examining the effects of a ctb mutation on the NO transcriptome and cgb gene expression during normoxia and hypoxia. Mutation of ctb does not elicit major compensatory transcriptomic changes but relatively minor changes in genes involved in intermediary metabolism, solute transport and signal transduction. We present and test the hypothesis that, by binding NO or O(2), Ctb dampens the response to NO under hypoxic conditions and limits cgb expression, perhaps because Cgb function (i.e. NO detoxification) requires O(2)-dependent chemistry. We report the purification of NssR and specific binding to the ctb promoter. GSNO does not affect the high affinity of NssR for the ctb promoter.
Nitric Oxide 01/2011; 25(2):234-41. · 3.55 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Nitric oxide (NO(·)) is a toxin, but bacteria have evolved various strategies to detoxify this harmful radical to nitrate, the best known mechanism being the dioxygenase reaction of bacterial flavohaemoglobins. In addition, globins can form oxoferryl (Fe(IV)=O) species through the reaction of the ferric haem with hydrogen peroxide: these species can also detoxify NO(·) to nitrite and nitrate. During infection, Campylobacter is exposed to both NO(·) and hydrogen peroxide. A question therefore arises: does Campylobacter jejuni utilize its single domain globin (Cgb) to detoxify NO(·) via the oxoferryl route, or via the more conventional dioxygenase or denitroxylase routes? The data herein demonstrate that the reaction between Cgb and hydrogen peroxide is much slower than for other globins, and subsequent reaction between the oxoferryl species and NO(·) is unfavourable. Furthermore, NO(·) may bind to Cgb in the oxyferrous, ferrous and ferric states. The ample opportunity for NO(·) to interact with ferrous and ferric Cgb, and the unfavourable reaction of ferric Cgb with hydrogen peroxide, suggests that NO(·) detoxification in C. jejuni proceeds via a dioxygenase or denitroxylase route requiring the haem iron to exist only in the Fe(II) or Fe(III) redox states.
Nitric Oxide 01/2011; 25(2):229-33. · 3.55 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Oxygen availability is the major determinant of the metabolic modes adopted by Escherichia coli. Although much is known about E. coli gene expression and metabolism under fully aerobic and anaerobic conditions, the intermediate oxygen tensions that are encountered in natural niches are understudied. Here, for the first time, the transcript profiles of E. coli K-12 across the physiologically significant range of oxygen availabilities are described. These suggested a progressive switch to aerobic respiratory metabolism and a remodeling of the cell envelope as oxygen availability increased. The transcriptional responses were consistent with changes in the abundance of cytochrome bd and bo' and the outer membrane protein OmpW. The observed transcript and protein profiles result from changes in the activities of regulators that respond to oxygen itself or to metabolic and environmental signals that are sensitive to oxygen availability (aerobiosis). A probabilistic model (TFInfer) was used to predict the activity of the indirect oxygen-sensing two-component system ArcBA across the aerobiosis range. The model implied that the activity of the regulator ArcA correlated with aerobiosis but not with the redox state of the ubiquinone pool, challenging the idea that ArcA activity is inhibited by oxidized ubiquinone. The amount of phosphorylated ArcA correlated with the predicted ArcA activities and with aerobiosis, suggesting that fermentation product-mediated inhibition of ArcB phosphatase activity is the dominant mechanism for regulating ArcA activity under the conditions used here.
Journal of Biological Chemistry 01/2011; 286(12):10147-54. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Many bacteria undergo transitions between environments with differing O₂ availabilities as part of their natural lifestyles and during biotechnological processes. However, the dynamics of adaptation when bacteria experience changes in O₂ availability are understudied. The model bacterium and facultative anaerobe Escherichia coli K-12 provides an ideal system for exploring this process.
Time-resolved transcript profiles of E. coli K-12 during the initial phase of transition from anaerobic to micro-aerobic conditions revealed a reprogramming of gene expression consistent with a switch from fermentative to respiratory metabolism. The changes in transcript abundance were matched by changes in the abundances of selected central metabolic proteins. A probabilistic state space model was used to infer the activities of two key regulators, FNR (O₂ sensing) and PdhR (pyruvate sensing). The model implied that both regulators were rapidly inactivated during the transition from an anaerobic to a micro-aerobic environment. Analysis of the external metabolome and protein levels suggested that the cultures transit through different physiological states during the process of adaptation, characterized by the rapid inactivation of pyruvate formate-lyase (PFL), a slower induction of pyruvate dehydrogenase complex (PDHC) activity and transient excretion of pyruvate, consistent with the predicted inactivation of PdhR and FNR.
Perturbation of anaerobic steady-state cultures by introduction of a limited supply of O₂ combined with time-resolved transcript, protein and metabolite profiling, and probabilistic modeling has revealed that pyruvate (sensed by PdhR) is a key metabolic signal in coordinating the reprogramming of E. coli K-12 gene expression by working alongside the O₂ sensor FNR during transition from anaerobic to micro-aerobic conditions.
PLoS ONE 01/2011; 6(9):e25501. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Nitric oxide and related nitrogen species (reactive nitrogen species) now occupy a central position in contemporary medicine, physiology, biochemistry, and microbiology. In particular, NO plays important antimicrobial defenses in innate immunity but microbes have evolved intricate NO-sensing and defense mechanisms that are the subjects of a vast literature. Unfortunately, the burgeoning NO literature has not always been accompanied by an understanding of the intricacies and complexities of this radical and other reactive nitrogen species so that there exists confusion and vagueness about which one or more species exert the reported biological effects. The biological chemistry of NO and derived/related molecules is complex, due to multiple species that can be generated from NO in biological milieu and numerous possible reaction targets. Moreover, the fate and disposition of NO is always a function of its biological environment, which can vary significantly even within a single cell. In this review, we consider newer aspects of the literature but, most importantly, consider the underlying chemistry and draw attention to the distinctiveness of NO and its chemical cousins, nitrosonium (NO(+)), nitroxyl (NO(-), HNO), peroxynitrite (ONOO(-)), nitrite (NO(2)(-)), and nitrogen dioxide (NO(2)). All these species are reported to be generated in biological systems from initial formation of NO (from nitrite, NO synthases, or other sources) or its provision in biological experiments (typically from NO gas, S-nitrosothiols, or NO donor compounds). The major targets of NO and nitrosative damage (metal centers, thiols, and others) are reviewed and emphasis is given to newer "-omic" methods of unraveling the complex repercussions of NO and nitrogen oxide assaults. Microbial defense mechanisms, many of which are critical for pathogenicity, include the activities of hemoglobins that enzymically detoxify NO (to nitrate) and NO reductases and repair mechanisms (e.g., those that reverse S-nitrosothiol formation). Microbial resistance to these stresses is generally inducible and many diverse transcriptional regulators are involved-some that are secondary sensors (such as Fnr) and those that are "dedicated" (such as NorR, NsrR, NssR) in that their physiological function appears to be detecting primarily NO and then regulating expression of genes that encode enzymes with NO as a substrate. Although generally harmful, evidence is accumulating that NO may have beneficial effects, as in the case of the squid-Vibrio light-organ symbiosis, where NO serves as a signal, antioxidant, and specificity determinant. Progress in this area will require a thorough understanding not only of the biology but also of the underlying chemical principles.
Advances in Microbial Physiology 01/2011; 59:135-219. · 9.88 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Oxidative and nitrosative stresses including nitric oxide (NO), superoxide (O2⁻) and peroxynitrite play key roles in determining the outcome of bacterial infections. In order to survive within the host and allow proliferation within immune cells such as macrophages, Salmonella isolates have a number of inducible proteins that are able to detoxify these highly reactive species, notably the anoxically functioning NO reductase NorVW, and the aerobically functioning flavohaemoglobin, Hmp, which catalyses the reaction between oxygen and NO to produce relatively inert nitrate. However, in the absence of NO but in the presence of reducing substrates and oxygen, O2⁻ is generated from Hmp-mediated electron transfer to bound oxygen and may form a variety of further oxidative species. Hence, Hmp expression is under tight negative regulation by the transcription factor NsrR, abolition of which causes an increase in the production of Hmp. In a previous study, this increase in Hmp levels conferred resistance to the nitrosating agent S-nitrosoglutathione but, perhaps surprisingly, the organism became more sensitive to killing by macrophages. Here, we report that an nsrR mutant that constitutively overexpresses Hmp is also hypersensitive to peroxynitrite in vitro. This sensitivity is alleviated by deletion of the hmp gene or pre-incubation of growing bacteria with NO-releasing agents. We hypothesize that Hmp-expressing cells, in the absence of NO, generate reactive oxygen species, the toxicity of which is exacerbated by peroxynitrite in vitro and in macrophages. RT-PCR confirmed that peroxynitrite causes oxidative stress and upregulation of katG and ahpC, whilst hmp and norV expression are affected very little. The katG gene upregulated by peroxynitrite encodes a catalase peroxidase enzyme with well-established roles in detoxifying peroxides. Here, we report that KatG is also able to enhance the breakdown of peroxynitrite, suggesting that the protective role of this enzyme may be wider than previously thought. These data suggest that spatial and temporal fluctuations in the levels of NO and reactive oxygen species will have important consequences for bacterial survival in the macrophage.
Microbiology 12/2010; 156(Pt 12):3556-65. · 3.06 Impact Factor
