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Journal of Bacteriology, June 2005, p. 4077-4085, Vol. 187, No. 12
0021-9193/05/$08.00+0     doi:10.1128/JB.187.12.4077-4085.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Regulation and Function of Cytochrome c' in Rhodobacter sphaeroides 2.4.3

Peter S. Choi,1,{dagger} Vladimir M. Grigoryants,3 Hector D. Abruña,2 Charles P. Scholes,3 and James P. Shapleigh1*

Department of Microbiology, Wing Hall, Cornell University, Ithaca, New York 14853,1 Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, New York 14853,2 Department of Chemistry, Center for Biophysics and Biochemistry, University at Albany, State University of New York at Albany, Albany, New York 122223

Received 19 November 2004/ Accepted 9 March 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cytochrome c' (Cyt c') is a c-type cytochrome with a pentacoordinate heme iron. The gene encoding this protein in Rhodobacter sphaeroides 2.4.3, designated cycP, was isolated and sequenced. Northern blot analysis and ß-galactosidase assays demonstrated that cycP transcription increased as oxygen levels decreased and was not repressed under denitrifying conditions as observed in another Rhodobacter species. CO difference spectra performed with extracts of cells grown under different conditions revealed that Cyt c' levels were highest during photosynthetic denitrifying growth conditions. The increase in Cyt c' under this condition was higher than would be predicted from transcriptional studies. Electron paramagnetic resonance analysis of whole cells demonstrated that Cyt c' binds NO during denitrification. Mass spectrometric analysis of nitrogen oxides produced by cells and purified protein did not indicate that Cyt c' has NO reductase activity. Taken together, these results suggest a model where Cyt c' in R. sphaeroides 2.4.3 may shuttle NO to the membrane, where it can be reduced.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Rhodobacter sphaeroides expresses a large number of periplasmic c-type cytochromes (6, 24). One of these is cytochrome c' (Cyt c'), a c-type cytochrome with pentacoordinated heme iron. Cyt c' orthologs are found in many bacteria, most of which are photosynthetic and/or denitrifying (1, 42, 43). Due to their unique heme properties, cytochromes c' from many organisms have been structurally characterized through X-ray crystallography (2, 20, 32, 36). Although the sequence similarity between the cytochromes c' is quite low, their overall structure is generally the same (36). Most cytochromes c' exist as a dimer, with each monomer consisting of a left-twisted four-{alpha}-helix bundle (36). The covalent c heme attachment motif, CXXCH, is located near the C terminus of the protein, and the axial sixth position of the heme iron is unoccupied (18, 36). Previous work has shown that Cyt c' can also bind nitric oxide (NO). NO binds to the proximal side of the heme through a six-coordinate intermediate which is distinct from the distal binding site of CO that leads to a six-coordinate complex (2, 18, 19). Cyt c' does not bind oxygen (1, 43).

Some strains of R. sphaeroides are able to reduce nitrate and nitrite to dinitrogen gas through the process of denitrification (25). One of the intermediates generated during denitrification is NO, a potentially toxic gas that can inhibit the growth of bacteria (10, 28). NO has been shown to be a freely diffusible intermediate, which allows it to interact with targets both inside and outside the cell. Nitrite reductase (Nir), the enzyme that generates NO, is located in the periplasm of R. sphaeroides, whereas the enzyme that reduces NO, nitric oxide reductase (Nor), is located in the inner membrane (46). It has been suggested that Nor is the enzyme primarily responsible for minimizing NO accumulation and limiting the impact of NO on cellular targets, although other proteins might also assist in minimizing NO damage (11, 33).

Although many Cyt c' homologues are structurally well characterized, the physiological function of these proteins is unresolved. Electron paramagnetic resonance (EPR) studies that detected the presence of a Cyt c'-NO adduct in several denitrifying bacteria demonstrated that Cyt c' binds denitrification-derived NO in vivo (43, 44). This finding led to the suggestion that Cyt c' is involved in minimizing NO damage during denitrification. More recent results from studies with Rhodobacter capsulatus are consistent with the proposal that Cyt c' protects against NO damage (8, 9). Mutations in cycP, which encodes Cyt c', increased the sensitivity to both S-nitrosoglutathione (GSNO) and free NO (8). Inactivation of cycP also decreased the rate of removal of exogenously provided NO from cultures (8). A similar result has been observed with Neisseria meningitidis (3). However, a catalytic activity has not been directly demonstrated for any purified cytochrome c'.

In R. sphaeroides, expression of Cyt c' is influenced by the presence of terminal oxidants. In the partial denitrifier R. sphaeroides 2.4.1, Cyt c' protein levels were repressed 10-fold under aerobic growth conditions compared to those under anaerobic, photosynthetic growth conditions, suggesting that oxygen levels are a key factor in controlling Cyt c' expression (6). In the denitrifier R. sphaeroides IL-106, Cyt c' was also found at high levels in photosynthetically grown cells, but when nitrate was added to cells grown under these conditions, Cyt c' was undetectable, indicating that nitrate respiration leads to a repression of Cyt c' expression (26).

This study was undertaken to examine how Cyt c' function and expression are related to denitrification in R. sphaeroides 2.4.3. As in other R. sphaeroides strains, expression of the gene encoding Cyt c' increased as oxygen levels decreased. In contrast, denitrifying conditions did not repress Cyt c' expression as observed in IL-106 but instead induced a higher level of Cyt c' expression. Visible and EPR spectroscopy demonstrate that Cyt c' binds NO both in vitro and under denitrifying conditions in vivo. However, no significant NO reductase activity could be attributed to Cyt c'.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and culture conditions. R. sphaeroides 2.4.3 and 2.4.1 are the wild-type denitrifying and partially denitrifying strains, respectively. R213 and R125 are FnrL- and NnrR-deficient strains of 2.4.3, respectively (5, 38). PC12 is a Cyt c'-deficient mutant of 2.4.3. Escherichia coli strains DH5{alpha} and JM109 ({lambda}pir) were used for transformations, and E. coli strains S17-1 and S17-1 ({lambda}pir) were used for conjugations (34).

All Rhodobacter strains were grown in Sistrom's medium (20) at 30°C, and when necessary, antibiotics were added at the following concentrations: tetracycline, 1.0 µg/ml; kanamycin, 25 µg/ml; streptomycin-spectinomycin, each 50 µg/ml; and trimethoprim, 30 µg/ml. For denitrifying conditions, sodium nitrate was added to a final concentration of 12 mM. Photosynthetic cultures for ß-galactosidase assays were grown in 14 ml of Sistrom's medium in 15-ml crimped vials. Photosynthetic cultures used for visible and EPR spectroscopy and gas chromatography-mass spectrometry (GC/MS) analysis were grown in 100 ml or 150 ml of Sistrom's medium in rubber-stoppered 250-ml Erlenmeyer flasks and placed on a rotary shaker approximately 50 cm away from an incandescent light source. In both cases, anaerobic conditions were achieved by relying on the bacteria to consume available oxygen in the medium. For microaerobic growth on solid media, plates were incubated at 30°C in an anaerobic jar that was evacuated and refilled with nitrogen gas two times. All other growth conditions have been previously described (37). E. coli strains were grown aerobically in Luria-Bertani medium at 30°C (22).

Cloning of cycP operon from 2.4.3. All cloned and partially sequenced open reading frames (ORFs) from 2.4.3 were given the same designation as in the annotated 2.4.1 genome, except that an asterisk denotes ORFs from 2.4.3 with the exception of cycP and cybP. cycP from 2.4.3 was amplified by nested PCR. The two degenerate oligonucleotides, FUCIP 5' and PLD3' (Table 1), were designed based on the putative amino acid sequences of two ORFs flanking 2.4.1 cycP and used for PCR amplification with 2.4.3 genomic DNA. Five microliters of the resultant amplification was used as a template with the two degenerate oligonucleotides cytoc'5' and cytoc'3' in a second round of PCR amplification (Table 1). The resultant 353-bp PCR product was cloned blunt end into the HincII site of pUC19 and sequenced to verify its identity as a portion of cycP from 2.4.3.


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  		TABLE 1. Oligonucleotides used in this studya

 
The remainder of cycP and its flanking genes were isolated from 2.4.3 by plasmid rescue. To do this, an internal portion of 2.4.3 cycP was amplified by PCR using the oligonucleotides c'5'X and c'3'E (Table 1). The resultant 353-bp PCR product was cloned into the EcoRI and XbaI sites of the suicide vector pJP4603 (30) to yield plasmid pPC002, which was transformed into S17-1 ({lambda}pir) and subsequently conjugated into 2.4.3. A Kmr ex-conjugant in which pPC002 had integrated into the genome was selected and designated PC3. The plasmid was rescued by digesting genomic DNA of PC3 with BamHI and self-ligating the restriction-digested DNA. This ligation was transformed into JM109 ({lambda}pir), and transformants were screened for Kmr. The rescued plasmid, designated pPC003, contained a BamHI fragment with approximately 2.7 kb of DNA upstream of cycP and 710 bp of DNA downstream of cycP (Fig. 1A). The 2.7-kb fragment was cloned into the EcoRI and HindIII restriction sites of pUC19 and designated pPC004. The 1.1-kb fragment was cloned into the EcoRI and BamHI sites of pUC19 and designated pPC004.5. Both constructs were partially sequenced.



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  		FIG. 1. (A) cycP region of the 2.4.3 genome. (B) lacZ fusion constructs cloned into pRK415. Color scheme: light grey, sequenced DNA; white, unsequenced DNA; dark grey, lacZ. ORF descriptions: RSP0470*, encodes a conserved hypothetical protein of unknown function; (RSP6116*, encodes a hypothetical protein of unknown function; RSP0472*, encodes a conserved hypothetical protein of unknown function; RSP0473*, encodes a hypothetical transmembrane protein similar to cardiolipin synthase/phospholipase D; cycP, encodes cytochrome c'; cybP, encodes a putative membrane-bound b-type cytochrome of unknown function. Dark box indicates location of putative FNR binding site. Restriction sites: E, EcoRI; B, BamHI.

 
Additional DNA upstream of the 2.7-kb fragment was amplified by PCR with the oligonucleotides uma1 and c'7 (Table 1). The resultant product, which contains a naturally occurring BamHI, site was cloned into EcoRI and BamHI sites of pUC19 and sequenced (41).

Construction of lacZ fusions and ß-galactosidase assays. A PCR product containing 207 bp of intergenic DNA upstream of 2.4.3 cycP and the first 234 bp of the cycP coding region was amplified using oligonucleotides 243c'up5.3 and 243c'3'2up (Table 1). This product was cloned into the BamHI and KpnI sites of pRK415 (13). A lacZ cassette (Kmr) from pKOK6 (14) was then ligated into the BamHI site to create pC'LACZ1 (Fig. 1B). pC'LACZ1 was conjugated into 2.4.3 for use in ß-galactosidase assays.

The PCR product generated by the oligonucleotides uma1 and c'7 was digested with EcoRI and BamHI and ligated into the corresponding sites in pPC004 to yield pPC009. The 3.6-kb insert of pPC009 was ligated into the EcoRI and XbaI sites of pRK415. A lacZ cassette from pKOK6 was then ligated into the SalI site to create pC'LACZ4 (Fig. 1B). pC'LACZ4 was then conjugated into 2.4.3 and R213 for ß-galactosidase assays.

ß-Galactosidase activities were determined for at least three independently grown cultures as previously described (22). Reported values are the average of three measurements, and error bars represent one standard deviation.

Construction of Cyt c'-deficient mutants. cycP was disrupted with an Smr/Spr cassette through a double-crossover event between the 2.4.3 chromosome and the construct pPC008, which was constructed by taking the amplicon used to construct pC'LACZ1 (Fig. 1B) and cloning it into the KpnI and BamHI sites of pUC19 to yield pPC005. The oligonucleotides c'3 and M13 reverse were used to amplify a product, which contained the last 147 bp of cycP and all of cybP (Table 1). A naturally occurring PstI site in the product and the BamHI site in the oligonucleotide were used to clone this product into the PstI and BamHI sites of pPC005 to yield pPC006. An Smr/Spr cassette from pHP45{Omega} (31) was then ligated into the BamHI site in pPC006 between the upstream portion of cycP and the downstream portion of cycP to yield pPC007. The DNA fragment containing the disrupted cycP gene was isolated by digestion of pPC007 with KpnI and PstI and ligated into the suicide vector pSUP202 (Tcr) (34) to yield pPC008, which was conjugated into 2.4.3. Mutants in which a double-crossover event led to an insertional inactivation of cycP in the 2.4.3 chromosome were isolated by selecting for exconjugants that were Smr/Spcr and Tcs. Correct insertion of the Smr/Spcr cassette was verified by Southern blot analysis (data not shown).

His-tag fusion construction and purification of His-tagged protein. The coding region of cycP was amplified by PCR with the oligonucleotides c'8 and cycPhis (Table 1). The resultant PCR product has six His codons and a stop codon attached to the 3' end of cycP. This PCR product was digested with EcoRI and BamHI and ligated into pYSW35 (17) to yield pC'HIS, which has the amplicon encoding the six-His-tagged Cyt c' (c'His) fused to the rrnB promoter. The 2.4.3 strain containing pC'HIS was designated C'HIS. For purification of c'His, C'HIS was grown aerobically in unmodified Sistrom's medium. The medium was centrifuged at 10,000 x g to pellet the cells, which were then washed in 100 ml of 20 mM sodium phosphate, pH 7.0, and resuspended in 50 ml of the same buffer. C'HIS cells were disrupted by passage through a French pressure cell at 1,280 lb/in2. High-speed supernatants of the extracts were obtained by an initial centrifugation for 30 min at 17,000 x g followed by a centrifugation for 2 h at 250,000 x g to pellet the membranes. The c'His in the high-speed supernatants was purified on a nickel-nitrilotriacetic acid agarose column according to the manufacturer's protocol.

Cyt c' overexpression construct. To create a 2.4.3 strain that overexpressed non-His-tagged Cyt c', oligonucleotides c'8 and c'9 were used to amplify cycP. The resultant PCR product was cloned into the EcoRI and BamHI sites of pYSW35 to yield pC'89, which was conjugated into 2.4.3 to yield strain C'89. The fusion of cycP to the rrnB promoter allows for the high expression of non-His-tagged Cyt c' in this strain.

RNA isolation and analysis. RNA was isolated from 10-ml cultures of 2.4.3 using an RNeasy Mini kit (QIAGEN) and quantified by UV spectrophotometry. The probe was a 353-bp internal fragment of cycP amplified by PCR using the oligonucleotides c'5'X and c'3'E. The probe was labeled with [{alpha}-32P]dCTP (ICN) using the Megaprime DNA labeling system (Amersham Biosciences). RNA samples containing 20 µg/ml ethidium bromide (EtBr) were run on a 0.8% agarose gel according to the manufacturer's protocol (Ambion). RNA was blotted onto Genescreen Plus membranes (Perkin-Elmer Life Sciences) according to the manufacturer's protocol. As a loading control, EtBr-stained rRNA was visualized by exposure to UV light subsequent to blotting. Blots were hybridized with the cycP probe in a 125-ml hybridization tube in 3.0 ml ULTRAhyb (Ambion) at 42°C for 18 h in a rotary oven. Northern blots were then washed as previously described (22) and visualized on a STORM phosphorimager (Molecular Dynamics).

Visible spectroscopy. Protein samples for spectroscopy were prepared from 100-ml or 150-ml overnight R. sphaeroides cultures grown under aerobic, microaerobic, or photosynthetic conditions. Cells were harvested by centrifugation for 10 min at 10,000 x g at 4°C, washed in 50 ml 20 mM sodium phosphate, pH 7.0, containing 1 mM EDTA, and resuspended in 10 ml of the same buffer. The cells were then disrupted by passage through a French pressure cell at 1,280 lb/in2. High-speed supernatants were obtained as described for purification of c'His. Solid ammonium sulfate was added to the supernatants to 55% saturation, and then the extracts were centrifuged for 10 min at 10,000 x g at 4°C to remove the precipitated proteins. The ammonium sulfate concentration was then raised to 85% saturation, and the precipitated proteins were pelleted by centrifugation at 10,000 x g and resuspended in 20 mM sodium phosphate and the pH adjusted to 7.5. Visible spectra were obtained using 0.8 ml of extract adjusted to 2.6 mg protein/ml using a DU 640 spectrophotometer (Beckman). CO difference spectra were obtained by scanning samples reduced with dithionite from 400 to 700 nm. Samples were then bubbled with CO for 2 to 5 min and scanned again. Difference spectra were calculated by subtracting the absorbance of dithionite-reduced samples from the absorbance of CO-reduced samples. The same procedure was used to perform difference spectra with purified c'His. The procedure for NO difference spectra with c'His was identical to that for CO difference spectra except that the sample was bubbled with NO.

GSNO disk assays. R. sphaeroides strains were plated on Sistrom's medium, pH 6.8. GSNO was synthesized from acidified nitrite and glutathione as previously described (12). Fifteen microliters of 500 mM GSNO was spotted onto a Whatman paper disk, 7 mm in diameter. The GSNO-saturated disks were placed in the center of the plated cultures and incubated at 18 h under microaerobic conditions.

Growth curves. R. sphaeroides strains were grown to stationary phase microaerobically in Sistrom's medium and diluted into 100 ml of fresh Sistrom's medium to yield a final optical density at 600 nm (OD600) of 0.25. Cultures were amended with 2 mM GSNO and incubated microaerobically for 8 h at 30°C. Samples were removed for OD600 measurements at each hour. Reported values are the average for three independent experiments.

DMS. Differential mass spectrometry (DMS) is a variation of the technique differential electrochemical mass spectrometry, which was initially developed by Bruckenstein (7) and modified by Wolter (40). The apparatus used in this work has been recently described in detail by Smith et al. (35). Differential pressures facilitate the diffusion of gaseous or volatile compounds across a Teflon membrane, which allows for detection of these compounds by a quadrupole mass spectrometer. In this study, the ions with m/z 30 and 44, which correspond to NO and N2O, respectively, were continuously monitored. For real-time DMS with 2.4.3 high-speed supernatants, 2.4.3 cells grown microaerobically with nitrate were centrifuged at 10,000 x g, washed in 50 ml Sistrom's medium and resuspended in 10 ml 20 mM phosphate buffer, pH 7.0. High-speed supernatants were produced as previously described. For these experiments, 66 µg of 2.4.3 high-speed supernatant was mixed with 0.5 mM nitrite in the DMS cell. To initiate Nir activity, ascorbate and phenazine methosulfate (PMS) were provided at final concentrations of 10 mM and 0.5 mM, respectively, at scan 100. At scan 250, 31 µg of c'His was then added, and NO levels were monitored for another 250 scans.

Gas chromatography/mass spectrometry analysis. Cells used in these experiments were from 100-ml cultures of 2.4.3 and PC12 grown photosynthetically with nitrate that had been concentrated by centrifugation and resuspended in 10 ml fresh Sistrom's medium to a final OD600 of 2.0 in crimped 15-ml vials. Samples were then incubated at 30°C for 1 h. Analysis of 100 µl of headspace samples was carried out using a Hewlett-Packard 5971A GC/MS equipped with a Hewlett-Packard Pora Plot Q column (25 m by 0.32 mm, 10-µm film thickness with He as the carrier gas). Splitless mode was used to separate gaseous components. The detector was operated at 1 x 10–5 torr, 70 eV (39). The GC oven was isothermal 100°C. Nitrous oxide (N2O) (m/z 44) was found to elute at 1.45 min, while CO2 (m/z 44) eluted at 1.39 min. Detection of the fragmentation ion of m/z 30 allowed for identification of which peak corresponded to N2O. N2O peak areas were integrated for comparison of N2O production by various R. sphaeroides cultures. Reported values are the average for 54 samples.

Whole-cell EPR analysis. For these experiments, 100-ml cultures of 2.4.3, PC12 and C'89 were grown under photosynthetic denitrifying conditions or aerobically in Sistrom's medium. Cultures were centrifuged and washed in 50 mM phosphate buffer, pH 7.0. Concentrated cells were packed into 3-mm-inside-diameter, 4-mm-outside-diameter quartz Wilmad tubes by centrifugation at 1,750 x g to yield a cell suspension of approximately 0.2 ml containing roughly 2.5 x 1011 bacteria. Samples were immediately frozen in liquid nitrogen and stored at –70°C. For ferric cytochrome detection, samples were thawed, supplemented with 2.5 mM ferricyanide, and refrozen (27).

The EPR system was a Bruker ER-200 D-SRC X-band spectrometer interfaced to a Gateway PC equipped with an IBM analog-to-digital converter and Scientific Services Systems (Bloomington, IL) EW 2.4A software for collecting and averaging EPR data. The EPR frequency was 9.51 GHz, and the magnetic field was swept from 500 to 4,500 G in 100 s with each spectrum the result of two such sweeps. The microwave power was 2.0 mW, and the field modulation was 9.5 G. Experiments were performed at 15 K. An EPR baseline obtained from an EPR tube containing 200 µl of frozen buffer was subtracted from each sample trace.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of the cycP region in 2.4.3. A 3.6-kb fragment of DNA from 2.4.3 containing the cycP region was isolated and partially sequenced (Fig. 1A). Comparison of the partial sequence of this segment of DNA shows that the order of genes in the cycP region in 2.4.3 is identical to that in the genome of 2.4.1 (http://genome.ornl.gov/microbial/rsph/). The deduced protein products of cycP and cybP, which encode for Cyt c' and a putative membrane-bound b-type cytochrome hydrogenase, respectively, are 86% and 75% identical to their orthologs in 2.4.1. The deduced amino acid sequence of 2.4.3 cybP contains four predicted transmembrane segments, suggesting that CybP is an integral membrane protein. cycP and cybP are separated by 6 bp and therefore are most likely cotranscribed. The pairing of these genes is a common feature in the genomes of other bacteria, which suggests that Cyt c' and the b-type cytochrome may form an abbreviated electron transport chain.

Immediately upstream of cycP in R. sphaeroides 2.4.1 are RSP6116, RSP0472, and RSP0471, which are transcribed in the same direction as cycP. The order and transcriptional orientation of these ORFs are conserved between 2.4.3 and 2.4.1. RSP6116* is upstream of cycP, and its predicted product is 63% identical to its 2.4.1 ortholog. The first 411 bp of the next ORF, RSP0472*, is 86% identical to the equivalent region of the 2.4.1 chromosome. RSP0472 is predicted to encode a conserved metal-dependent hydrolase of unknown function. The final 80 bp of RSP0473*, which encodes a phospholipase D/cardiolipin synthase, was sequenced and is 90% identical to the equivalent region in 2.4.1.

Analysis of cycP transcription using promoter fusions. Two reporter fusion constructs were made to analyze cycP transcription. The first was pC'LACZ1, which fused lacZ to 207 bp of DNA upstream of the putative translation start of cycP (Fig. 1B). The second was pC'LACZ4, in which lacZ was fused to a DNA fragment that begins 277 bp upstream of RSP6116* and terminates within the cycP ORF (Fig. 1B). In a wild-type background, ß-galactosidase expression from pC'LACZ1 increased about four to fivefold under microaerobic conditions compared to that under aerobic conditions (Fig. 2A). Growth under illuminated, anaerobic conditions induced 10- to 15-fold-higher transcription of cycP than under aerobic conditions. Interestingly, the highest ß-galactosidase activity from pC'LACZ1 was obtained in cells grown under illuminated, anaerobic conditions in medium containing nitrate (Fig. 2A). Like pC'LACZ1, pC'LACZ4 facilitated higher levels of ß-galactosidase activity under microaerobic and illuminated, anaerobic conditions, with the highest level of expression occurring in nitrate-supplemented medium under illuminated, anaerobic conditions (Fig. 2B).



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  		FIG. 2. lacZ fusion analysis of cycP transcription. (A) ß-Galactosidase activity of pC'LACZ1 in 2.4.3 and R213 (FnrL–) backgrounds grown under various conditions. (B) ß-Galactosidase activity of pC'LACZ4 in 2.4.3 and R213 (FnrL–) backgrounds grown under various conditions. Microaerobic/N and photosynthetic/N indicate cultures grown in media supplemented with nitrate.

 
The increase in cycP expression in response to decreasing oxygen concentrations suggests that a regulatory protein sensitive to changes in oxygen tension controls cycP expression. FnrL has been shown to activate the expression of a number of genes in R. sphaeroides in response to a lower oxygen concentration (45). To determine if cycP expression is under the control of FnrL, both lacZ fusion constructs were moved into the FnrL-deficient strain R213. In an R213 background, ß-galactosidase activity produced by pC'LACZ1 was two- to sixfold lower than in a 2.4.3 background (Fig. 2A). Similarly, pC'LACZ4 in a 213 background produced four to fivefold-lower ß-galactosidase activity than in 2.4.3 (Fig. 2B). These results demonstrate that cycP transcription is FnrL dependent under microaerobic growth conditions. Since the FnrL-deficient mutant cannot grow photosynthetically, ß-galactosidase activity could not be measured under this growth condition.

Northern blot analysis of cycP transcription. The pattern of cycP transcriptional regulation observed with both lacZ fusion constructs is inconsistent with previous studies with R. sphaeroides IL-106, which does not express Cyt c' under photosynthetic denitrifying conditions (26). In order to confirm that cycP is transcribed under denitrifying conditions in 2.4.3, cycP transcript levels were assessed by Northern blot analysis. In agreement with the results of the ß-galactosidase assays, decreases in oxygen concentrations in the cells increased transcription of cycP (Fig. 3A). Microaerobic conditions increased the level of the transcript about fourfold over that with aerobic conditions. Cells grown under photosynthetic conditions produced approximately ninefold-higher transcript levels than aerobically grown cells. The addition of nitrate did not affect the intensity of the hybridization band significantly (Fig. 3A). The FnrL dependence of cycP transcription was also confirmed by Northern blot analysis. cycP transcription was 2.5- to 5-fold lower in microaerobically grown R213 than in photosynthetically grown 2.4.3 (Fig. 3B).



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  		FIG. 3. Northern blot analysis using a 32P-labeled internal fragment of cycP as a probe. Microaerobic/N and photosynthetic/N indicate cultures grown in media supplemented with nitrate. Twenty micrograms of total RNA was loaded in each lane. Unless noted, all RNA was isolated from wild-type cells. (A) RNA from cells grown under various conditions and the corresponding EtBr-stained 23S rRNA as a loading control. Numbers under each lane represent the fold change in cycP transcription relative to that in aerobic cultures. (B) Comparison of cycP transcript levels between the FnrL-deficient mutant R213, wild type, and PC12 and the corresponding EtBr-stained 23S rRNA as a loading control.

 
Variations in Cyt c' levels. Both ß-galactosidase and Northern analysis demonstrate that cycP expression does not decrease under denitrifying conditions (Fig. 2 and 3). To confirm that Cyt c' is present under denitrifying conditions, Cyt c' protein levels in 2.4.3 were assessed in cells grown under various culture conditions using CO difference spectroscopy. While there are a number of soluble c-type cytochromes expressed by R. sphaeroides, only a few are pentacoordinate. Previous work suggests that Cyt c' is the most highly expressed of these pentacoordinate cytochromes (6), which makes it possible to use CO difference spectroscopy to detect its presence. However, since Cyt c' from 2.4.3 has not been previously characterized, an overexpressed His-tagged version of 2.4.3 Cyt c' (c'His) was purified and examined spectroscopically. The CO difference spectra of c'His were nearly identical to those of purified Cyt c' from IL-106 and showed a maxima at 416 nm and a minimum at 431 nm (Fig. 4A) (26).



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  		FIG. 4. CO or NO difference spectra of purified protein and cell extracts. Spectra were obtained by scanning samples reduced with dithionite from 400 to 700 nm. Samples were then bubbled with CO or NO and scanned again. The difference spectrum for each sample was calculated by subtracting the absorbance of dithionite-reduced samples from the absorbance of CO/NO-reduced samples. (A) CO difference spectra of purified c'His; (B) CO difference spectra of concentrated protein extracts of 2.4.3 cultures grown under aerobic conditions (gray scan), PC12 grown under microaerobic conditions (solid scan), 2.4.3 grown under microaerobic conditions (dashed scan), and 2.4.3 grown under microaerobic conditions in nitrate-supplemented medium (dotted scan); (C) CO difference spectra of concentrated protein extracts of 2.4.3 grown under photosynthetic conditions in unmodified medium (dotted scan) or nitrate-supplemented medium (solid scan); (D) NO difference spectra of purified c'His.

 
To determine the levels of Cyt c' in 2.4.3, the proteins in high-speed supernatants were concentrated using ammonium sulfate precipitation. Concentrated protein samples from Cyt c'-deficient cells and 2.4.3 aerobically grown cells exhibited a maxima at 416 nm but lacked the characteristic minimum at 431 nm (Fig. 4B). This indicates that the CO-absorbing protein in these samples was not Cyt c'. Samples from 2.4.3 cells grown microaerobically and photosynthetically in the presence or absence of nitrate displayed the characteristic CO difference spectra maxima and minima, demonstrating that Cyt c' is expressed in 2.4.3 under these conditions (Fig. 4B and C). The increases in Cyt c' levels as measured by CO difference spectra were consistent with the expression trends observed in cycP transcription with one exception. In both Northern blot analysis and ß-galactosidase assays, there was little or no difference in the levels of cycP transcription between unamended and nitrate-amended photosynthetic cultures. In contrast, CO difference spectra revealed that a much higher level of Cyt c' was present under photosynthetic denitrifying conditions (Fig. 4C). A similar result, although to a smaller degree, is seen in microaerobically grown cells (Fig. 4B). These results demonstrate that Cyt c' is not repressed under denitrifying conditions as observed in IL-106 (26). In fact, Cyt c' levels increased when cells were grown under denitrifying conditions in spite of the observation that these conditions did not cause a significant increase in cycP transcription (Fig. 3A).

NO binding by Cyt c'. The observation that Cyt c' is present in denitrifying cells suggests that it might serve as a sink for free NO (18, 42, 43). In order to confirm that purified Cyt c' from 2.4.3 is capable of binding NO, NO difference spectroscopy was performed with purified c'His (Fig. 4D). The appearance of a maxima at 416 nm and a minima at 431 nm in the difference spectra shows that the reduced form of 2.4.3 Cyt c' does bind NO. As was observed with IL-106 Cyt c', the NO difference spectra and CO difference spectra for c'His have similar absorption maxima and minima (26).

To determine if Cyt c' in denitrifying cells can bind NO produced during denitrification, X-band EPR was used to detect the formation of Cyt c'-NO complexes in denitrifying 2.4.3 whole cells. The spectral signal characteristic of a Cyt c'-NO complex was undetectable in both wild-type and Cyt c'-deficient cells grown under photosynthetic denitrifying conditions (Fig. 5A, spectra 1 and 3). However, when C'89, which constitutively expresses Cyt c', was grown under photosynthetic denitrifying conditions, the characteristic three-line signal in the g = 2.01 region was readily detected (Fig. 5A, spectrum 4). To confirm that overexpression of Cyt c' does increase Cyt c' production in C'89, ferricyanide was added to the whole cells of the various strains. Ferricyanide will oxidize Cyt c', allowing it to be directly detected due to its unique mixed-spin signal at approximately g = 5. Comparison of spectra 1 and 5, from cells of the overexpressing and wild-type strains, respectively, shows there is a significantly higher level of Cyt c' in the overexpressing strain. This is consistent with the relative levels of Cyt c'-NO detected in this strain (Fig. 5A, compare spectra 3 and 4). Comparison of oxidized and as-isolated cells (Fig. 5B, spectra 1 and 2, respectively) reveals that most of the Cyt c' in whole cells is reduced and would therefore likely form stable NO complexes.



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  		FIG. 5. Cyt c' in denitrifying whole cells binds NO. (A) X-band EPR spectra of isolated whole cells of (1) Cyt c'-deficient strain grown photosynthetically with nitrate; (2) C'89 grown aerobically; (3) 2.4.3 grown photosynthetically with nitrate; (4) C'89 grown photosynthetically with nitrate. (B) Spectra of isolated whole cells of (1) C'89 grown photosynthetically with nitrate treated with 2.5 mM ferricyanide; (2) same as (1), no ferricyanide treatment; (3) Cyt c'-deficient strain, grown photosynthetically with nitrate, ferricyanide treatment; (4) C'89 grown aerobically, ferricyanide treatment; (5) 2.4.3 grown photosynthetically with nitrate, ferricyanide treatment.

 
The aerobically grown C'89 sample also exhibited a small signal at g = 2.01, suggesting the formation of a Cyt c'-NO complex under nondenitrifying conditions (Fig. 5A, spectrum 2). However, the precise origin of this signal is unclear. A comparison of the levels of ferricyanide-oxidized Cyt c' (g = 5) of aerobically grown C'89 cells with that of C'89 grown under denitrifying conditions shows there is much less Cyt c' in the aerobically grown cells (Fig. 5B, spectra 1 and 4). There is also an additional signal of unknown origin at g = 4.3 and an abnormally strong signal at g = 2 not seen in denitrifying C'89 cells, indicating that the spectral properties of Cyt c' are perturbed under aerobic conditions (Fig. 5B, spectrum 4). The reduced levels of Cyt c' in the aerobic cells coupled with the unusual spectral features suggest the protein may be unstable under these conditions and the signal at g = 2.01 may not be due to binding of NO.

Does Cyt c' reduce NO? Cross et al. have suggested that R. capsulatus Cyt c' functions as a NO reductase that reduces NO to N2O (9). To test if Cyt c' in 2.4.3 also contributes to N2O production, GC/MS was used to compare the N2O produced during denitrification by the wild-type strain 2.4.3 to that of the Cyt c'-deficient strain PC12. 2.4.3 cultures grown photosynthetically with nitrate produced N2O peaks with an average integrated area of 6.8 x 105 ± 3.4 x 105 (relative abundance). Under the same conditions, PC12 produced N2O peaks with an average integrated area of 6.2 x 105 ± 3.1 x 105 (relative abundance). These results suggest that a deficiency in Cyt c' does not result in a significant decrease in N2O production. Similar results were obtained with microaerobically grown cells.

To more directly address if Cyt c' could generate N2O when supplied with NO, real-time differential mass spectrometry (DMS) was used to test if c'His could reduce the NO produced by 2.4.3 high-speed supernatants (Fig. 6). Similar to membrane inlet mass spectrometry, DMS allows the continuous detection of gaseous compounds that diffuse through a Teflon membrane, such as NO or N2O. Since Nor is a membrane-bound protein and nitrous oxide reductase (Nos) activity is lost upon cell lysis, 2.4.3 high-speed supernatants are free of Nor and Nos activity. This means that under the experimental conditions, NO produced by Nir is not consumed by Nor, and that if Cyt c' reduces NO to N2O, this N2O is not consumed by Nos. As expected, generation of NO (m/z 30) occurs immediately upon addition of ascorbate-PMS to the 2.4.3 high-speed supernatants supplemented with 0.5 mM nitrite (Fig. 6). Upon addition of 31 µg of c'His, the NO signal decreased instantaneously but was not completely abolished (Fig. 6, scan 250). The decrease in the NO signal was short lived, and NO production soon increased again (Fig. 6, scan 350). Analysis of the m/z 44 signal did not show an increase in N2O production upon addition of Cyt c' (data not shown), suggesting that addition of c'His decreased the NO signal by binding to NO but not by reducing it to N2O.



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  		FIG. 6. Differential mass spectrometry detection of NO (m/z 30) signal after addition of cell extracts and Cyt c'. At scan 0, 66 µg of high-speed supernatant of 2.4.3 and 0.5 mM sodium nitrite was added to the DMS cell and followed until scan 100, when the electron donors ascorbate/PMS were adding, resulting in an increase in the m/z 30 signal. At scan 250, 31 µg of c'His was added, and production of the m/z 30 signal followed for another 250 scans.

 
Cyt c' and GSNO resistance. Previous work with R. capsulatus has indicated that Cyt c' plays a role in resistance to GSNO (8). To test if Cyt c' in 2.4.3 plays a similar role, disk diffusion assays were used to compare the sensitivities of the wild-type and Cyt c'-deficient strains. When grown on solid medium under microaerobic conditions, the cycP mutant PC12 was not any more susceptible to GSNO than 2.4.3 (data not shown). R. sphaeroides 2.4.1, which is naturally more sensitive to GSNO than 2.4.3, was also tested to verify that GSNO was toxic under these conditions. As expected, 2.4.1 exhibited a zone of growth inhibition around the disk that is absent in 2.4.3 and PC12 (data not shown).

As a further test of GSNO sensitivity, the growth rates of 2.4.3 and PC12 were monitored in liquid culture in the presence and absence of GSNO (Fig. 7). Both 2.4.3 and PC12 grew at equivalent rates in the presence of 2 mM GSNO under microaerobic conditions, which is consistent with the results of disk diffusion assays. Comparison of the growth of the two strains in unamended medium revealed that the Cyt c'-deficient PC12 grew faster than 2.4.3 under microaerobic conditions. A similar finding has been reported for R. capsulatus grown under anaerobic conditions (8).



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  		FIG. 7. Comparative effect of GSNO on growth of the Cyt c'-deficient strain PC12 and wild-type strain 2.4.3. {blacklozenge}, 2.4.3 microaerobic; {blacksquare}, 2.4.3 microaerobic plus 2 mM GSNO; {blacktriangleup}, cycP mutant microaerobic; x, cycP mutant microaerobic plus 2 mM GSNO. Reported values are the average for three independent experiments. Error bars represent one standard deviation.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In R. sphaeroides 2.4.3, both oxygen and nitrate are important in regulating cycP expression and Cyt c' levels. The finding that the promoter activity of both pC'LACZ1 and pC'LACZ4 was FnrL dependent demonstrates that FnrL activates cycP transcription under low-oxygen conditions (Fig. 2). Northern blot analysis also demonstrated the role of FnrL as an activator of cycP transcription (Fig. 3B). A putative FnrL binding site is located 62 bp upstream of RSP6116* and might serve as the operator for cycP transcription. An identical sequence is found upstream of RSP6116 in R. sphaeroides 2.4.1. This is consistent with the previous observations showing that Cyt c' protein levels were 10-fold lower in aerobic cultures than in phototrophic cultures of 2.4.1 and with gene array analysis detecting oxygen-dependent transcription of cycP (6, 29).

While the effect of oxygen on Cyt c' levels is the same in all the strains of R. sphaeroides in which Cyt c' expression has been studied, the influence of nitrate is not. In the complete denitrifier R. sphaeroides IL-106, Cyt c' levels were found to decrease significantly under photosynthetic denitrifying conditions (26). In contrast, these same conditions resulted in maximal Cyt c' levels in 2.4.3 (Fig. 4C). Interestingly, Cyt c' levels also decreased in the presence of nitrate in R. capsulatus BK5DNIT (9). However, Cyt c' regulation in BK5DNIT differs from that in R. sphaeroides strains in that Cyt c' expression is also observed under aerobic conditions. The physiological reasons underlying the differences in Cyt c' levels remain unclear, but it is obvious that even in closely related bacteria similar environmental factors influence Cyt c' expression in different ways.

It has been proposed that Cyt c' in R. capsulatus PAS100 will reduce NO to N2O (9). Since Cyt c' is most highly expressed under denitrifying conditions in 2.4.3, the ability to reduce NO could be physiologically significant; however, attempts to detect NO reductase activity from the 2.4.3 Cyt c' using GC/MS were unsuccessful. Experiments in this study comparing activities in the wild type and cycP mutants were complicated by the fact that unlike R. capsulatus PAS100, 2.4.3 expresses a membrane-bound Nor complex (4). If Cyt c' has a weak NO reducing activity relative to that of Nor, then its activity would likely be undetectable when comparing N2O production between 2.4.3 and a Cyt c'-deficient mutant. The results reported here, therefore, do not eliminate the possibility that Cyt c' has some NO reductase activity. However, the simultaneous expression of Nor and Cyt c' observed in 2.4.3 does seem to eliminate any physiological requirement for Cyt c' to have this activity. Nor is obviously the most active NO reductase in the cell, since the inactivation of the genes encoding Nor prevent growth in nitrate-amended medium, whereas inactivation of cycP has no observable effect on NO reduction in 2.4.3 (4).

If Cyt c' can bind NO but does not significantly affect NO reduction during denitrification, what physiological role might it have in R. sphaeroides? It could be argued that Cyt c' is useful in protecting cells against exogenous sources of NO. This would explain why cycP is expressed under nondenitrifying conditions and its presence in 2.4.1, which lacks Nir. However, NO also induces Nor expression (15). Thus, when either 2.4.3 or 2.4.1 is exposed to exogenous NO, Nor will be expressed and mitigate potential NO toxicity. Cyt c' also does not appear to play a role in the resistance of 2.4.3 to GSNO, in contrast to R. capsulatus, in which the loss of Cyt c' increased sensitivity to this NO derivative (8).

Perhaps the ability to bind NO is the basis for the primary physiological function of Cyt c' in 2.4.3 and 2.4.1. A model in which Cyt c' serves to shuttle NO has been previously proposed (23). In this model, NO binds to reduced Cyt c'. The protein then diffuses to an inner membrane component that oxidizes the Cyt c'-NO complex. Oxidation favors the release of NO either into or near the inner membrane. NO is more soluble in hydrophobic solvents than in water and consequently accumulates in the lipid bilayer (21). This accumulation would facilitate a more efficient transfer of substrate to Nor in 2.4.1 and 2.4.3, since Nor is an integral inner membrane protein. Another potential advantage of having Cyt c' shuttle NO molecules to the membrane is that the accumulation of NO in the lipid bilayer likely increases the diffusion of NO into the intracellular environment. NO is an important signal molecule that is required for expression of the genes encoding Nir and Nor (15, 16). The increased diffusion of NO into the intracellular environment would thereby allow the cell to respond more rapidly to NO.

The benefit of Cyt c' acting as a NO shuttle is likely limited under laboratory conditions. When nitrate is added to media at millimolar levels, any loss of NO by diffusion would be negligible due to the large reservoir of nitrate present and the fact that the culture is a closed system. This possibly accounts for the lack of a detectable phenotype in cycP mutants under the experimental conditions. However, in the natural environment, nitrate is nearly always present at much lower concentrations than those available in laboratory cultures. Such low concentrations would mean that any NO lost by diffusion would likely represent a significant fraction of the available oxidant pool and is unlikely to be recovered. By increasing the efficiency of NO transfer to the membrane, Cyt c' becomes advantageous to cells growing in an environment low in nitrogen oxides. This proposed role is supported by the observation that in R. sphaeroides 2.4.3, Cyt c' can bind NO produced during denitrification (Fig. 5) and is most highly expressed when NO is present (Fig. 2 and 3). This is the first study to demonstrate a positive relationship between NO binding by Cyt c' and Cyt c' expression during denitrification. Further investigation is required to determine if this relationship exists in other denitrifiers.


   ACKNOWLEDGMENTS
 
This work was supported by the Department of Energy (95ER20206 to J.P.S.) and NIH (GM-35103 to C.P.S.).


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Cornell University, Wing Hall, Ithaca, NY 14853. Phone: (607) 255-8535. Fax: (607) 255-3904. E-mail: jps2{at}cornell.edu. Back

{dagger} Present address: NIDDK, Genetics and Biochemistry Branch, National Institutes of Health, Bethesda, MD 20892-0538. Back


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Bacteriology, June 2005, p. 4077-4085, Vol. 187, No. 12
0021-9193/05/$08.00+0     doi:10.1128/JB.187.12.4077-4085.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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