INTRODUCTION

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Many bacteria that live in soil, sewage or sludge possess a genome that encodes a highly branched respiratory network. The encoded network comprises various types of dehydrogenase and of terminal oxidoreductase, which are capable of electron input and output flow, respectively, during oxidative phosphorylation (4). Such versatility allows these bacteria to use a variety of electron donors and terminal electron acceptors once they become available in their natural habitats. The genes or gene clusters that encode the different redox enzymes are usually tightly regulated. They are expressed only under the growth conditions at which they are required. One of the best-studied organisms in that respect is Paracoccus denitrificans. This gram-negative bacterium is able to grow heterotrophically with a variety of organic carbon and free-energy sources, as well as autotrophically, by using hydrogen, or so-called C1 substrates such as methylamine or methanol, as free-energy sources (2, 3, 17, 41, 42, 46). The electron transport chain used for aerobic heterotrophic growth possesses a full complement of proteins with counterparts in the mitochondrial respiratory chain (20, 41). Upon depletion of the organic substrates, the bacterium induces hydrogenase and methanol or methylamine dehydrogenases, as well as their dedicated electron acceptors, provided that their substrates are present (16, 49).

P. denitrificans is able to reduce molecular oxygen at a wide range of oxygen concentrations. This flexibility is the result of its potential to synthesize three types of terminal oxidase (9, 10, 32, 33, 35), which all belong to the superfamily of heme copper oxidases (15, 38, 44). The one that is most expressed at atmospheric oxygen concentrations is the aa₃-type cytochrome c oxidase, which has a relatively low affinity for oxygen (31, 36). Its expression level decreases with decreasing oxygen concentrations (5). The second type of oxidase is the cbb₃-type cytochrome c oxidase, which has a relatively high affinity for oxygen and which is increasingly synthesized at decreasing oxygen concentrations (10, 31). The third type of oxidase is a ba₃-type quinol oxidase, which is the counterpart of the bo₃-type quinol oxidase found in Escherichia coli (8, 35). The ba₃-type oxidase receives electrons from ubiquinol, is expressed and increasingly active under conditions that give rise to high reduction levels of the Q-pool, and apparently serves to prevent that by rapidly transferring electrons from ubiquinol to oxygen (27).

P. denitrificans is also able to synthesize four types of N-oxide oxidoreductase, which catalyze the sequential reduction of nitrate to dinitrogen gas via the intermediates nitrite, nitric oxide, and nitrous oxide. This denitrification process takes place at nearly anoxic conditions, during which nitrate substitutes for oxygen as a terminal electron acceptor. Nitrate, nitrite, nitric oxide, and nitrous oxide reductases are expressed when nitrate is present and oxygen is virtually absent (2, 3, 14, 42).

Much of our knowledge on how electrons are transferred from the dehydrogenases to the terminal oxidoreductases stems from the analyses of mutants with mutations in genes encoding electron carriers that make up the respiratory network (9, 11, 50, 51, 52, 53). However, it has thus far been difficult to understand the exact role of the different types of cytochrome c in situ since mutations in their corresponding genes did not give clear phenotypes if at all. Apparently, cytochromes c or other small redox carriers can substitute for each other. The present study sought to elucidate the roles of the cytochromes c₅₅₀ and c₅₅₂ and the cytochrome bc₁ complex in electron transport in P. denitrificans. The approach was to isolate P. denitrificans strains with mutations in the genes encoding these three types of cytochrome c and in combinations of these genes in order to study the resultant effects of these mutations on their growth characteristics.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and growth conditions. The strains and plasmids used in this study are listed in Table 1. E. coli strains were cultivated in yeast-tryptone medium at 37°C. P. denitrificans strains were grown at 34°C in batch cultures either aerobically, i.e., in 2-liter flasks filled with 300 ml of medium and shaken vigorously; semiaerobically, i.e., in 1-liter flasks filled with 500 ml of medium and slowly shaken; or anaerobically, i.e., in flasks almost completely filled with medium and not shaken. The P. denitrificans strains were grown on mineral batch medium containing Lawford trace solution and methylamine (100 mM), succinate (25 mM), or butyrate (25 mM) as carbon and free-energy sources at pH 7.0 (7, 24). Anaerobic cultures were supplemented with 100 mM potassium nitrate as an electron acceptor. Inhibition of the cytochrome bc₁ complex was achieved by addition of 5 µM myxothiazol to the cultures (24). Cells were harvested in the mid-exponential phase of growth when the turbidity at 660 nm was ca. 1.0 cm¹. When appropriate, antibiotics were added, i.e., rifampin (40 mg liter¹), streptomycin (50 mg liter¹), kanamycin (50 mg liter¹), or ampicillin (100 mg liter¹). Residual nitrite in denitrifying cultures was determined by using nitrite strips from Boehringer.

Isolation of intact cells and membrane fractions. Cells were washed twice in 0.1 M of Tris (Tris hydroxymethyl aminomethaan)-HCl at pH 7.5 at 4°C and then suspended in the same buffer to a turbidity of 50 at 660 nm. After the addition of MnCl₂ to 10 mM and DNase I to 2 mg ml¹, cell extracts were obtained by using a French pressure cell (American Instrument Company, Silver Spring, Md.) at 10,000 lb/in². Cell debris was spun down for 5 min at 17,000 × g, and the resulting supernatant was centrifuged at 438,000 × g for 30 min at 4°C in order to separate the soluble and membrane fractions. The pellet containing the membrane faction was resuspended in 0.1 M Tris-HCl at pH 7.5. Protein concentrations were determined by using the BCA Kit (Pierce) with bovine serum albumin as a standard. Samples (at 10 mg of protein ml¹) were routinely stored at 80°C.

PAGE and heme staining. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out by using the Bio-Rad Mini-Protean II Gel System with 13% slab gels (23). The samples (100 µg of protein each) were diluted in sample buffer (60 mM Tris-HCl, pH 6.8; 25% glycerol [vol/vol]; 2% SDS; 50 mM 2-mercaptoethanol; 50 mM dithiothreitol) and incubated at room temperature for 10 min. The heme-binding proteins were detected by using chemiluminescent substrate "Lumi-Light Plus" (Boehringer Mannheim) on a high-performance chemiluminescence film (Amersham) (29). The program NIH-Image/ppc 1.56b61 was used for densitometric analyses.

Spectral analyses. Spectra of dithionite (5 mg/ml) reduced cell suspensions (turbidity at 660 nm of 50) were recorded in the dual-wavelength mode of an automated Aminco/SLM DW-2 UV/Vis spectrophotometer with the reference set at 578 nm. During kinetic measurements, the reduction level of cytochromes c as a function of time was recorded at 552 nm with the reference set at 578 nm. Cuvettes were thermostated at 20°C. Oxygen was released from 20 µl of 0.3% hydrogen peroxide by intracellular catalase activity. Dithionite was added to a final concentration of 5 mg/ml.

Oxygen consumption. Oxygen consumption rates of cells were measured polarographically with a Clark-type oxygen electrode (Yellow Springs, Ohio) in 1 ml of Tris-HCl (pH 7.5) at 25°C. Succinate (10 mM) was added as an electron donor. Where indicated, the electron flow via cytochrome bc₁ was inhibited by adding antimycin A (6 µM) and myxothiazol (6 µM).

DNA manipulations. Routine cloning procedures were performed according to standard protocols (1). The construction of marked and unmarked mutations in chromosomal genes is described in Results and was performed as described earlier (52). E. coli TG1 was used routinely for cloning procedures. E. coli HB101(pRK2020) was used as the helper strain in the conjugative transfer of plasmids via triparental mating.

Construction of cytochrome c single and multiple mutant strains. The following strains were constructed in an earlier study (11): Pd21.21 (cycA::Km^r) with an interrupted cytochrome c₅₅₀ encoding gene, Pd21.31 (cycA) with an unmarked mutation in the cytochrome c₅₅₀ encoding gene, Pd24.21 (fbcC::Km^r) with an interrupted cytochrome c₁ encoding gene, and the double mutant strain Pd92.06 (cycA fbcC::Km^r). In this study, an internal part of the chromosomal fbcC gene that contained the gene cartridge encoding kanamycin resistance (Km^r) in strains Pd24.21 and Pd92.06 was removed in order to make the fbcC mutations in these strains unmarked. For this, plasmid pBL250, which is a pBR322 derivative containing the fbcC gene and flanking regions (22), was cut with XhoI and SalI and then religated. As a result, a 1,060-bp fragment within the fbcC gene was removed. The fragment with the mutated gene was then cloned in suicide vector pRVS1, yielding plasmid pRTd24.31. This plasmid was used in the gene exchange technique described earlier (52) in order to isolate strains Pd24.31 (fbcC) and Pd92.07 (cycA fbcC), which have unmarked mutations in their chromosomal fbcC genes. In this gene exchange technique, mutants with unmarked mutations are selected by using the E. coli lacZ gene as a suicide gene (52). Plasmid pAT8 harbors the cytochrome c₅₅₂ encoding cycM gene in which the gene cartridge encoding kanamycin resistance was inserted in the internal StuI site (43). A 4.6-kb NotI fragment of this plasmid, which contains the mutated cycM gene, was cloned in suicide vector pRVS2, yielding vector pRTd28.22. This plasmid was used in the gene exchange technique described earlier (51) in order to isolate strains Pd28.22 (cycM::Km^r), Pd92.26 (cycA cycM::Km^r), Pd92.28 (fbcC cycM::Km^r), and Pd93.14 (cycA fbcC cycM::Km^r), which all have marked mutations in their chromosomal cycM genes. The correctnesses of the mutations were confirmed by Southern analyses of their chromosomal DNA. As a result of these constructions, we had mutant strains with mutations in one, two, or three of the genes encoding cytochromes c₅₅₀, c₅₅₂, and c₁.

	RESULTS

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Spectral analyses of the cytochrome c single and multiple mutant strains. P. denitrificans wild-type and mutants with single and multiple mutations in the cycA, cycM, and fbcC genes were cultured aerobically in mineral medium containing succinate as the sole carbon and free-energy source. Cell suspensions from these cultures exhibited composite -bands of ca. 555 nm at which cytochromes b (at ca. 560 nm) and c (at ca. 550 nm) have their maximum absorbance (Fig. 1). It should be noted that the absorbance at that wavelength reflects not only all three cytochromes c that are important here, i.e., cytochromes c₅₅₀, c₅₅₂, and c₁, but also further contributions from other cytochromes c as well. Both the shape and the height of that composite band differed between the strains. Only the cytochrome c₅₅₂-deficient mutant displayed a spectrum very similar to that of the wild-type strain. Paradoxically, strains Pd24.31 (c₁ deficient) and Pd92.28 (c₅₅₂/c₁ deficient) had a higher cytochrome c peak than the wild-type strain, whereas that peak is lower in strains Pd21.31 and Pd92.26, which are cytochrome c₅₅₀ and cytochrome c₅₅₀/c₅₅₂-deficient mutants, respectively. A sharp decrease in the height of that band is observed in the spectra of strains Pd92.07 and Pd93.14, which are cytochrome c₅₅₀/c₁- and cytochrome c₅₅₀/c₅₅₂/c₁-deficient mutants. Another remarkable observation is that the peak at ca. 605 nm where hemes a of cytochrome aa₃ have their maximum absorbance is decreased to ca. 20% of the wild-type signal in the spectra of strains Pd21.31 (cytochrome c₅₅₀ deficient), Pd92.26 (cytochrome c₅₅₀/c₅₅₂ deficient), and Pd93.14 (cytochrome c₅₅₀/c₅₅₂/c₁ deficient).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Dithionite-reduced absorption spectra at room temperature of cell suspensions from the wild-type strain (Pd1222 [C]) and from mutant strains deficient in cytochrome c1 (Pd24.31 [A]), cytochromes c552/c1 (Pd92.28 [B]), cytochrome c552 (Pd28.22 [D]), cytochromes c550/c1 (Pd92.07 [E]), cytochrome c550 (Pd21.31 [F]), cytochromes c550/c552 (Pd92.26 [G]), or cytochromes c550/c552/c1 (Pd93.14 [H]). The cells had been grown aerobically to their mid-exponential phase of growth (optical density at 660 nm [OD660] = ~1.0 cm1) in batch cultures with succinate as the carbon and free-energy source.

PAGE and heme staining of cytochrome c single and multiple mutant strains. In order to study the cytochrome c composition of the mutants in more detail, cells from cultures grown aerobically on succinate were broken and centrifuged to yield soluble and membrane fractions. For each fraction, the proteins were separated by PAGE, after which the cytochromes c were visualized by a heme staining approach. Two important conclusions can be drawn from the results (Fig. 2). First, they confirm that the mutations in the cycA, cycM, and fbcC genes of the mutants had resulted in the inability to express the corresponding cytochromes c as judged by the absence of heme-stained proteins of 14 kDa (cytochrome c₅₅₀) in the periplasmic fractions (Fig. 2A) and of 22 kDa (cytochrome c₅₅₂) and ca. 60 kDa (cytochrome c₁) in the membrane fractions (Fig. 2B) of the corresponding mutants. The faint band at ca. 14 kDa observed in the lane of the cytochrome c₅₅₀/c₅₅₂-deficient double mutant in Fig. 2A probably originates from a cytochrome c different from cytochrome c₅₅₀ and which is specifically expressed in this mutant. This band is not present in the lane of its parent strain Pd21.31 (Fig. 2A, lane 2). We monitored the staining of the cytochromes c present in that part of the gel over time (Fig. 3) and noticed that cytochrome c₅₅₀ stained much faster than the unknown one (maximally stained after 1 min and after 15 min, respectively). The second conclusion is that the knockout of cytochromes c₅₅₀, c₅₅₂, and bc₁ singly and in various combinations had resulted in the increased expression of other types of cytochrome c. The periplasmic fractions of the wild-type strain and the cytochrome c₅₅₂-deficient strain contained moderate levels of cytochrome c₅₅₀, but its concentration was more than 10-fold increased in the cytochrome c₁- and cytochrome c₅₅₂/c₁-deficient mutant strains. In addition, all of the strains that lacked the cytochrome bc₁ complex induced high levels of cytochrome c peroxidase (45 kDa), as well as two cytochromes c of ca. 25 and 35 kDa. The 35-kDa cytochrome c was abundant in the cytochrome c₅₅₀/c₅₅₂-deficient mutant strain. Since this abundance correlated with the appearance of the unknown cytochrome c of 14 kDa, the latter might be a heme containing breakdown product of the former. In contrast to the cytochrome bc₁ mutants, the cytochrome c₅₅₀/c₅₅₂-deficient mutant strain did not show a significantly increased induction of cytochrome c peroxidase nor of the 25-kDa cytochrome c. This correlation could indicate that the 25-kDa cytochrome c was a heme-containing breakdown product of cytochrome c peroxidase. Inspection of the cytochromes c in the membrane fraction indicated that the concentrations of the cytochrome bc₁ complex and of cytochrome c₅₅₂ in the mutants still able to express them were not significantly different from those of the wild-type strain. In contrast, the intensities of the bands containing the cytochrome c containing subunits of the cbb₃-type oxidase, CcoO and, to a lesser extent, CcoP, were up to five times higher in the cytochrome bc₁ mutants compared to the wild-type strain.

View larger version (105K): [in this window] [in a new window]   FIG. 2.   SDS-PAGE and subsequent heme staining of cytochromes c in the soluble fractions (A) and membrane fractions (B) of P. denitrificans wild-type (lanes 1) and mutant strains deficient in cytochrome c550 (lanes 2), cytochrome c552 (lanes 3), cytochrome c1 (lanes 4), cytochromes c550/c1 (lanes 5), cytochromes c550/c552 (lanes 6), cytochromes c552/c1 (lanes 7), and cytochromes c550/c552/c1 (lanes 8). Growth and harvesting conditions were as shown in the legend of Fig. 1. The fractions, containing 100 µg of protein each, were obtained from one set of experiments and analyzed in parallel. The relative concentrations of heme c-containing proteins within each gel were determined by densitometric analyses. The c-type cytochromes indicated are cytochromes c550 and c552 (CycA and CycM), cytochrome c peroxidase (Ccp), subunits of the cbb3-type oxidase (CcoO and CcoP), and cytochrome c1 (FbcC). Sizes of the molecular mass markers are indicated on the right of the gels in kilodaltons.

View larger version (117K): [in this window] [in a new window]   FIG. 3.   Time-dependent staining of soluble cytochromes c run into the gel to the position of the 14-kDa marker. Lane 1, the cytochrome c550/c552-deficient mutant; lane 2, the cytochrome c1-deficient mutant. The exposure times of the film are indicated at the left in minutes.

Growth characteristics of the cytochrome c mutants. P. denitrificans wild-type strain and the cytochrome c₅₅₀, c₅₅₂, and bc₁ single and multiple mutants were grown in mineral medium under three different growth conditions. The increase in cell density with time was determined, as well as their final turbidity at 660 nm. The growth conditions were (i) aerobic with succinate as the sole electron donor, (ii) aerobic with methylamine as the sole electron donor, and (iii) anaerobic with succinate as the electron donor and nitrate as the electron acceptor. The data were used to estimate the maximum specific growth rates and the maximum cell numbers (as determined from turbidity measurements) of the strains. These values are presented in Table 2.

Kinetics of electron transport in cytochrome c mutants. P. denitrificans wild-type strain and cytochrome c mutants were grown under aerobic conditions with succinate as the sole carbon and free-energy source. Cells grown to their mid-exponential phase of growth were harvested, washed, and resuspended. The oxygen consumption rates of these suspensions were determined with succinate as the electron donor (Table 3). The data show that the oxygen consumption rates of the strains that still had the potential to synthesize the cytochrome bc₁ complex were comparable. Those that lacked the complex had rates ca. 15% higher than that of the wild-type strain. In the presence of antimycin A and myxothiazol, inhibitors of the cytochrome bc₁ complex, the oxygen consumption rates of the wild-type and cytochrome c₅₅₂-deficient mutant were almost halved compared to the uninhibited condition, whereas they were ca. 25% decreased in cytochrome c₅₅₀- and cytochrome c₅₅₀/c₅₅₂-deficient mutants. The respiratory inhibitor hardly if at all affected the oxygen consumption rates of the cytochrome c₁-deficient strains.

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	DISCUSSION

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The studies presented in this study have shed more light on the roles of cytochromes c₅₅₀, c₅₅₂, and c₁ in controlling the rates of electron transfer in P. denitrificans grown at various growth conditions. The data also indicate that cytochrome c deficiencies by the mutations are in many cases accompanied by changed levels of expression of other respiratory enzymes.

Much of our knowledge on the makeup and function of the P. denitrificans respiratory system at various growth conditions has been described in earlier studies (reviewed in references 2, 3, and 46). The roles of cytochromes c₅₅₀, c₅₅₂, and c₁ in that electron transport have now been defined by careful examination of the growth characteristics of mutant strains lacking one or more of these types of cytochrome. A scheme of the flexible respiratory network of methylamine grown cells as based on these studies is presented in Fig. 4. It shows that cytochrome c₅₅₀ is a potential electron donor to any of the cytochrome c oxidases. Cytochrome c₅₅₂ is a dedicated donor to only the aa₃-type oxidase, in agreement with the observation that cytochrome c₅₅₂ made part of a purified supercomplex containing the cytochrome bc₁-complex and the aa₃-type oxidase, which was functional in electron transport (4). Our results further show that cytochrome c₅₅₂ is not a donor to the cbb₃-type oxidase in agreement with an earlier observation that a mutant strain lacking cytochromes c₁ and c₅₅₀, as well as the aa₃-type oxidase, was unable to grow with methylamine as the sole free-energy source, whereas it could still express cytochrome c₅₅₂ and the cbb₃-type oxidase (11).

View larger version (22K): [in this window] [in a new window]   FIG. 4.   Scheme of the respiratory network of P. denitrificans during growth with methylamine as sole source of free energy. The sequential oxidation of methylamine to carbon dioxide (dashed arrows) is mediated by methylamine dehydrogenase (MADH), formaldehyde dehydrogenase (FyDH), and formate dehydrogenase (FDH). Physiologically important electron transfer routes (straight arrows) from NADH dehydrogenase (NADHDH) and MADH to the terminal oxidases were deduced from the studies in this study. amic, amicyanin.

The studies described here present a qualitative view on all possible electron transfer pathways during methylamine oxidation as they operate in the various mutant strains. Our observation do not prove that all of these pathways operate to the same extent in the wild-type cells as they do in these mutants. The deletions of cytochromes c₁ and c₅₅₀ resulted in decreased maximum specific growth rates of the mutant strains by 40 and 20%, respectively. These cytochromes are apparently essential for optimal performance of the methylamine oxidizing pathways since they have multiple tasks in connecting quinol and amicyanin oxidation to cytochrome c reduction. However, it is not possible to deduce from these numbers the relative electron fluxes through the parallel electron transfer pathways in the wild-type cells. This is because every mutation in these mutants gives rise to changes in the kinetics of the remaining electron transfer reactions as a consequence of (i) changes in the expression levels of the respiratory components (28) and (ii) changes in the reduction levels of them (27).

The respiratory network of methanol-grown cells is quite similar to that of methylamine-grown cells. The difference is that methylamine dehydrogenase and its dedicated electron acceptor amicyanin (19, 53) are replaced by a methanol dehydrogenase and its dedicated electron acceptor cytochrome c_551i (18, 50). Just like amicyanin, this cytochrome requires another cytochrome c as electron acceptor and is unable to donate to any of the cytochrome c oxidases.

When the cytochrome c-deficient strains were grown with succinate as the carbon and free-energy source, their maximum specific growth rates were quite comparable to that of the wild-type strain, indicating that these mutant cells adapt completely in that respect to compensate for the loss of any of these cytochromes c. That adaptation involves higher electron transfer fluxes through the ba₃-type quinol oxidases since these mutants display lower maximum cell numbers (since this pathway is less efficient with respect to free-energy transduction) and higher maximum oxygen uptake activity via the ba₃-type oxidase. This result is in agreement with earlier studies on cytochrome bc₁-deficient mutants (27).

The analyses of the mutants grown under denitrifying conditions indicated that cytochrome c₁ is essential for the reduction of nitrite to dinitrogen gass. This result is in agreement with the view that the cytochrome bc₁ complex is a compulsory intermediate in electron transfer from the Q-pool to the nitrite, nitric oxide, and nitrous oxide reductases (reviewed in reference 34). At least up to and including nitrite reductase, the pathway also involves cytochrome c₅₅₀, which appeared to be an essential electron donor to this enzyme during copper limitation. In the presence of copper, however, the mutation in the gene encoding cytochrome c₅₅₀ had hardly any effect on the denitrifying potential of that mutant strain, indicating that a copper-dependent electron carrier substituted for cytochrome c₅₅₀ (51). A likely candidate is pseudoazurin, which is a type I blue copper protein and able to mediate electron transfer between the cytochrome bc₁ complex and the nitrite, nitric oxide, and nitrous oxide reductases in vitro (21, 25, 26). Indeed, it has been shown that a mutant strain deficient in cytochrome c₅₅₀ and pseudoazurin failed to reduce nitrite (30; Stuart Ferguson, unpublished data). Cytochrome c₅₅₂ is apparently redundant in the denitrifying pathways since we observed virtually no effect of its absence on the denitrifying potential of the mutant cells and since it was unable to compensate for the loss of cytochrome c₅₅₀ under copper limitation.

In the course of our studies, we noticed that the expression levels of certain types of cytochrome c was increased in aerobically grown mutant strains, especially in those that lacked the cytochrome bc₁ complex. Apparently, the mutations in the cytochrome c-deficient mutants cause metabolic changes that result in changes in transcription activation of the genes encoding these cytochromes c. One of these metabolic changes affects the activity of the FnrP protein, which is a transcription activator regulating the expression of genes in response to oxygen limitation (47). This conclusion is based on the observation that the concentrations of the cytochrome c-containing subunits of the cbb₃-type oxidase and cytochrome c peroxidase, the genes of which are regulated by FnrP (47), are increased in the mutant strains. Some of the mutants, i.e., the cytochrome c₅₅₀ single mutant, the cytochrome c₅₅₀/c₅₅₂ double mutant, and the cytochrome c₅₅₀/c₅₅₂/c₁ triple mutant, also displayed lower levels of the aa₃-type oxidase as judged by spectroscopic analyses. That phenomenon has been described earlier for the cytochrome c₅₅₀-deficient mutant (51). The reason for the observed decrease is not clear at present but cannot be merely ascribed to the cycA mutation alone since the cytochrome c₅₅₀/c₁-deficient double mutant has wild-type levels of the aa₃-type oxidase.

Taken together, these observations indicate that the performance of the respiratory network is continuously monitored and that its makeup adapted in response to changes in that performance apparently in order to maintain an optimal electron flux at each given growth condition.