H(2)O(2) generation in Saccharomyces cerevisiae respiratory pet mutants: effect of cytochrome c

Impaired electron transport chain function has been related to increases in reactive oxygen species (ROS) generation. Here we analyzed different pet mutants of Saccharomyces cerevisiae in order to determine the relative contribution of respiratory chain components in ROS generation and removal. We found that the maintenance of respiration strongly prevented mitochondrial H(2)O(2) release and increased cellular H(2)O(2) removal. Among all respiratory-deficient strains analyzed, cells lacking cytochrome c (cyc3 point mutants) presented the highest level of H(2)O(2) synthesis, indicating that the absence of functional cytochrome c in mitochondria leads to oxidative stress. This finding was supported by the presence of high levels of catalase and peroxidase activity despite the lack of respiration. Furthermore, the addition of exogenous cytochrome c to isolated yeast mitoplasts significantly reduced H(2)O(2) detection in a manner enhanced by cytochrome c reduction and the presence of a functional respiratory chain. Together, our results indicate that the maintenance of electron transport by cytochrome c prevents ROS generation by the respiratory chain.     

Mitochondrial complex I impairment and differential carbon monoxide sensitivity of cytochrome c oxidase in wild type and CMS II mutants of Nicotiana sylvestris

Plant mitochondria unlike their animal counterpart have some unique features with highly branched respiratory chain. The present work was undertaken in order to investigate the effect of loss/dysfunction of plant mitochondrial complex I on the relative flux of electrons through alternative oxidase (AOX) and cytochrome oxidase. Loss of a major subunit of mitochondrial complex I in cytoplasmic male sterile II (CMS II) mutant of Nicotiana sylvestris caused respiratory redox perturbations, as evident from the differential CO sensitivity of cytochrome oxidase. The leaf segments of CMS II mutant when exposed to CO under dark aerobic condition were insensitive to the inhibition of cytochrome oxidase, as against the wild type (WT). The differential CO response of WT and CMS II mutants appeared to be due to differences in the redox state of cytochrome a3 (cyt a3), the terminal electron acceptor during in situ respiration. Cyt a3 appeared to be more in its oxidized form in CMS II and hence unable to form cyt a3-CO complex. Pre-treatment of CMS II leaves with 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation increased the CO response. The slight increase in rotenone-insensitive respiration of CMS II could be attributed partly to enhanced flux of electrons through cytochrome pathway to compensate for the loss of phosphorylation site and partly through AOX, which was induced by nitrate.     

The respiratory chain of Corynebacterium glutamicum

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

Antimycin-insensitive mutants of Candida utilis II. The effects of antimycin on Cytochrome b.

1. Cytochrome b-562 is more reduced in submitochondrial particles of mutant 28 during the aerobic steady-state respiration with succinate than in particles of the wild type. When anaerobiosis is reached, the reduction of cytochrome b is preceded by a rapid reoxidation in the mutnat. A similar reoxidation is observed in the wild type in the present of low concentrations of antimycin. 2. In contrast to the wild type, inhibition of electron transport in the mutant has a much higher antimycin titre than effects on cytochromes b (viz., aerobic steady-state reduction; reduction in the presence of substrate, cyanide and oxygen; the 'red shift' and lowering of E'-o of cytochrome b-562). Moreover, the titration curve of electron transport is hyperbolic whereas the curves for the reduction are sigmoidal. The conclusion is, that in both mutant and wild type, the actions of antimycin on electron transport and cytochromes b are separable. 3. The red shift in the mutant is more extensive than in the wild type. 4. Cytochrome b-558 and cytochrome b-566 (that absorbs in mutant and wild type at 564.5 nm) do not respond simultaneously to addition of antimycin, indicating that they are two separate cytochromes. 5. The difference between the effect of antimycin on electron transport and cytochromes b reduction is also found in intact cells of the mutant. 6. A model is suggested for the wild-type respiratory chain in which (i) the cytochromes b lie, in an uncoupled system, out of the main electron-transfer chain, (ii) antimycin induces a conformation change in QH-2-cytochrome c reductase resulting in effects on cytochrome b and inhibition of electron transport, (iii) a second antimycin-binding site with low affinity to the antibiotic is present, capable of inhibiting electron transport.     

Energy transduction in photosynthetic bacteria. X. Composition and function of the branched oxidase system in wild type and respiration deficient mutants of Rhodopseudomonas capsulata.

The respiratory chain of Rhodopseudomonas capsulata, strain St. Louis and of two respiration deficient mutants (M6 and M7) has been investigated by examining the redox and spectral characteristics of the cytochromes and their response to substrates and to specific respiratory inhibitors. Since the specific lesions of M6 and M7 have been localized on two different branches of the multiple oxidase system of the wild type strain, the capability for aerobic growth of these mutants can be considered as a proof of the physiological significance of both branched systems "in vivo". Using M6 and M7 mutants the response of the branched chain to respiratory inhibitors could be established. Cytochrome oxidase activity, a specific function of an high potential cytochrome b (E'0 = +413 mV) is sensitive to low concentrations of KCN (5-10(-5) M); CO is a specific inhibitor of an alternative oxidase, which is also inhibited by high concentrations of KCN (10(-3) M). Antimycin A inhibits preferentially the branch of the chain affected by low concentrations of cyanide. Redox titrations and spectral data indicate the presence in the membrane of three cytochromes of b type (E'0 = +413, +260, +47 vM) and two cytochromes of c type (E'0 = +342, +94 mV). A clear indication of the involvement in respiration of cytochrome b413, cytochrome c342 and cytochrome b47 has been obtained. Only 50% of the dithionite reducible cytochrome b can be reduced by respiratory substrates also in the presence of high concentrations of KCN or in anaerobiosis. The presence and function of quinones in the respiratory electron transport system has been clearly demonstrated. Quinones, which are reducible by NADH and succinate to about the same extent can be reoxidized through both branches of the respiratory chain, as shown by the response of their redox state to KCN. The possible site of the branching of the electron transport chain has been investigated comparing the per cent level of reduction of quinones and of cytochromes b and c as a function of KCN concentrations in membranes from wild type and M6 mutants cells. The site of the branching has been localized at the level of quinones-cytochrome b47. A tentative scheme of the respiratory chains operating in Rhodopseudomonas capsulata, St. Louis and in the two respiration deficient mutants, M6 and M7 is presented.     

Azorhizobium caulinodans respires with at least four terminal oxidases.

In culture, Azorhizobium caulinodans used at least four terminal oxidases, cytochrome aa3 (cytaa3), cytd, cyto, and a second a-type cytochrome, which together mediated general, respiratory electron (e-) transport to O2. To genetically dissect physiological roles for these various terminal oxidases, corresponding Azorhizobium apocytochrome genes were cloned, and three cytaa3 mutants, a cytd mutant, and a cytaa3, cytd double mutant were constructed by reverse genetics. These cytochrome oxidase mutants were tested for growth, oxidase activities, and N2 fixation properties both in culture and in symbiosis with the host plant Sesbania rostrata. The cytaa3 mutants grew normally, fixed N2 normally, and remained fully able to oxidize general respiratory e- donors (NADH, succinate) which utilize a cytc-dependent oxidase. By difference spectroscopy, a second, a-type cytochrome was detected in the cytaa3 mutants. This alternative a-type cytochrome (Amax = 610 nm) was also present in the wild type but was masked by bona fide cytaa3 (Amax = 605 nm). In late exponential-phase cultures, the cytaa3 mutants induced a new, membrane-bound, CO-binding cytc550, which also might serve as a cytc oxidase (a fifth terminal oxidase). The cloned Azorhizobium cytaa3 genes were strongly expressed during exponential growth but were deactivated prior to onset of stationary phase. Azorhizobium cytd mutants showed 40% lower N2 fixation rates in culture and in planta, but aerobic growth rates were wild type. The cytaa3, cytd double mutant showed 70% lower N2 fixation rates in planta. Pleiotropic cytc mutants were isolated by screening for strains unable to use N,N,N',N'-tetramethyl-p-phenylenediamine as a respiratory e- donor. These mutants synthesized no detectable cytc, excreted coproporphyrin, grew normally in aerobic minimal medium, grew poorly in rich medium, and fixed N2 poorly both in culture and in planta. Therefore, while aerobic growth was sustained by quinol oxidases alone, N2 fixation required cytc oxidase activities. Assuming that the terminal oxidases function as do their homologs in other bacteria, Azorhizobium respiration simultaneously employs both quinol and cytc oxidases. Because Azorhizobium terminal oxidase mutants were able to reformulate their terminal oxidase mix and grow more or less normally in aerobic culture, these terminal oxidases are somewhat degenerate. Its extensive terminal oxidase repertoire might allow Azorhizobium spp. to flourish in wide-ranging O2 environments.     

Localization of cytochrome b6f complexes implies an incomplete respiratory chain in cytoplasmic membranes of the cyanobacterium Synechocystis sp. PCC 6803

The cytochrome b₆f complex is an integral part of the photosynthetic and respiratory electron transfer chain of oxygenic photosynthetic bacteria. The core of this complex is composed of four subunits, cytochrome b, cytochrome f, subunit IV and the Rieske protein (PetC). In this study deletion mutants of all three petC genes of Synechocystis sp. PCC 6803 were constructed to investigate their localization, involvement in electron transfer, respiration and photohydrogen evolution. Immunoblots revealed that PetC1, PetC2, and all other core subunits were exclusively localized in the thylakoids, while the third Rieske protein (PetC3) was the only subunit found in the cytoplasmic membrane. Deletion of petC3 and both of the quinol oxidases failed to elicit a change in respiration rate, when compared to the respective oxidase mutant. This supports a different function of PetC3 other than respiratory electron transfer. We conclude that the cytoplasmic membrane of Synechocystis lacks both a cytochrome c oxidase and the cytochrome b₆f complex and present a model for the major electron transfer pathways in the two membranes of Synechocystis. In this model there is no proton pumping electron transfer complex in the cytoplasmic membrane.Cyclic electron transfer was impaired in all petC1 mutants. Nonetheless, hydrogenase activity and photohydrogen evolution of all mutants were similar to wild type cells. A reduced linear electron transfer and an increased quinol oxidase activity seem to counteract an increased hydrogen evolution in this case. This adds further support to the close interplay between the cytochrome bd oxidase and the bidirectional hydrogenase.     

Antimycin-insensitive mutants of Candida utilis. II. The effects of antimycin on cytochrome b

1. Cytochrome b-562 is more reduced in submitochondrial particles of mutant 28 during the aerobic steady-state respiration with succinate than in particles of the wild type. When anaerobiosis is reached, the reduction of cytochrome b is preceded by a rapid reoxidation in the mutant. A similar reoxidation is observed in the wild type in the presence of low concentrations of antimycin.

2. In contrast to the wild type, inhibition of electron transport in the mutant has a much higher antimycin titre than effects on cytochromes b (viz., aerobic steadystate reduction; reduction in the presence of substrate, cyanide and oxygen; the ‘red shift’ and lowering of E′₀ of cytochrome b-562). Moreover, the titration curve of electron transport is hyperbolic whereas the curves for the reduction are sigmoidal. The conclusion is, that in both mutant and wild type, the actions of antimycin on electron transport and cytochromes b are separable.

3. The red shift in the mutant is more extensive than in the wild type.

4. Cytochrome b-558 and cytochrome b-566 (that absorbs in mutant and wild type at 564.5 nm) do not respond simultaneously to addition of antimycin, indicating that they are two separate cytochromes.

5. The difference between the effect of antimycin on electron transport and cytochromes b reduction is also found in intact cells of the mutant.

6. A model is suggested for the wild-type respiratory chain in which (i) the cytochromes b lie, in an uncoupled system, out of the main electron-transfer chain, (ii) antimycin induces a conformation change in QH₂-cytochrome c reductase resulting in effects on cytochrome b and inhibition of electron transport, (iii) a second antimycinbinding site with low affinity to the antibiotic is present, capable of inhibiting electron transport.     

Identification of subunit g of yeast mitochondrial F1F0-ATP synthase, a protein required for maximal activity of cytochrome c oxidase.

By means of a yeast genome database search, we have identified an open reading frame located on chromosome XVI of Saccharomyces cerevisiae that encodes a protein with 53% amino acid similarity to the 11.3-kDa subunit g of bovine mitochondrial F1F0-ATP synthase. We have designated this ORF ATP20, and its product subunit g. A null mutant strain, constructed by insertion of the HIS3 gene into the coding region of ATP20, retained oxidative phosphorylation function. Assembly of F1F0-ATP synthase in the atp20-null strain was not affected in the absence of subunit g and levels of oligomycin-sensitive ATP hydrolase activity in mitochondria were normal. Immunoprecipitation of F1F0-ATP synthase from mitochondrial lysates prepared from atp20-null cells expressing a variant of subunit g with a hexahistidine motif indicated that this polypeptide was associated with other well-characterized subunits of the yeast complex. Whilst mitochondria isolated from the atp20-null strain had the same oxidative phosphorylation efficiency (ATP : O) as that of the control strain, the atp20-null strain displayed approximately a 30% reduction in both respiratory capacity and ATP synthetic rate. The absence of subunit g also reduced the activity of cytochrome c oxidase, and altered the kinetic control of this complex as demonstrated by experiments titrating ATP synthetic activity with cyanide. These results indicate that subunit g is associated with F1F0-ATP synthase and is required for maximal levels of respiration, ATP synthesis and cytochrome c oxidase activity in yeast.     

Physiological importance of cytochrome c peroxidase in ethanologenic thermotolerant Zymomonas mobilis

Zymomonas mobilis ZmCytC as a peroxidase bearing three heme c-binding motifs was investigated with ΔZmcytC constructed. The mutant exhibited filamentous shapes and reduction in growth under a shaking condition at a high temperature compared to the parental strain and became hypersensitive to exogenous H(2)O(2). Under the same condition, the mutation caused increased expression of genes for three other antioxidant enzymes. Peroxidase activity, which was detected in membrane fractions with ubiquinol-1 as a substrate but not with reduced horse heart cytochrome c, was almost abolished in ΔZmcytC. Peroxidase activity was also detected with NADH as a substrate, which was significantly inhibited by antimycin A. NADH oxidase activity of ΔZmcytC was found to be about 80% of that of the parental strain. The results suggest the involvement of ZmCytC in the aerobic respiratory chain via the cytochrome bc(1) complex in addition to the previously proposed direct interaction with ubiquinol and its contribution to protection against oxidative stress.     

Inhibitor studies of dissimilative Fe(III) reduction by Pseudomonas sp. strain 200 ("Pseudomonas ferrireductans")

Aerobic respiration and dissimilative iron reduction were studied in pure, batch cultures of Pseudomonas sp. strain 200 ("Pseudomonas ferrireductans"). Specific respiratory inhibitors were used to identify elements of electron transport chains involved in the reduction of molecular oxygen and Fe(III). When cells were grown at a high oxygen concentration, dissimilative iron reduction occurred via an abbreviated electron transport chain. The induction of alternative respiratory pathways resulted from growth at low oxygen tension (less than 0.01 atm [1 atm = 101.29 kPa]). Induced cells were capable of O2 utilization at moderately increased rates; dissimilative iron reduction was accelerated by a factor of 6 to 8. In cells grown at low oxygen tension, dissimilative iron reduction appeared to be uncoupled from oxidative phosphorylation. Models of induced and uninduced electron transport chains, including a mathematical treatment of chemical inhibition within the uninduced, aerobic electron transport system, are presented. In uninduced cells respiring anaerobically, electron transport was limited by ferrireductase activity. This limitation may disappear among induced cells.     

Mitochondrial biogenesis during fungal spore germination: respiration and cytochrome c oxidase in Neurospora crassa.

The germination of conidiospores of wild-type Neurospora crassa was found to be dependent upon the function of the cytochrome-mediated electron transport pathway. The cyanide-insensitive alternate oxidase did not contribute significantly to the respiration of these germinating spores. The dormant spores contained all of the cytochrome components and a catalytically active cytochrome c oxidase required for the activity of the standard respiratory pathway, and these preserved components were responsible for the accelerating rates of oxygen uptake which began immediately upon suspension of the spores in an incubation medium. Mitochondria of the dormant spores contained all of the subunit peptides of the functional cytochrome c oxidase; nevertheless, de novo synthesis of these subunits began at low rates in the first stages of germination. Reactivation of the respiratory system of germinating N. crassa spores seems not to be dependent initially upon the function of either the mitochondrial or cytoplasmic protein-synthesizing systems. The respiratory activity of spores of three mutant cytochrome c oxidase-deficient strains of N. crassa also was found to depend upon the function of the cytochrome electron transport pathway; the dormant and germinating spores of these strains contained a catalytically active cytochrome c oxidase. Cytochrome c oxidase may be present in the dormant and germinating spores of these strains as the result of a developmental-phase-specific synthesis of and requirement for the enzyme.     

ATP-regulation of cytochrome oxidase in yeast mitochondria: role of subunit VIa.

The role of the nuclear-encoded subunit VIa in the regulation of cytochrome oxidase by ATP was investigated in isolated yeast mitochondria. As the subunit VIa-null strain possesses a fully active and assembled cytochrome oxidase, multiple ATP-regulating sites were characterized with respect to their location and their kinetic effect: (a) intra-mitochondrial ATP inhibited the complex IV activity of the null strain, whereas the prevailing effect of ATP on the wild-type strain, at low ionic strength, was activation on the cytosolic side of complex IV, mediated by subunit VIa. However, at physiological ionic strength (i.e. approximately 200 mM), activation by ATP was absent but inhibition was not impaired; (b) in ethanol-respiring mitochondria, when the electron flux was modulated using a protonophoric uncoupler, the redox state of aa3 cytochromes varied with respect to activation (wild-type) or inhibition (null-mutant) of the cytochrome oxidase by ATP; (c) consequently, the control coefficient of cytochrome oxidase on respiratory flux, decreased (wild-type) or increased (null-mutant) in the presence of ATP; (d) considering electron transport from cytochrome c to oxygen, the response of cytochrome oxidase to its thermodynamic driving force was increased by ATP for the wild-type but not for the mutant subunit. Taken together, these findings indicate that at physiological concentration, ATP regulates yeast cytochrome oxidase via subunit-mediated interactions on both sides of the inner membrane, thus subtly tuning the thermodynamic and kinetic control of respiration. This study opens up new prospects for understanding the feedback regulation of the respiratory chain by ATP.     

Respiratory chain analysis of Zymomonas mobilis mutants producing high levels of ethanol

We previously isolated respiratory-deficient mutant (RDM) strains of Zymomonas mobilis, which exhibited greater growth and enhanced ethanol production under aerobic conditions. These RDM strains also acquired thermotolerance. Morphologically, the cells of all RDM strains were shorter compared to the wild-type strain. We investigated the respiratory chains of these RDM strains and found that some RDM strains lost NADH dehydrogenase activity, whereas others exhibited reduced cytochrome bd-type ubiquinol oxidase or ubiquinol peroxidase activities. Complementation experiments restored the wild-type phenotype. Some RDM strains seem to have certain mutations other than the corresponding respiratory chain components. RDM strains with deficient NADH dehydrogenase activity displayed the greatest amount of aerobic growth, enhanced ethanol production, and thermotolerance. Nucleotide sequence analysis revealed that all NADH dehydrogenase-deficient strains were mutated within the ndh gene, which includes insertion, deletion, or frameshift. These results suggested that the loss of NADH dehydrogenase activity permits the acquisition of higher aerobic growth, enhanced ethanol production, and thermotolerance in this industrially important strain.     

d-Glucose Transport System of Zymomonas mobilis

The properties of the d-glucose transport system of Zymomonas mobilis were determined by measuring the uptake of nonmetabolizable analogs (2-deoxy-d-glucose and d-xylose) by wild-type cells and the uptake of d-glucose itself by a mutant lacking glucokinase. d-Glucose was transported by a constitutive, stereospecific, carrier-mediated facilitated diffusion system, whereby its intracellular concentration quickly reached a plateau close to but not above the external concentration. d-Xylose was transported by the d-glucose system, as evidenced by inhibition of its uptake by d-glucose. d-Fructose was not an efficient competitive inhibitor of d-glucose uptake, indicating that it has a low affinity for the d-glucose transport system. The apparent K(m) of d-glucose transport was in the range of 5 to 15 mM, with a V(max) of 200 to 300 nmol min mg of protein. The K(m) of Z. mobilis glucokinase (0.25 to 0.4 mM) was 1 order of magnitude lower than the K(m) for d-glucose transport, although the V(max) values for transport and phosphorylation were similar. Thus, glucose transport cannot be expected to be rate limiting at concentrations of extracellular glucose normally used in fermentation processes, which greatly exceed the K(m) for the transport system. The low-affinity, high-velocity, nonconcentrative system for d-glucose transport described here is consistent with the natural occurrence of Z. mobilis in high-sugar environments and with the capacity of Z. mobilis for rapid conversion of glucose to metabolic products with low energetic yield.     

Isolation and characterization of an Escherichia coli mutant lacking the cytochrome o terminal oxidase.

A respiration-deficient mutant of Escherichia coli has been isolated which is unable to grow aerobically on nonfermentable substrates such as succinate and lactate. Spectroscopic and immunological studies showed that this mutant lacks the cytochrome o terminal oxidase of the high aeration branch of the aerobic electron transport chain. This strain carries a mutation in a gene designated cyo which is cotransducible with the acrA locus. Mutations in cyo were obtained by mutagenizing a strain that was cyd and, thus, was lacking the cytochrome d terminal oxidase. Strain RG99, which carries both the cyd- and cyo- alleles, grows normally under anaerobic conditions in the presence of nitrate. Introduction of the cyd+ allele into the strain restores the respiration function of the strain, indicating that the cytochrome o branch of the respiratory chain is dispensable under normal laboratory growth conditions.     

Function of the cytochrome bc1-aa3 branch of the respiratory network in mycobacteria and network adaptation occurring in response to its disruption

The aerobic electron transport chain in Mycobacterium smegmatis can terminate in one of three possible terminal oxidase complexes. The structure and function of the electron transport pathway leading from the menaquinol-menaquinone pool to the cytochrome bc1 complex and terminating in the aa3-type cytochrome c oxidase was characterized. M. smegmatis strains with mutations in the bc1 complex and in subunit II of cyctochome c oxidase were found to be profoundly growth impaired, confirming the importance of this respiratory pathway for mycobacterial growth under aerobic conditions. Disruption of this pathway resulted in an adaptation of the respiratory network that is characterized by a marked up-regulation of cydAB, which encodes the bioenergetically less efficient and microaerobically induced cytochrome bd-type menaquinol oxidase that is required for the growth of M. smegmatis under O2-limiting conditions. Further insights into the adaptation of this organism to rerouting of the electron flux through the branch terminating in the bd-type oxidase were revealed by expression profiling of the bc1-deficient mutant strain using a partial-genome microarray of M. smegmatis that is enriched in essential genes. Although the expression profile was indicative of an increase in the reduced state of the respiratory chain, blockage of the bc1-aa3 pathway did not induce the sentinel genes of M. smegmatis that are induced by oxygen starvation and are regulated by the DosR two-component regulator.