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Journal of Bacteriology, April 2008, p. 2933-2938, Vol. 190, No. 8
0021-9193/08/$08.00+0     doi:10.1128/JB.01409-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Evidence for Involvement of Copper Ions and Redox State in Regulation of Butane Monooxygenase in Pseudomonas butanovora

D. M. Doughty,¹ E. G. Kurth,² L. A. Sayavedra-Soto,² D. J. Arp,² and P. J. Bottomley^1,3*

Department of Microbiology,¹ Department of Botany and Plant Pathology,² Department of Crop and Soil Science, Oregon State University, Corvallis, Oregon 97331-3804³

Received 30 August 2007/ Accepted 5 February 2008

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Pseudomonas butanovora possesses an alcohol-inducible alkane monooxygenase, butane monooxygenase (BMO), that initiates growth on C₂-C₉ alkanes. A lacZ transcriptional reporter strain, P. butanovora bmoX::lacZ, in which the BMO promoter controls the expression of β-galactosidase activity, was used to show that 1-butanol induced the BMO promoter in the presence or absence of O₂ when lactate-grown, BMO-repressed cells were washed free of lactate and incubated in NH₄Cl-KNa phosphate buffer. In contrast, when lactate-grown cells of the reporter strain were incubated in phosphate buffer containing the mineral salts of standard growth medium, 1-butanol-dependent induction was significantly repressed at low O₂ (1 to 2% [vol/vol]) and totally repressed under anoxic conditions. The repressive effect of the mineral salts was traced to its copper content. In cells exposed to 1% (vol/vol) O₂, CuSO₄ (0.5 µM) repressed 1-butanol-dependent induction of β-galactosidase activity. Under oxic conditions (20% O₂ [vol/vol]), significantly higher concentrations of CuSO₄ (2 µM) were required for almost complete repression of induction in lactate-grown cells. A combination of the Cu²⁺ reducing agent Na ascorbate (100 µM) and CuSO₄ (0.5 µM) repressed the induction of β-galactosidase activity under oxic conditions to the same extent that 0.5 µM CuSO₄ alone repressed it under anoxic conditions. Under oxic conditions, 2 µM CuSO₄ repressed induction of the BMO promoter less effectively in butyrate-grown cells of the bmoX::lacZ strain and of an R8-bmoX::lacZ mutant reporter strain with a putative BMO regulator, BmoR, inactivated. Under anoxic conditions, CuSO₄ repression remained highly effective, regardless of the growth substrate, in both BmoR-positive and -negative reporter strains.

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A model has been developed for the regulation of butane monooxygenase (BMO) in the C₂-C₉ alkane-utilizing bacterium Pseudomonas butanovora (6-8, 23, 24). The salient features of this model are as follows. (i) BMO transcription is up-regulated by alcohol, aldehyde, and epoxide products of alkane and alkene oxidation (7, 8, 24). (ii) Recently, a gene (bmoR) upstream of the bmo operon that is involved in induction of BMO by alcohols of chain length C₂ to C₈ was identified (15). (iii) BMO transcription is down-regulated in response to the presence of the organic acid propionate, a downstream product of odd-chain-length alkane oxidation (8). (iv) Both even- and odd-chain-length organic-acid products of alkane oxidation, e.g., propionate and butyrate, are potent in vivo inhibitors and inactivators of the BMO enzyme in the absence of alkane substrates (6). The net result is the close coupling of alkane oxidation to the cells' demands for C and energy and the ability of the cell to consume the products of alkane oxidation.

Under anoxic conditions, P. butanovora can utilize various alcohols, aldehydes, and organic acids for growth using nitrate as a terminal electron acceptor (D. M. Doughty, D. J. Arp, and P. J. Bottomley, unpublished data). Because the activity of the BMO enzyme is dependent upon O₂ as a cosubstrate for catalytic activity, the induction of BMO in response to alcohols and aldehydes under anoxic conditions would be superfluous. Our studies were extended to determine if alcohol induction of BMO was overridden in the absence of O₂. During the course of the work, 1-butanol-dependent induction of BMO transcription was found to be unaffected when aerobically grown cells were suspended in buffer and transitioned into low-O₂ conditions. In contrast, when cells were resuspended in the complete mineral salts of standard growth medium under low-O₂ conditions, BMO induction was repressed. Because of the novelty of this observation, the objectives of this study were (i) to identify the component(s) of the mineral salts causing the repressive effect, (ii) to determine if the effect was redox influenced, and (iii) to eliminate the possibility that the repression was due to metal toxicity. Subsequently, copper ions were identified as the causative agent of the repressive effect. Because copper ions are involved in repression of the soluble form of methane monooxygenase (sMMO) in methanotrophs (4, 11, 19, 22), and since sMMO is structurally and biochemically similar to BMO (3, 13, 26), our findings are discussed in the context of a recently published model that links the role of copper ions in sMMO expression with the redox state of a regulatory protein, MmoS (28).

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Bacterial strains, growth conditions, and routine assays. P. butanovora (ATCC 43655) is a gram-negative bacterium that grows on n-alkanes C₂ to C₉ (8, 27) and is closely related to the betaproteobacterial genera Azoarcus and Thauera (1). Liquid cultures were grown as previously described (7), with NH₄Cl (2 mM) supplied as an N source and KNO₃ (30 mM) added where indicated to serve as the electron acceptor. For anoxic growth, the medium was autoclaved, degassed by aspiration, flushed with N₂ for 30 min to remove residual O₂, and autoclaved a second time for sterilization. When organic acids or alcohols served as C sources for growth, concentrations were balanced to 12 mM carbon equivalents, sufficient to support the growth of P. butanovora to an optical density at 600 nm (OD₆₀₀) of 0.6 (0.3 mg protein·ml^–1).

Determination of induction and repression of β-galactosidase expression in lacZ transcriptional reporter strains of P. butanovora. The P. butanovora bmoX::lacZ::kan strain contains a bicistronic expression system in which kanamycin resistance is constitutive and the BMO promoter controls the expression of β-galactosidase activity (24). bmoR, a gene encoding a putative transcriptional regulator, was studied by mutational analysis and was determined to be involved in the induction of the BMO promoter (15). A BmoR-deficient variant of the bmoX::lacZ::kan strain (an R8-bmoX::lacZ strain) was constructed (15). Although β-galactosidase activity was not induced by alcohols in either the acetate or lactate-grown R8-bmoX::lacZ strain, when the R8-bmoX::lacZ strain was grown on C₃, C₄, or C₅ fatty acids to repress BMO expression, β-galactosidase was induced by C₂-C₅ alcohols. These data imply that a second BMO regulatory system exists in P. butanovora, albeit one that is expressed only during growth on propionate, butyrate, or valerate. Experiments were carried out to determine to what extent BmoR-dependent and -independent induction of the BMO promoter was sensitive to CuSO₄. Both of the lacZ reporter strains (bmoX::lacZ and R8-bmoX::lacZ strains) were grown on organic acids or alcohols under the same conditions described for the wild-type cells. Induction assays were performed as described previously (5), with the modification that the cells were resuspended either in 50 mM KNa phosphate-2 mM NH₄Cl, pH 7.1; in 50 mM KNa phosphate-2 mM NH₄Cl, pH 7.1, plus the complete mineral salts of standard growth medium; or in 50 mM KNa phosphate-2 mM NH₄Cl, pH 7.1, plus individual trace metal salts of the growth medium. Preliminary experiments were conducted to ensure that the trace metal salts tested for BMO repression were not inhibitory to β-galactosidase activity at the concentrations tested. For experiments carried out under subambient O₂ concentrations, O₂ was flushed from 10-ml vials with N₂ (30 ml/s for 30 s). Anoxic growth medium or buffer was prepared as described above, and aliquots (1 ml) were transferred with an airtight syringe to induction vials. Anoxic solutions of the individual trace element compounds used in the growth medium (MnCl₂·4H₂O, FeSO₄·7H₂O, CoSO₄·7H₂O, NiCl₂·6H₂O, CuSO₄·5H₂O, and ZnSO₄·7H₂O) and others not used in the growth medium (CdCl₂, HgCl₂, AgCl, and PtCl₂) were prepared as 100 µM stock solutions and made anoxic by flushing them with N₂. The salt solutions were transferred into assay vials using an airtight syringe. Specific amounts of O₂ were added to each vial, and the values are expressed as percent O₂ (volume per total volume of headspace in the vial). A high concentration (approximately 10 mg protein·ml^–1) of washed cells was resuspended in 50 mM KNa phosphate buffer, pH 7.1, and was made anoxic by flushing them with N₂. Aliquots of the concentrated cell suspension (5 µl) were injected into the induction vials to a final OD₆₀₀ of 0.2 (0.1 mg protein·ml^–1). Additional replicates of low-O₂ and anoxic treatments were set up to confirm the presence or absence of O₂ at the beginning and end of each experiment by the methylene blue oxidation assay (18). A series of experiments were conducted to assess whether the effect of CuSO₄ under oxic conditions could be enhanced by the addition of the Cu²⁺ reducing agent ascorbic acid (20, 30). Stock solutions of 10 mM Na ascorbate were prepared daily in KNa phosphate buffer and used at a final concentration of 100 µM.

Induction of propionate consumption in lactate-grown P. butanovora. The effects of CuSO₄ and O₂ on the induction of propionate metabolism were examined. Cells grown under oxic conditions in the presence of lactate were harvested, washed three times, resuspended in NH₄Cl-KNa phosphate buffer, and made anoxic by flushing them with N₂. The cells were injected into 160-ml vials containing 50 ml of anoxic NH₄Cl-KNa phosphate buffer. O₂ (1% [vol/total volume of headspace]) was added to the headspace of the vials, and CuSO₄ (0.5 µM) and rifampin (40 µg·ml^–1) were added as indicated. Propionate consumption was induced by the addition of propionate (2 mM, final concentration). To confirm that propionate metabolism was not induced in the absence of propionate, control vials that did not receive propionate were included. Following 2 h of incubation, the cells were harvested and washed three times in phosphate buffer, and propionate consumption was determined as described previously (8, 13).

Determination of the effect of the CuSO₄ concentration on O₂ consumption in lactate-grown P. butanovora. Endogenous and 1-butanol-dependent O₂ uptake were followed using a Clark style O₂ electrode in a glass reaction vial (YSI model 5300 Biological Oxygen Monitor; Yellow Springs Co., Yellow Springs, OH) as described previously (7). Cells were grown aerobically on lactate, harvested, and resuspended in NH₄Cl-KNa phosphate buffer containing either 0, 0.1, 0.3, 1, 3, 10, or 30 µM CuSO₄. After 2 h, the cells were washed, resuspended in buffer without CuSO₄, and assayed for lactate-dependent O₂ consumption.

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Repression of β-galactosidase in the bmoX::lacZ reporter strain of P. butanovora during growth on 1-butanol under nitrate-respiring conditions. Preliminary data were obtained showing that P. butanovora grown under oxic conditions on 1-butanol as the sole C source expressed BMO activity approximately 20% that of butane-grown cells (data not shown). In contrast, BMO activity was not detected in cells grown under anoxic conditions on 1-butanol as the C source and with KNO₃ as an electron acceptor (data not shown). Because these data do not distinguish an effect of anoxic growth on transcription of the bmo operon from conditions unfavorable for the development of functional BMO per se, a transcriptional bmoX::lacZ reporter strain was used to study the effect of anoxia on transcription of the bmo operon. β-Galactosidase activity was detected at high levels in the bmoX::lacZ reporter strain during oxic growth on 1-butanol and was not significantly affected by the presence or absence of nitrate (Fig. 1A and B). In contrast, β-galactosidase activity was much lower during growth on 1-butanol under anoxic conditions with KNO₃ supplied as the electron acceptor (Fig. 1C). These data show that 1-butanol induction of bmo transcription is repressed during growth of P. butanovora under anoxic denitrifying conditions and that KNO₃ does not repress β-galactosidase under oxic conditions.

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FIG. 1. The effects of O2 and KNO3 on expression of β-galactosidase activity in the bmoX::lacZ reporter strain of P. butanovora during growth on 1-butanol. The open symbols represent growth measured by OD600, and the closed symbols represent β-galactosidase activity expressed in Miller units. (A) bmoX::lacZ reporter strain grown under oxic conditions on 1-butanol (3 mM) in standard growth medium. (B) bmoX::lacZ reporter strain grown under oxic conditions on 1-butanol (3 mM) with KNO3 (30 mM) added to the growth medium. (C) bmoX::lacZ reporter strain grown on 1-butanol (3 mM) under anoxic conditions with KNO3 (30 mM) added to the growth medium. Inocula were grown under the conditions described for each panel. The data points are representative of the means of three replicates, and the error bars are proportional to the standard deviations of the means.

Effect of percent O₂ on 1-butanol-dependent β-galactosidase expression in the bmoX::lacZ reporter strain. The sensitivity of 1-butanol-dependent induction of β-galactosidase to O₂ levels was explored. When cells of the lactate-grown bmoX::lacZ reporter strain were resuspended in NH₄Cl/KNa phosphate buffer with or without KNO₃, the level of 1-butanol-dependent induction of β-galactosidase activity did not change significantly as O₂ decreased from 20% to 0%; the induction of the BMO promoter was reduced somewhat by the presence of KNO₃ at 2% O₂ (Fig. 2A and B). When cells were resuspended in the phosphate-buffered complete mineral salts of standard growth medium, there was a striking reduction in the expression of β-galactosidase activity in the absence of O₂ regardless of the presence or absence of KNO₃ (Fig. 2C and D). These data suggest that transcriptional repression of the bmo promoter under anoxic conditions requires a component of the growth medium and that low O₂ concentrations per se, with or without the presence of KNO₃, were insufficient to cause transcriptional repression.

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FIG. 2. Influences of buffer and growth medium mineral salts solution on the effects of percent O2 and KNO3 on 1-butanol-dependent induction of β-galactosidase activity in the bmoX::lacZ reporter strain of P. butanovora. Lactate-grown cells were washed and resuspended in NH4Cl-NaK phosphate buffer (A), NH4Cl-NaK phosphate buffer plus 30 mM KNO3 (B), NH4Cl-NaK phosphate-buffered complete mineral salts solution (C), and NH4Cl-NaK phosphate-buffered complete mineral salts solution plus 30 mM KNO3 (D). The values represent the means of three replicates, and the error bars represent the standard deviations of the means.

Effect of transition metals on 1-butanol-dependent induction of BMO at 20% and 1% O₂. Preliminary experiments pointed to the trace element mixture of the growth medium being responsible for the repression effect. We examined the effects of individual transition metal components of the growth medium on 1-butanol-dependent induction of β-galactosidase activity at low (1%) and ambient (20%) O₂ levels (Table 1). Although CuSO₄ (0.5 µM) had no effect on 1-butanol-dependent induction of β-galactosidase at ambient O₂, induction was reduced to 25% of the buffer control at 1% O₂. However, CuSO₄ exerted a repressive effect of similar magnitude under oxic conditions when its concentration was raised to 2 µM (Fig. 3A). Other transition metals included in the growth medium did not significantly enhance or diminish the ability of 1-butanol to induce BMO transcription under either O₂ level. An analysis of other transition metals that are not components of the growth medium revealed that 0.5 µM AgCl was as effective as CuSO₄ at repressing β-galactosidase induction (data not shown). Repression by Ag¹⁺ was observed only under ambient O₂ and not under low-O₂ conditions; Zn²⁺, Cd²⁺, Hg²⁺, and Pt²⁺ had no effect on induction. All subsequent experiments were limited to CuSO₄, since it is an essential component of the standard growth medium.

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TABLE 1. Effects of percent O2 and transition metals on 1-butanol-dependent induction of β-galactosidase activity in lactate-grown cells of the bmoX::lacZ reporter strain of P. butanovora

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FIG. 3. Effects of O2 and CuSO4 on 1-butanol-dependent induction of β-galactosidase activity in lactate-grown (A and C) and butyrate-grown (B and D) bmoX::lacZ and R8-bmoX::lacZ reporter strains of P. butanovora. Experiments were conducted in NH4Cl-KNa phosphate buffer in the presence of 20% O2 (closed symbols) or 1% O2 (open symbols). (A and B) The lactate- and butyrate-grown bmoX::lacZ reporter strains, respectively. (C and D) The lactate- and butyrate-grown BmoR-deficient R8-bmoX::lacZ reporter strains, respectively. The data points are the means of three replicates, and the error bars represent the standard deviations of the means.

Effects of ascorbate and CuSO₄ on 1-butanol-dependent induction of β-galactosidase in the bmoX::lacZ reporter strain under oxic conditions. We examined the effect of the Cu²⁺ reducing agent ascorbate on 1-butanol-dependent β-galactosidase induction in the lactate-grown bmoX::lacZ reporter strain under oxic conditions. In NH₄Cl-KNa phosphate buffer, 1-butanol induced β-galactosidase to similar levels (300 Miller units) in the presence or absence of 100 µM Na ascorbate. In contrast, when CuSO₄ (0.5 µM) was mixed with 100 µM Na ascorbate and added to the induction assay, β-galactosidase activity was induced by 1-butanol to 18% of the level (55 Miller units) that was observed in the absence of ascorbate. These data show that the Cu²⁺ reducing agent Na ascorbate enhanced the sensitivity of β-galactosidase repression to CuSO₄ under oxic conditions.

Expression of a substrate-dependent, BmoR-independent BMO regulatory system influences the sensitivity of 1-butanol-dependent induction of β-galactosidase to CuSO₄ repression under oxic conditions. As mentioned above, we recently identified a putative ⁵⁴ factor (BmoR) that was hypothesized to be involved in expression of the bmo operon (15). Although β-galactosidase activity could not be induced by 1-butanol in lactate-grown cells of a BmoR-deficient variant of the bmoX::lacZ reporter strain (the R8-bmoX::lacZ strain), 1-butanol partially induced β-galactosidase activity in the R8-bmoX::lacZ strain after growth on propionate, butyrate, or valerate (15). These data suggested the existence of a second, BmoR-independent BMO regulatory system. In this work, we describe further studies with the two lacZ reporter strains (the bmoX::lacZ and R8-bmoX::lacZ strains) that illustrate that the 1-butanol-dependent induction of BMO in butyrate-grown cells is less sensitive to repression by CuSO₄ than in lactate-grown cells, regardless of whether BmoR is functional or inactive. As shown in Fig. 3A, CuSO₄ (2 µM) almost completely repressed 1-butanol-dependent induction of β-galactosidase under oxic conditions in lactate-grown cells of a BmoR-positive bmoX::lacZ strain (Fig. 3A). In butyrate-grown cells, however, about 50% of the 1-butanol-induced β-galactosidase activity (180 to 200 Miller units) was insensitive to repression by up to 2 µM CuSO₄ (Fig. 3B). An identical experiment was conducted with the BmoR-deficient R8-bmoX::lacZ lacZ reporter strain (Fig. 3C and D). In the case of lactate-grown cells, β-galactosidase activity was not induced by 1-butanol regardless of the presence or absence of CuSO₄ and confirmed the essentiality of BmoR for the induction of lactate-repressed cells (Fig. 3C). In the case of butyrate-grown cells, however, 1-butanol induced β-galactosidase activity to about one-half of the level (250 Miller units) induced in the BmoR-positive bmoX::lacZ reporter strain under oxic conditions (Fig. 3B versus D), and about 60 to 75% of this activity (150 to 180 Miller units) was insensitive to repression by 2 µM CuSO₄ (Fig. 3D). In contrast, under 1% O₂, 1-butanol-dependent induction of β-galactosidase activity in butyrate-grown cells of both the BmoR-positive (Fig. 3B) and the BmoR-deficient (Fig. 3D) reporter strains was completely or almost completely repressed by CuSO₄. These data suggest that the BmoR-independent regulation of the BMO promoter is less sensitive to CuSO₄ under oxic conditions but remains quite sensitive to 0.5 µM CuSO₄ under low-oxygen or anoxic conditions. The lack of CuSO₄-resistant induction in the lactate-grown bmoX::lacZ reporter strain (Fig. 3A) implies that the BmoR-independent system is not expressed during growth on lactate. At this time, it is unknown if both BMO regulatory systems are expressed in butyrate-grown cells.

Effects of CuSO₄ on growth, O₂ consumption, and the induction of propionate consumption in lactate-grown P. butanovora. Several experiments were conducted to determine if CuSO₄ repression of 1-butanol-dependent induction of BMO might be caused by general copper toxicity on P. butanovora. Generally, there was no evidence of copper toxicity at concentrations that are effective in the repression of BMO. First, although P. butanovora did not grow in medium without supplemental CuSO₄, it grew on butyrate as a sole C source in the presence of 2 µM supplemental CuSO₄ at a rate comparable to that achieved in normal growth medium (0.2 µM CuSO₄). Second, endogenous and 1-butanol-dependent O₂ consumption rates were both unaffected by exposure of cells to CuSO₄ at 2 µM. Third, CuSO₄ (2 µM) had no significant inhibitory effect on preexisting BMO activity as determined on butane-grown cells treated with chloramphenicol to stop further protein synthesis. Fourth, the induction of propionate consumption under 1% O₂ was unaffected by the presence of 0.5 µM CuSO₄ (Fig. 4), suggesting that the integrated processes of transcription and translation associated with the development of a catabolic pathway in P. butanovora were insensitive to CuSO₄ at concentrations that affected BMO induction.

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FIG. 4. Effects of CuSO4 and rifampin on the induction of propionate consumption under 1% O2. Lactate-grown cells of wild-type P. butanovora were washed and resuspended in NH4Cl-KNa phosphate-buffered complete mineral salts solution under 1% O2. Propionate (2 mM), rifampin (40 µg/ml), or CuSO4 (0.5 µM) was added as indicated. The values represent the means of three replicates, and the error bars represent the standard deviations of the means.

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The results of this study clearly demonstrate that P. butanovora has a previously uncharacterized ability to repress transcription from the BMO promoter when O₂ is limiting. BMO repression occurs even in the presence of an inducer (e.g., 1-butanol), and copper ions play a critical role. Copper was an effective repressor at low concentrations (<0.2 to 2 µM), similar to those used by researchers of the well-studied "copper switch" phenomenon of copper repression of sMMO in methanotrophs (4, 11, 19, 22), an enzyme genetically, biochemically, and structurally related to BMO (3, 13, 26). The fact that the repressive effect of copper occurs at lower concentrations of copper ions under anoxic than under oxic conditions and that the presence of the Cu²⁺ reducing agent ascorbate enables lower concentrations of copper ions to be effective under oxic conditions indicates that regulation by copper ions is linked to a redox-sensitive system.

Recent research from our laboratory identified a ⁵⁴-type transcriptional regulator, BmoR, that is encoded by a gene upstream of the bmo operon and that is required for complete alcohol-dependent induction of BMO by lactate- and acetate-grown P. butanovora, but not for alcohol-dependent induction by propionate-, butyrate-, or valerate-grown cells (15). Data presented in the study unequivocally show that the copper repression of 1-butanol-dependent induction occurs under both oxic and anoxic incubation conditions, regardless of whether cells are grown on lactate or butyrate and whether or not BmoR is actively involved in the regulation of BMO expression. Clearly, our research is in its infancy, and more work is needed to identify the molecular mechanisms associated with BmoR-dependent and -independent induction of BMO. Interestingly, a substantial percentage of the induced activity is copper insensitive in butyrate-grown cells and an even larger percentage in the absence of a functional BmoR. Given the relatively large errors associated with analyzing β-galactosidase activity in replicate samples, further detailed work is needed to more accurately determine the size of the fraction of activity that is copper insensitive, particularly in the BmoR-deficient mutant.

There is some precedent in the literature for ⁵⁴-type transcriptional regulators to be able to function through redox mechanisms. For example, up-regulation of the nitrogenase (nif) operon in many diazotrophic bacteria under anoxic conditions is controlled by the activity of a ⁵⁴ promoter under the control of the redox-influenced NifA/NifL proteins (5, 14, 16). Recently, Ukaegbu and colleagues proposed a model of copper repression of the sMMO operon in which the relative amounts of the oxidized and reduced forms of a regulatory protein, MmoS, are controlled by the level of Cu²⁺ ions. In the absence of Cu²⁺ ions, the reduced form of MmoS dominates and expresses a kinase activity that phosphorylates MmoR, which then up-regulates the transcription of sMMO (28). Obviously, much more work is needed to rigorously determine if the effect of copper ions on BMO expression follows a model similar to that proposed for sMMO. A particularly interesting aspect of the putative relationship between copper and redox in regulation will be the way in which ascorbate under oxic conditions, or anoxic conditions without ascorbate, can lower the effective concentration of copper ions required for BMO repression. For example, this could be achieved by anoxic conditions facilitating transformation of Cu²⁺ to Cu¹⁺, with the latter being the active ionic species. In this context, two points are worthy of note (1). We showed that Ag¹⁺ is a potent repressor of BMO induction under oxic conditions, and in contrast to copper, Ag¹⁺ is the normal oxidation state of silver found under oxic and anoxic conditions (2). It has been shown that methanobactin, the copper-binding molecule secreted by methanotrophs, binds Cu²⁺, which is subsequently reduced to Cu¹⁺ (2, 12). It remains unclear if Cu¹⁺ plays any role in the regulation of sMMO.

In methanotrophs, copper ions are also involved in up-regulation of genes encoding the copper-containing protein particulate MMO. By analogy, copper might be involved in up-regulating genes encoding copper-containing proteins needed for growth of P. butanovora on alcohols under anoxic nitrate-respiring conditions. For example, there are at least two 1-butanol dehydrogenases in P. butanovora, one of which is a quinohemoprotein (29), and electron transport from the latter type of alcohol dehydrogenase often occurs via a copper-containing protein, azurin (10, 17). A blue redox-sensitive azurin-like protein has been detected in extracts of P. butanovora (L. A. Sayavedra-Soto and D. J. Arp, unpublished data). Furthermore, the copper ion is an essential cofactor in two of the enzymes involved in nitrate respiration (NirK and NosZ) under anoxic conditions (31). Although we do not know if P. butanovora contains a NirK- or NirS-like nitrite reductase, its growth under denitrifying conditions is inhibited by the copper chelator allylthiourea and by acetylene, a potent inhibitor of Cu containing nitrous oxide reductase (NosZ). Finally, we know that cells of P. butanovora grown under denitrifying conditions do not induce BMO in response to a short incubation with alcohols under oxic conditions, indicating that the alcohol-dependent inducing system of BMO is inactivated after anoxic metabolism becomes established (Doughty et al. unpublished). It is known that facultative aerobes, such as Escherichia coli and Pseudomonas aeruginosa, possess molecular regulatory systems that enable them to switch from aerobic to anoxic growth by nitrate respiration under O₂-limited conditions (9, 21, 25). More work is needed to determine how the copper ion repression of BMO induction is linked to the molecular mechanisms of regulation that permit P. butanovora to transition from oxic to anoxic growth conditions and what is involved in its inactivation.

 
This research was supported by grants from the U.S. Environmental Protection Agency-sponsored Western Region Hazardous Substance Research Center under agreement R-828772, National Institutes of Health Grant no. 5RO1 GM56128-06, and the Oregon Agricultural Experiment Station.

We thank K. H. Halsey for insightful discussion, constructive comments, and editing of the manuscript.

 
* Corresponding author. Mailing address: Department of Microbiology, Nash Hall Rm. 220, Oregon State University, Corvallis, OR 97331-3804. Phone: (541) 737-4441. Fax: (541) 737-0496. E-mail: Peter.Bottomley{at}oregonstate.edu

Published ahead of print on 15 February 2008.

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Journal of Bacteriology, April 2008, p. 2933-2938, Vol. 190, No. 8
0021-9193/08/$08.00+0     doi:10.1128/JB.01409-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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