Mutations in the Escherichia coli fnr and tgt genes: control of molybdate reductase activity and the cytochrome d complex by fnr.

In eubacteria, the tRNA transglycosylase (Tgt) in specific tRNAs exchanges a guanine in the anticodon for 7-aminomethyl-7-deazaguanine, which is finally converted to queuosine. The tgt gene of Escherichia coli has been mapped at 9 min on the genome, and mutant pairs containing an intact or mutated tgt allele were obtained after transduction of the tgt locus by P1 bacteriophages into a genetically defined E. coli strain (S. Noguchi, Y. Nishimura, Y. Hirota, and S. Nishimura, J. Biol. Chem. 257:6544-6550, 1982). These tgt mutants grew anerobically with fumarate as an electron acceptor, while nitrate or trimethylamine N-oxide could not be reduced. Furthermore, molybdate reductase activity was almost lacking and the characteristic absorption maxima, corresponding to cytochrome a1 and the cytochrome d complex, were not detectable in low-temperature reduced-minus-oxidized difference spectra in anaerobically grown cells. Transduction of the mutated tgt locus into another E. coli recipient resulted in tgt mutants without anaerobic defects. Transformation of the original tgt mutants with an fnr gene-containing plasmid reversed the anaerobic defects. Clearly, the original tgt mutants harbor a second mutation, affecting the anaerobic regulator protein Fnr. The results suggest that fnr is involved in anaerobic control of components of the cytochrome d complex and of the redox system that transfers electrons to molybdate. F' plasmids containing a fused lacI-lacZ gene with the nonsense codon UAG at different positions in the lacI part were transferred to E. coli strains with a mutated or nonmutated tgt locus but intact in fnr. A twofold increase in the frequency of incorrect readthrough of the UAG codon, dependent on the codon context, was observed in the tgt mutant and is suggested to be caused by a tRNA(Tyr) with G in place of queuosine.     

New MADS box domains in Asparagus officinalis L.

New MADS box domains have been cloned from Asparagus officinalis L. using PCR technology. Several clones share high homology with the Arabidopsis agamous gene while other clones appear to represent novel MADS box domains. These results show that extended PCR primers are useful for selectively amplifying conserved DNA binding domains across widely divergent plant taxa.Asparagus MADS box sequences are listed in GenBank with the following accession numbers: UO7330 (Asp 13), UO7331 (Asp 16), UO7332 (Asp 22), UO7333 (Asp 23), UO7334 (Asp 3), UO7335 (Asp 39), UO7336 (Asp 8), UO7337 (Asp 4)     

Characterization and phenotypic control of the cytochrome content of Escherichia coli

1. Electron-transport particles derived from Escherichia coli grown aerobically contain three b-type cytochromes with mid-point oxidation-reduction potentials at pH7 of +260mV, +80mV and -50mV, with n=1 for each. The variation of these values with pH was determined. 2. E. coli develops a different set of b-type cytochromes when grown anaerobically on glycerol with fumarate or nitrate as terminal electron acceptor. Electron-transport particles of fumarate-grown cells contain b-type cytochromes with mid-point potentials at pH7 of +140mV and +250mV (n=1). These two cytochromes are also present in cells grown with nitrate as terminal acceptor, where an additional cytochrome b with a mid-point potential of +10mV (n=1) is developed. 3. The wavelengths of the alpha-absorption-band maxima of the b-type cytochromes at 77K were: (a) for aerobically grown cells, cytochrome b (E(m7) +260mV), 556nm and 563nm, cytochrome b (E(m7) +80mV), 556nm and cytochrome b (E(m7)-50mV), 558nm; (b) for anaerobically grown cells, cytochrome b (E(m7) +250mV), 558nm, cytochrome b (E(m7) +40mV), 555nm and cytochrome b (E(m7) +10mV), 556nm. 4. Cytochrome d was found to have a mid-point potential at pH7 of +280mV (n=1). 5. Cytochrome a(1) was resolved as two components of equal magnitude with mid-point potentials of +260mV and +160mV (n=1). 6. Redox titrations performed in the presence of CO showed that one of the b-type cytochromes in the aerobically grown cultures was reduced, even at the upper limits of our range of electrode potentials (above +400mV). Cytochrome d was also not oxidizable in the presence of CO. Neither of the cytochromes a(1) was affected by the presence of CO.     

Regulation of reductase formation in Proteus mirabilis

Summary The levels of several redox enzymes in a chlorate-resistant mutant of Proteus mirabilis, which is partially affected in the formation of formate hydrogenlyase, thiosulfate reductase and tetrathionate reductase, were compared with those of the wild type. The composition of the electron transport system of both strains was almost the same in cells grown aerobically, but very different in cells grown anaerobically. In the mutant, the cytochrome content increased twofold, whereas the level of the anaerobic enzymes is strongly diminished. The anaerobic formation of electron transport components in the mutant was, in contrast to that of the wild type, not influenced significantly by azide. During anaerobic growth with nitrate low levels of a functional nitrate reductase system were formed in the mutant. Under these conditions the formation of formate dehydrogenase, formate hydrogenlyase, formate oxidase, thiosulfate reductase, tetrathionate reductase, cytochrome b_563,5 and partly that of cytochrome a₂, was repressed. The repressive effect of nitrate, however, was completely abolished by azide. Therefore, it seems likely that a functional nitrate reductase system, rather than nitrate, controls the formation of the enzymes repressible by nitrate.     

Nitrate Reductase and Soluble Cytochrome c in Spirillum itersonii

The addition of nitrate to cultures of Spirillum itersonii incubated under low aeration produced a diauxic growth pattern in which the second exponential phase was preceded by the appearance of nitrite in the medium. The organism also grew anaerobically in the presence of nitrate. Nitrate reductase activity could be demonstrated in cell-free extracts by use of reduced methyl viologen as the electron donor. The enzyme was located in the supernatant fraction after centrifugation of extracts for 2 hr at 40,000 x g, and it sedimented as a single peak when centrifuged in a sucrose gradient. Nitrate reductase activity was found in cells grown with low aeration without nitrate, but was increased about twofold by addition of nitrate. Enzyme activity was negligible in cells grown with high aeration. The proportion of soluble cytochrome c was increased two- to threefold in cells grown with nitrate. The specific activities of nitrate reductase and soluble cytochrome c rose when nitrate or nitrite was added to cell suspensions incubated with low aeration; nitrite was more effective than nitrate during the early stages of incubation. A nitrate reductase-negative mutant synthesized increased amounts of soluble cytochrome c in response to nitrate or to nitrite in the cell suspension system. It is concluded that enhanced synthesis of soluble cytochrome c does not require the presence of a functional nitrate reductase.     

Influence of nitrate on fermentation pattern, molar growth yields and synthesis of cytochrome b in Propionibacterium pentosaceum.

Under anaerobic conditions, Propionibacterium pentosaceum reduces nitrate to nitrite until nitrate is exhausted from the medium when nitrite is converted into N2 or N2O. In the presence of nitrate, fermentation patterns for lactate, glycerol and pyruvate were different from those obtained during anaerobic growth without an inorganic electron acceptor. In the presence of these substrates, a drastic decrease in propionate formation was observed, some pyruvate accumulated during growth with lactate, and acetate was produced from glycerol. Acetate production from lactate and pyruvate was not influenced by the presence of nitrate. Furthermore, CO2 was produced by citric acid cycle activity. The fermentation pattern during nitrite reduction resembled that of P. pentosaceum grown anaerobically without an inorganic electron acceptor. Nitrits has a toxic effect, since bacteria inoculated into a medium with 9 mM-nitrite failed to grow. The cytochrome spectrum of anaerobically grown P. pentosaceum was similar with and without nitrate. In membrane fractions of bacteria grown anaerobically with nitrate, cytochrome b functioned in the transfer of electrons from lactate, glycerol I-phosphate and NADH to nitrate. Molar growth yeilds were increased in the presence of nitrate, indicating an increased production of ATP. This could be explained by citric acid cycle activity, and by ocidative phosphorylation coupled to nitrate reduction. Assuming that I mol ATP is formed in the electron transfer from lactate or glycerol I-phosphate to nitrate, and that 2 mol ATP are formed in the electron transfer from NADH to nitrate, YATP values (g dry wt bacteria/mol ATP) were obtained of between 5-0 and 12-6. The higher YATP values were similar to those obtained during anaerobic growth without an inorganic electron acceptor. This supports the assumptions about the efficiency of oxidative phosphorylation for electron transport to nitrate. Low YAPT values were found when high concentrations of nitrite (15 to 50 mM) accumulated, and were probably due to the toxic effect of nitrite.     

Nitric oxide formation by Escherichia coli. Dependence on nitrite reductase,the NO-sensing regulator Fnr,and flavohemoglobin Hmp

Nitric oxide (NO) is a key signaling and defense molecule in biological systems. The bactericidal effects of NO produced, for example, by macrophages are resisted by various bacterial NO-detoxifying enzymes, the best understood being the flavohemoglobins exemplified by Escherichia coli Hmp. However, many bacteria, including E. coli, are reported to produce NO by processes that are independent of denitrification in which NO is an obligatory intermediate. We demonstrate using an NO-specific electrode that E. coli cells, grown anaerobically with nitrate as terminal electron acceptor, generate significant NO on adding nitrite. The periplasmic cytochrome c nitrite reductase (Nrf) is shown, by comparing Nrf+ and Nrf- mutants, to be largely responsible for NO generation. Surprisingly, an hmp mutant did not accumulate more NO but, rather, failed to produce detectable NO. Anaerobic growth of the hmp mutant was not stimulated by nitrate, and the mutant failed to produce periplasmic cytochrome(s) c, leading to the hypothesis that accumulating NO in the absence of Hmp inactivates the global anaerobic regulator Fnr by reaction with the [4Fe-4S]2+ cluster (Cruz-Ramos, H., Crack, J., Wu, G., Hughes, M. N., Scott, C., Thomson, A. J., Green, J., and Poole, R. K. (2002) EMBO J. 21, 3235-3244). Fnr thus failed to up-regulate nitrite reductase. The model is supported by the inability of an fnr mutant to generate NO and by the restoration of NO accumulation to hmp mutants upon introducing a plasmid encoding Fnr* (D154A) known to confer activity in the presence of oxygen. A cytochrome bd-deficient mutant retained NO-generating activity. The present study reveals a critical balance between NO-generating and -detoxifying activities during anaerobic growth.     

Proton translocation coupled to nitrite reduction in anaerobically grown Escherichia coli

Proton translocation coupled to the reduction of nitrite was studied in anaerobically grown Escherichia coli. Extrusion of protons occurred by adding nitrite to an anaerobic suspension of wild-type cells. This extrusion was sensitive to a proton conductor, 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847) or carbonylcyanide-p-trifluoromethoxyphenylhydrazone. Dicyclohexylcarbodiimide, an inhibitor of H+-ATPase, prevented the proton extrusion linked to nitrite reduction, whereas this reagent had no effect on respiratory nitrate reduction to nitrite. Proton extrusion was undetectable when nitrite was added to a suspension of mutant cells defective in H+-ATPase. These results indicate that the proton extrusion associated with nitrite reduction to ammonia is not by redox pumps but by H+-ATPase. From the results obtained by the measurement of proton extrusion in nitrite reductase-deficient mutants, NADH-nitrite reductase system is suggested to involve the proton extrusion in whole cells of E. coli.     

Evidence for a Second Nitrate Reductase Activity That Is Distinct from the Respiratory Enzyme in Salmonella typhimurium

Significant nitrate reductase activity was detected in mutants of Salmonella typhimurium which mapped at or near chlC and which were incapable of growth with nitrate as electron acceptor. The same mutants were sensitive to chlorate and performed sufficient nitrate reduction to permit anaerobic growth with nitrate as the sole nitrogen source in media containing glucose. The mutant nitrate-reducing protein did not migrate with the wild-type nitrate reductase in polyacrylamide electrophoretic gels. Studies of the electrophoretic mobility in gels of different polyacrylamide concentration revealed that the wild-type and mutant nitrate reductases differed significantly in both size and charge. The second enzyme also differed from the wild-type major enzyme in its response to repression by low pH and its lack of response to repression by glucose. The same mutants were found to be derepressed for nitrite reductase and for a cytochrome with a maximal reduced absorbance at 555 nm at 25°C. This cytochrome was not detected in preparations of the wild type grown under the same conditions. Extracts of these mutants contained normal amounts of the b-type cytochromes which, in the wild type, were associated with nitrate reductase and formate dehydrogenase, respectively, although they could not mediate the oxidation of these cytochromes with nitrate. They were capable of oxidizing the derepressed 555-nm peak cytochrome with nitrate. It is suggested that these mutants synthesize a nitrate-reducing enzyme which is distinct from the chlC gene product and which is repressed in the wild type during anaerobic growth with nitrate.     

The periplasmic nitrite reductase of Thauera selenatis may catalyze the reduction of selenite to elemental selenium

Thauera selenatis grows anaerobically with selenate, nitrate or nitrite as the terminal electron acceptor; use of selenite as an electron acceptor does not support growth. When grown with selenate, the product was selenite; very little of the selenite was further reduced to elemental selenium. When grown in the presence of both selenate and nitrate both electron acceptors were reduced concomitantly; selenite formed during selenate respiration was further reduced to elemental selenium. Mutants lacking the periplasmic nitrite reductase activity were unable to reduce either nitrite or selenite. Mutants possessing higher activity of nitrite reductase than the wild-type, reduced nitrite and selenite more rapidly than the wild-type. Apparently, the nitrite reductase (or a component of the nitrite respiratory system) is involved in catalyzing the reduction of selenite to elemental selenium while also reducing nitrite. While periplasmic cytochrome C ₅₅₁ may be a component of the nitrite respiratory system, the level of this cytochrome was essentially the same in mutant and wild-type cells grown under two different growth conditions (i.e. with either selenate or selenate plus nitrate as the terminal electron acceptors). The ability of certain other denitrifying and nitrate respiring bacteria to reduce selenite will also be described.     

Nitrate,fumarate, and oxygen as electron acceptors for a late step in microbial heme synthesis

Nitrate can serve as anaerobic electron acceptor for the oxidation of protoporphyrinogen to protoporphyrin in cell-free extracts of Escherichia coli grown anaerobically in the presence of nitrate. Two kinds of experiments indicated this: anaerobic protoporphyrin formation from protoporphyrinogen, followed spectrophotometrically, was markedly stimulated by addition of nitrate; and anaerobic protoheme formation from protoporphyrinogen, determined by extraction procedures, was markedly stimulated by addition of nitrate. In contrast, anaerobic protoheme formation from protoporphyrin was not dependent upon addition of nitrate. This was the first demonstration of the ability of nitrate to serve as electron acceptor for this late step of heme synthesis. Previous studies with mammalian and yeast mitochondria had indicated an obligatory requirement for molecular oxygen at this step.In confirmation of our previous preliminary report, fumarate was also shown to be an electron acceptor for anaerobic protoporphyrinogen oxidation in extracts of E. coli grown anaerobically on fumarate. For the first time, anaerobic protoheme formation from protoporphyrinogen, but not from protoporphyrin, was shown to be dependent upon the addition of fumarate.The importance of these findings is 2-fold. First, they establish that enzymatic protoporphyrinogen oxidation can occur in the absence of molecular oxygen, in contrast to previous observations using mammalian and yeast mitochondria. Secondly, these findings help explain the ability of some facultative and anaerobic bacteria to form very large amounts of heme compounds, such as cytochrome pigments, when grown anaerobically in the presence of nitrate or fumarate. In fact, denitrifying bacteria are known to form more cytochromes when grown anaerobically than during aerobic growth.An unexpected finding was that extracts of another bacterium, Staphylococcus epidermidis, exhibited very little ability to oxidize protoporphyrinogen to protoporphyrin as compared to E. coli extracts. This finding suggests some fundamental differences in these two organisms in this key step in heme synthesis. It is known that these two facultative organisms also differ in that E. coli synthesizes cytochrome during both aerobic and anaerobic growth, while Staphylococcus only synthesizes cytochromes when grown aerobically.     

Anaerobic induction of trimethylamine N-oxide reductase and cytochromes by dimethyl sulfoxide inEscherichia coli

Reduction of trimethylamine N-oxide is catalyzed by at least two enzymes inEscherichia coli: trimethylamine N-oxide reductase, which is anaerobically induced by trimethylamine N-oxide, and the constitutive enzyme dimethyl sulfoxide reductase. In this study, an increase in the specific activity of trimethylamine N-oxide reduction was observed in the anaerobic culture with dimethyl sulfoxide, but the specific activity of dimethyl sulfoxide reduction was not changed. The inducible enzyme trimethylamine N-oxide reductase was found in this culture. A marked expression of the structural genetorA for trimethylamine N-oxide reductase was also observed in atorA-lacZ gene fusion strain under anaerobic conditions with either trimethylamine N-oxide or dimethyl sulfoxide.l-Methionine sulfoxide and the N-oxides of adenosine, picolines, and nicotinamide slightly repressed expression of the gene. Membrane-boundb- andc-type cytochromes involved in the trimethylamine N-oxide reduction were also produced in a wild-type strain grown anaerobically with dimethyl sulfoxide. But thec-type cytochrome was not produced in thetorA-lacZ strain grown anaerobically with trimethylamine N-oxide or dimethyl sulfoxide; this suggests that there is a correlation between the expression oftorA and the synthesis of the cytochrome.     

Molybdenum cofactor: a compound in the in vitro activation of both nitrate reductase and trimethylamine-N-oxide reductase activities in Escherichia coli K12

Nitrate reductase (nitrite: (acceptor) oxidoreductase, EC 1.7.99.4) and trimethylamine N-oxide reductase (NADH : trimethylamine-N-oxide oxidoreductase, EC 1.6.6.9) activities were reconstituted by incubation of the association factor FA (the putative product of the chlB gene) with the soluble extract of the chlB mutant grown anaerobically in the presence of trimethylamine N-oxide. When soluble extracts of the chlB mutant grown on 10 mM sodium tungstate, a molybdenum competitor, were used in complementation systems, no enzymatic reactivation was observed. Heated extracts of the parental strain 541 were shown to contain a thermoresistant molybdenum cofactor by their ability to reactivate NADPH-nitrate reductase activity in the nit1 mutant of Neurospora crassa. By complementation of parental strain heated extract with association factor FA and soluble extract of the chlB mutant grown in the presence of sodium tungstate, we were able to show for the first time that the molybdenum cofactor is an activator common to the in vitro reconstitution of both nitrate reductase and trimethylamine-N-oxide reductase activities.     

Membrane cytochromes of Escherichia coli grown aerobically and anaerobically with nitrate

Redox titration has been coupled to spectroscopic techniques, enzyme fractionation, and the use of mutants to examine the cytochrome composition of the membranes from cells grown aerobically and anaerobically with nitrate. A combination of techniques was found to be necessary to resolve the cytochromes. At least six b-type cytochromes were present. Besides cytochromes bfdh and bnr, components of the formate dehydrogenase-nitrate reductase pathway, cytochromes b556, b555, b562, and o, characteristic of aerobic respiratory pathways, were present. The midpoint oxidation-reduction potentials of the aerobic b-type cytochromes suggested that the sequence of electron transfer is: cytochrome b556 leads to b555 leads to b562 leads to O2.     

NarK enhances nitrate uptake and nitrite excretion in Escherichia coli.

narK mutants of Escherichia coli produce wild-type levels of nitrate reductase but, unlike the wild-type strain, do not accumulate nitrite when grown anaerobically on a glucose-nitrate medium. Comparison of the rates of nitrate and nitrite metabolism in cultures growing anaerobically on glucose-nitrate medium revealed that a narK mutant reduced nitrate at a rate only slightly slower than that in the NarK+ parental strain. Although the specific activities of nitrate reductase and nitrite reductase were similar in the two strains, the parental strain accumulated nitrite in the medium in almost stoichiometric amounts before it was further reduced, while the narK mutant did not accumulate nitrite in the medium but apparently reduced it as rapidly as it was formed. Under conditions in which nitrite reductase was not produced, the narK mutant excreted the nitrite formed from nitrate into the medium; however, the rate of reduction of nitrate to nitrite was significantly slower than that of the parental strain or that which occurred when nitrite reductase was present. These results demonstrate that E. coli is capable of taking up nitrate and excreting nitrite in the absence of a functional NarK protein; however, in growing cells, a functional NarK promotes a more rapid rate of anaerobic nitrate reduction and the continuous excretion of the nitrite formed. Based on the kinetics of nitrate reduction and of nitrite reduction and excretion in growing cultures and in washed cell suspensions, it is proposed that the narK gene encodes a nitrate/nitrite antiporter which facilitates anaerobic nitrate respiration by coupling the excretion of nitrite to nitrate uptake. The failure of nitrate to suppress the reduction of trimethylamine N-oxide in narK mutants was not due to a change in the level of trimethylamine N-oxide reductase but apparently resulted from a relative decrease in the rate of anaerobic nitrate reduction caused by the loss of the antiporter system.     

Metabolic characteristics of an aerobe isolated from a methylotrophic methanogenic enrichment culture

An anaerobic methylotrophic methanogenic enrichment culture, with sustained metabolic characteristics, including that of methanation for over a decade, was the choice of the present study on interspecies interactions. Growth and methanation by the enrichment were suppressed in the presence of antibiotics, and no methanogen grown on methanol could be isolated using stringent techniques. The present study confirmed syntrophic metabolic interactions in this enrichment with the isolation of a strain ofPseudomonas sp. The organism had characteristic metabolic versatility in metabolizing a variety of substrates including alcohols, aliphatic acids, amino acids, and sugars. Anaerobic growth was favoured with nitrate in the growth medium. Cells grown anaerobically with methanol, revealed maximal nitrate reductase activity. Constitutive oxidative activity of the membrane system emerged from the high-specific oxygen uptake and nitrate reductase activities of the aerobically and anerobically grown cells respectively. Cells grown anaerobically on various alcohols effectively oxidized methanol in the presence of flavins, cofactor FAD and the methanogenic cofactor F₄₂₀, suggesting a constitutive alcohol oxidizing capacity. In cells grown anaerobically on methanol, the rate of methanol oxidation with F₄₂₀ was three times that of FAD. Efficient utilization of alcohols in the presence of F₄₂₀ is a novel feature of the present study. The results suggest that utilization of methanol by the mixed culture would involve metabolic interactions between thePseudomonas sp. and the methanogen(s). Methylotrophic, methanogenic partnership involving an aerobe is a novel feature hitherto unreported among anaerobic syntrophic associations and is of ecological significance.     

Isolation of an Escherichia coli mutant defective in cytochrome biosynthesis

Abstract A pleiotropic mutant of Escherichia coli affected in cytochrome biosynthesis was detected by anaerobic screening on a solid medium containing triphenyltetrazolium. When grown anaerobically on glycerol, nitrate and Casamino acids, this mutant exhibited a level of soluble cytochrome c ₅₅₂ which was ten times higher than that found in wild-type cells. The level of membrane-bound cytochrome b and the activity of nitrate reductase were about half the normal level. The mutant grew aerobically on succinate or d,l -lactate at a greatly reduced rate. The mutation impairing the growth ability at the locus sox (succinate oxidation) is also responsible for the deficiency of cytochrome b , nitrate reductase and formate dehydrogenase. Mapping by transduction placed sox at 86.7 min on the chromosome, very close to the glnA locus. Genetic analysis also indicated that the elevated level of cytochrome c ₅₅₂ was the result of a separate mutation, the location of which is yet to be determined.     

Effects of Molybdate and Selenite on Formate and Nitrate Metabolism in Escherichia coli

The effects of adding molybdate and selenite to a glucose-minimal salts medium on the formation of enzymes involved in the anaerobic metabolism of formate and nitrate in Escherichia coli have been studied. When cells were grown anaerobically in the presence of nitrate, molybdate stimulated the formation of nitrate reductase and a b-type cytochrome, resulting in cells that had the capacity for active nitrate reduction in the absence of formate dehydrogenase. Under the same conditions, selenite in addition to molybdate was required for forming the enzyme system which permits formate to serve as an effective electron donor for nitrate reduction. When cells were grown anaerobically on a glucose-minimal salts medium without nitrate, active hydrogen production from formate as well as formate dehydrogenase activity depended on the presence of both selenite and molybdate. The effects of these metals on the formation of formate dehydrogenase was blocked by chloramphenicol, suggesting that protein synthesis is required for the increases observed. It is proposed that the same formate dehydrogenase is involved in nitrate reduction, hydrogen production, and in aerobic formate oxidation.     

Requirement for terminal cytochromes in generation of the aerobic signal for the arc regulatory system in Escherichia coli: study utilizing deletions and lac fusions of cyo and cyd.

Escherichia coli has two terminal oxidases for its respiratory chain: cytochrome o (low O2 affinity) and cytochrome d (high O2 affinity). Expression of the cyo operon, encoding cytochrome o, is decreased by anaerobic growth, whereas expression of the cyd operon, encoding cytochrome d, is increased by anaerobic growth. We show by the use of lac gene fusion that the expressions of cyo and cyd are under the control of the two-component arc system. In a cyo+ cyd+ background, expression of phi(cyo-lac) is higher when the organism is grown aerobically than when it is grown anaerobically. A mutation in either the sensor gene arcB or the pleiotropic regulator gene arcA almost abolishes the anaerobic repression. In the same background, expression of phi(cyd-lac) is higher under anaerobic growth conditions than under aerobic growth conditions. A mutation in arcA or arcB lowers both the aerobic and anaerobic expressions, suggesting that ArcA plays an activating role instead of the typical repressing role. Under aerobic growth conditions, double deletions of cyo and cyd lower phi(cyo-lac) expression but enhance phi(cyd-lac) expression. The double deletions also prevent elevated aerobic induction of the lct operon (encoding L-lactate dehydrogenase), another target operon of the arc system. In contrast, these deletions do not circumvent aerobic repression of the nar operon (encoding the anaerobic respiratory enzyme nitrate reductase) under the control of the pleiotropic fnr gene product. It thus appears that ArcB senses the presence of O2 by level of an electron transport component in reduced form or that of an nonautoxidizable compound linked to the process by a redox reaction, whereas Fnr senses O2 by a different mechanism.