INTRODUCTION

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Aerobic and facultative aerobic bacteria can respond to changes within the environment by using different types of respiratory pathways (3, 23). During aerobic growth, the final step in the pathway, the four-electron reduction of dioxygen to two water molecules, is catalyzed by a group of membrane-bound enzymes called terminal oxidases. Many bacteria use more than one terminal oxidase (1, 3, 29). For example in the gram-negative bacterium Escherichia coli, there are two types of terminal oxidasescytochrome bo₃ and cytochrome bd. The former is used under aerobic growth conditions, whereas the latter is induced under microaerobic conditions (7, 30). In the soybean symbiont bacterium Bradyrhizobium japonicum, the main terminal oxidase under free-living conditions is an aa₃-type cytochrome c oxidase (20, 21). When B. japonicum lives endosymbiotically, it uses a cbb₃-type oxidase. This terminal oxidase has an extremely high affinity for oxygen, which allows it to operate under the low oxygen pressure of the root nodules (21). Another example is from the obligately aerobic, nitrogen-fixing bacterium Azotobacter vinelandii, which has two known terminal oxidases, a cytochrome bo₃ and a cytochrome bd. In A. vinelandii, cytochrome bd with its high oxygen affinity protects the oxygen-labile nitrogenase by keeping the oxygen levels sufficiently low (19). The gram-positive endospore-forming soil bacterium Bacillus subtilis synthesizes under aerobic growth conditions a branched electron transport chain comprising three or possibly four terminal oxidases (Fig. 1) (33, 34). The physiological role(s) of the specific terminal oxidases in B. subtilis is unknown. The long-term objective of our work is to define the physiological roles of the terminal oxidases in B. subtilis.

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Aerobic respiratory pathways in B. subtilis strain 168. Solid arrows, known electron pathways; dashed arrows, tentative pathways.

The electron transport chain in B. subtilis contains two major branches, one quinol oxidase branch and one cytochrome oxidase branch (Fig. 1). Three known terminal oxidases are present. Cytochrome caa₃ is a cytochrome c oxidase, whereas cytochrome aa₃ and cytochrome bd are quinol oxidases (16, 34). Both a-type oxidases belong to the well-characterized heme-copper oxidase superfamily of respiratory oxidases (4, 6, 34). Characteristic for the bacterial heme-copper oxidases is that they have a subunit homologous to subunit I of the mitochondrial cytochrome c oxidase, contain copper, and pump protons across the cytoplasmic membrane in response to electron transfer (4, 34).

Four structural genes, qoxABCD, are required for expression of B. subtilis cytochrome aa₃ (26). Cytochrome caa₃ is encoded by the ctaCDEF genes (27). Two additional genes, ctaA and ctaB, are also required for production of both cytochrome caa₃ and cytochrome aa₃ (28, 31). The ctaA and ctaB gene products are involved in the biosynthesis of the heme a prosthetic group (28). The bd-type of oxidases is a distinct group of terminal oxidases, not related to the heme-copper oxidases. They do not pump protons or contain copper (14). As there is no proton pumping, less energy is conserved by cytochrome bd compared to the heme-copper oxidases.

Expression of cytochrome bd requires cydA and cydB, which code for the two subunits of the enzyme as well as two additional genes, cydC and cydD (33). The latter two genes encode a putative ATP-binding-cassette (ABC) type of transporter. In B. subtilis, the presence of a fourth terminal oxidase can be predicted from the genome sequence (15, 33). A gene cluster containing three genes, ythA, ythB, and ythC, has been identified. The translated sequences of ythA and ythB are closely related to Bacillus stearothermophilus CbdA and CbdB, which constitute a terminal oxidase of bd type (24). No homologue of ythC has been found in B. stearothermophilus. The ythA and ythB genes might encode a terminal oxidase related to the bd-type oxidases. However, there is no direct experimental evidence for the presence of this terminal oxidase in B. subtilis. Throughout this article, the product of these genes is referred to as YthAB. In addition, there is spectroscopic evidence for a putative terminal oxidase of bb' type. The genes encoding this oxidase have not been identified, but it is not the product of ythA and ythB (2).

In this work, we show that, in B. subtilis, cytochrome aa₃ is the most important terminal oxidase during the exponential-growth phase. Moreover, we show that no single terminal oxidase is essential for aerobic growth of B. subtilis. However, the presence of one of the quinol oxidases, cytochrome aa₃ or cytochrome bd, is essential for aerobic growth. In addition, we show that one of the heme-copper oxidases, cytochrome caa₃ or cytochrome aa₃, is required for normal sporulation of B. subtilis.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used in this work are listed in Table 1. Escherichia coli strains were kept on Luria agar (25). B. subtilis strains grown aerobically were kept on tryptose blood agar base (TBAB) (Difco) plates, which when indicated were supplemented with 1% (wt/vol) glucose. Liquid media were inoculated with B. subtilis cells grown on TBAB plates over night. The cultures were grown at 37°C in an orbital shaker at 200 rpm in nutrient sporulation medium phosphate (NSMP) (5) or in NSMP supplemented with 0.5% (wt/vol) glucose (NSMPG) or in minimal medium supplemented with 0.5% (wt/vol) glucose (MM) (36). The doubling times in the exponential-growth phase were calculated as follows: doubling time equals (t₂  t₁) × log 2]/[log optical density at 600 nm (OD₆₀₀) at t₂  log OD₆₀₀ at t₁], where t₁ and t₂ are the times of measurement. B. subtilis cells were also grown on minimal medium plates supplemented with 0.5% (wt/vol) of one of the following carbon sources: glucose, malate, glutamate, or succinate. For the sporulation frequency experiment, strains were grown in NSMP at 37°C for 30 h. The number of viable cells per milliliter of culture was determined as the total number of CFUs on TBAB plates. The number of spores per milliliter of culture was determined as the number of CFUs after heat treatment at 80°C for 10 min.

DNA techniques. E. coli cells were transformed using the electroporation method described by Hanahan et al. (9). Chromosomal DNA was isolated and competent B. subtilis cells prepared essentially as described by Hoch (12). General DNA techniques were performed as described by Sambrook et al. (25). PCR was performed essentially as described previously (35), using Taq DNA polymerase. The primers used to amplify a 269-bp fragment of qoxA were QoxA1 (5'-GCAAGCTTTTGAGGAAGTATGCACTTCAGA-3') and QoxA2 (5'-GCTCTAGAGTCGCGGTATTTTACTAAAATAATGG-3'). Chromosomal DNA (0.1 ng) from B. subtilis 1A1 was used as a template. To construct double or triple mutants, B. subtilis strains were transformed with nonsaturating amounts of chromosomal DNA.

Spectral analysis on membranes. Membranes were prepared as described previously (10) and suspended in 20 mM sodium morpholinic propane sulphonic buffer (pH 7.4). Reduced minus oxidized difference light absorption spectra were recorded as described previously (33).

Construction of a cydCD expression plasmid. A plasmid containing the cydC and cydD genes under control of the cyd promoter was constructed by removing the 2-kb BglII and NdeI fragment containing cydA and cydB from plasmid pCYD23. The remaining part of pCYD23 was treated with the large (Klenow) fragment of E. coli DNA polymerase I, self-ligated, and used to transform B. subtilis 168A to chloramphenicol resistance. This resulted in plasmid pCYD25 containing cydC and cydD under the control of their native promoter (Fig. 2).

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Restriction map of the cyd region and plasmids carrying different parts of this region. At the top, the physical map of the B. subtilis cyd region is shown. The restriction sites are abbreviated as follows; B, BglII; N, NdeI; H, HindIII; P, PstI; and S, SphI. Plasmid pCYD24 is a derivative of pCYD13 (33), and plasmid pCYD25 is a derivative of pCYD23 (33). Construction of plasmids is described in Materials and Methods.

Construction of a cydCD null mutant. To make a cydCD deletion-insertion mutant, the 0.5-kbp EcoRI-HindIII fragment of pCYD13 was replaced by a 1-kb EcoRI-HindIII fragment of pCYD22 carrying a part of the cydC gene. The resulting plasmid, pCYD24 (Fig. 2), was used to transform strain 168A to chloramphenicol resistance. The deletion-insertion within the chromosomal cydC and cydD genes arising from a double-crossover recombination event was confirmed by Southern blot analysis (data not shown).

Construction of conditional qoxABCD mutant strains (P_spac-qoxABCD). A 269-base-pair fragment (qoxA') of the 5' region (48 to +221 relative to the putative qox translational start site) of qoxA was amplified by PCR. The resulting fragment contains a part of qoxA and includes a putative ribosome-binding site but lacks the promoter region. Plasmid pDH88 contains the artificial hybrid promoter spac, which can be induced by the addition of 1 mM isopropyl -D-thiogalactoside (IPTG) to the growth medium. The amplified qoxA' fragment was cleaved with restriction enzymes HindIII and XbaI and inserted into plasmid pDH88, cleaved with the same enzymes. The resulting plasmid was used to transform E. coli XL1-Blue to ampicillin resistance, creating plasmid pSPOX, containing the spac promoter followed by the qoxA' fragment (Fig. 3). When pSPOX was used to transform B. subtilis strains to chloramphenicol resistance, the plasmid was integrated into the chromosome by a single homologous recombination event in front of the qoxABCD genes. This resulted in strains in which expression of the qoxABCD operon could be controlled by IPTG (Fig. 3).

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Construction of B. subtilis strains with the qoxABCD operon under control of Pspac. The integrative plasmid pSPOX contains the inducible promoter spac and a 269-bp fragment of qoxA (qoxA'), including the ribosome-binding site but not the promoter region. Integration of pSPOX into the B. subtilis chromosome results in control of the qoxABCD genes by the spac promoter and control of the truncated qoxA by the native qox promoter. The genes for -lactamase, chloramphenicol resistance, and the lac repressor are indicated as bla, cat, and lacI, respectively.

	RESULTS AND DISCUSSION

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Growth properties of single oxidase mutants. Doubling times and growth yields of B. subtilis strains lacking one of the terminal oxidases, cytochrome caa₃, cytochrome aa₃, cytochrome bd, or YthAB, were compared with the doubling time and growth yield of the wild-type strain in different media (Table 2). In a broth medium (NSMP) or in MM, the doubling times and growth yields of the strains lacking cytochrome caa₃, cytochrome bd, or YthAB did not differ from those of the wild type. However, the strain lacking cytochrome aa₃ grew significantly more slowly and reached the stationary phase at a lower cell density compared to the wild type in both media (Table 2). When grown in NSMP supplemented with 0.5% glucose, all four mutant strains showed a doubling time and growth yield similar to those of the wild-type strain (Table 2). We concluded that none of the terminal oxidases are, by themselves, essential for aerobic growth. The significantly reduced growth rate of the cytochrome aa₃ mutant in NSMP and MM media suggests that cytochrome aa₃ in these growth media is the most important oxidase in exponentially growing B. subtilis cells.

Mutants defective in multiple terminal oxidases. Next, we attempted to make B. subtilis mutant strains lacking two, three, or four terminal oxidases. To make a strain lacking both cytochrome aa₃ and cytochrome bd, LUW20 (cydABCD::tet) was transformed with nonsaturating amounts of chromosomal DNA (0.2 mg/liter) from LUH14 (qoxABCD::kan), and transformants were selected on TBAB plates containing kanamycin, with and without glucose. However, no transformants were obtained. The reverse experiment, i.e., transformation of a strain lacking cytochrome aa₃ with chromosomal DNA from a strain lacking cytochrome bd, was performed with similar results. The experiment was also done with strains derived from 1A1 and JH642, with similar results. As a control, strain LUW20 (cydABCD::tet) harboring plasmid pCYD23, which carries a functional set of the cydABCD genes, was transformed with LUH14 (qoxABCD::kan) chromosomal DNA. Transformants (7.5 × 10⁶/mg of DNA) were obtained, showing that the actual transformation event works in this strain. From this we concluded that a strain lacking both cytochrome aa₃ and cytochrome bd is not viable under the conditions employed.

Anaerobic growth of double and triple mutants. Under anaerobic conditions, B. subtilis is able to utilize nitrate as a terminal electron acceptor (18). To find out if a strain lacking cytochrome aa₃ in combination with cytochrome bd is viable under anaerobic nitrate-respiratory conditions, the transformation experiments were carried out in an anaerobic atmosphere. LUH14 (qoxABCD::kan) was transformed with chromosomal DNA from LUW10 (cydABCD::cat). Transformants were selected on TBAB plates containing chloramphenicol, 20 mM KNO₃, and 1% (wt/vol) glucose, incubated at 37°C in an anaerobic cabinet for 24 h. Strain LUW29, lacking both cytochrome aa₃ and cytochrome bd, was obtained. A similar procedure was used to construct strain LUW33, lacking cytochrome caa₃, cytochrome aa₃, and cytochrome bd. The mutant strains LUW29 and LUW33 were streaked on two new plates of which one was incubated aerobically and the other one was incubated in the anaerobic cabinet. The strains grew well in the anaerobic cabinet but could not grow in an aerobic atmosphere. If the anaerobically incubated cells were exposed to oxygen, they could not resume growth in the anaerobic cabinet. The results showed that a strain lacking both cytochrome bd and cytochrome aa₃ grows under anaerobic, nitrate-respiratory conditions.

Construction of strains with the qoxABCD genes under control of an IPTG-inducible promoter. To be able to study the growth properties of mutants lacking both cytochrome aa₃ and cytochrome bd, we constructed strains in which expression of the qoxABCD operon was controlled by the IPTG-inducible spac promoter. An integrative plasmid (pSPOX) carrying P_spac was constructed and used to transform different B. subtilis strains to chloramphenicol resistance as described in Materials and Methods and in Figure 3.

ctaCD cydABCD

-qoxABCD cydABCD

-qoxABCD ctaCD cydABCD

-qoxABCD ctaCD

View larger version (17K): [in this window] [in a new window]   FIG. 4.   B. subtilis strains carrying pSPOX grown in NSMPG with and without IPTG. Cells were grown in NSMPG supplemented with IPTG. After 2.25 h (OD600 between 0.15 and 0.24, indicated by arrows), cells were harvested, washed, and resuspended in fresh media. Open circles show cells grown in NSMPG containing 1 mM IPTG. Solid circles show cells grown in NSMPG without IPTG. (A) LUW32 (Pspac-qoxABCD); (B) LUW42 (Pspac-qoxABCD ctaCD); (C) LUW22 (Pspac-qoxABCD cydABCD); (D) LUW24 (Pspac-qoxABCD ctaCD cydABCD).

Sporulation of oxidase mutants. Sporulation in B. subtilis is an energy-requiring process. To see whether the absence of any of the terminal oxidases affected sporulation, mutant strains were tested for sporulation efficiency. As shown in Table 4, the sporulation efficiency of the single-oxidase mutants did not differ from that of the wild-type strain. Mutants lacking both cytochrome caa₃ and cytochrome bd or both cytochrome caa₃ and YthAB or both cytochrome bd and YthAB showed normal sporulation (Table 4). The same was observed for the strain containing only cytochrome aa₃ (i.e., a strain lacking cytochrome caa₃, cytochrome bd, and YthAB). However, the strain containing only cytochrome bd showed an approximately 5,000-fold reduction of the sporulation frequency relative to that of the wild-type strain (Table 4). Sporulation was also inhibited in the strain lacking both cytochrome aa₃ and cytochrome caa₃. This is in line with previous data showing that a CtaA mutant strain, which is unable to make the heme a prosthetic group, is sporulation deficient (17). The strain lacking cytochrome aa₃ and YthAB showed a 24-fold decrease in the level of sporulation (Table 4). These results showed that one of the heme copper terminal oxidases, cytochrome aa₃ or cytochrome caa₃, is required for efficient sporulation of B. subtilis strain 168, probably because at least one proton-pumping oxidase is required to conserve enough energy for sporulation. In addition, our results suggested that YthAB may have a role in sporulation and can compensate for the loss of cytochrome aa₃.

The role of the CydCD transporter. The cydC and cydD gene products are likely to encode a heterodimeric, membrane bound ABC type of transporter that is required for assembly of cytochrome bd (33). To analyze whether the CydCD ABC transporter is also required for the assembly of an additional terminal oxidase in B. subtilis, we constructed a strain containing the CydCD transporter but lacking cytochrome bd. A plasmid, pCYD25, carrying cydCD under control of the cyd promoter, was introduced into LUW20, thus creating a strain lacking the chromosomal cydABCD operon but carrying cydCD on a plasmid. To confirm that pCYD25 contained a functional set of cydCD, strain LUW128 lacking cydCD was constructed, and pCYD25 was introduced into this strain. LUW128 carrying pCYD25 and LUW128 carrying pHP13 were grown in NSMPG, and membranes were studied by light absorption difference (reduced minus oxidized) spectroscopy. Membranes from strain 1A1 (wild type) grown in the same way were used as a control. No cytochrome bd was detected in membranes from LUW128(pHP13). Membranes from LUW128(pCYD25) showed a spectrum similar to that of the wild-type strain, showing that pCYD25 expressed a functional cydCD and that overexpression of cydCD did not result in an increased production of cytochrome bd (Fig. 5).

View larger version (17K): [in this window] [in a new window]   FIG. 5.   Light absorption difference (dithionite-reduced minus ferricyanide-oxidized) spectra of membranes (3 mg of protein per ml) from strains 1A1 and LUW128 carrying different plasmids. B. subtilis strains were grown in NSMPG and harvested in the stationary-growth phase. Line A, LUW128(pHP13); line B, LUW128(pCYD25); line C, 1A1.

Conclusion. The aerobic respiratory pathways in B. subtilis terminate with one of three or possibly four alternative terminal oxidases, as indicated in Fig. 1. Taken together, our data strongly indicate that one of the quinol oxidases, cytochrome aa₃ or cytochrome bd, is essential for aerobic growth of B. subtilis strain 168. The reason that the cytochrome oxidase branch cannot compensate for the loss of the quinol oxidase branch is most probably that the cytochrome oxidase branch is not expressed until the cells enter the stationary phase. This hypothesis is supported by observations that the genes encoding the bc complex and probably also cytochrome caa₃ are repressed by the transition state regulator AbrB in the exponential growth phase (37; L. Winstedt and C. von Wachenfeldt, unpublished data). We do not know under which conditions the ythAB genes are expressed. However, it seems likely that they are not expressed in exponentially growing cells. Deletion of ythAB in a strain lacking cytochrome aa₃ makes the strain sporulation deficient, indicating a physiological role for YthAB in B. subtilis.