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Journal of Bacteriology, February 2008, p. 954-962, Vol. 190, No. 3
0021-9193/08/$08.00+0     doi:10.1128/JB.01572-06
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification of Two Catalases in Azotobacter vinelandii: a KatG Homologue and a Novel Bacterial Cytochrome c Catalase, CCCAv{triangledown} ,{dagger}

James R. Sandercock and William J. Page*

Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G2E9

Received 10 October 2006/ Accepted 8 November 2007


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ABSTRACT
 
Azotobacter vinelandii produces two detectable catalases during growth on minimal medium. The heat-labile catalase expressed during exponential growth phase was identified as a KatG homologue by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a mixed protein sample. The second catalase was heat resistant and had substantial residual activity after treatment at 90°C. This enzyme was purified by anion-exchange and size exclusion chromatography and was found to exhibit strong absorption at 407 nm, which is often indicative of associated heme moieties. The purified protein was fragmented by proteinase K and identified by LC-MS/MS. Some identity was shared with the MauG/bacterial cytochrome c peroxidase (BCCP) protein family, but the enzyme exhibited a strong catalase activity never before observed in this family. Because two putative c-type heme sites (CXXCH) were predicted in the peptide sequence and were demonstrated experimentally, the enzyme was designated a cytochrome c catalase (CCCAv). However, the local organization of the CCCAv heme motifs differed significantly from that of the BCCPs as the sites were confined to the C-terminal half of the catalase. A possible Ca2+ binding motif, previously described in the BCCPs, is also present in the CCCAv peptide sequence. Some instability in the presence of EGTA was observed. Expression of the catalase was abolished in cccA mutants, resulting in a nearly 8,700-fold reduction in peroxide resistance in stationary phase.


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INTRODUCTION
 
Soil bacteria are said to endure a "feast or famine" existence (19), and many of them have acquired unique characteristics that allow them to survive prolonged periods of nutrient limitation. One such organism, the obligate aerobe Azotobacter vinelandii, is able to fix dinitrogen gas during periods of nitrogen source limitation (25) or to differentiate into quiescent cysts during periods of carbon source depletion (52). Because nitrogenases are oxygen labile, protective methods must be employed if the organism is to fix nitrogen. Two mechanisms are frequently invoked to explain how A. vinelandii nitrogenases remain functional during growth in aerobic environments: respiratory protection using the branched electron transport system (38) and the use of polysaccharides as a diffusional barrier to oxygen (2). The high respiration rate of A. vinelandii relative to other bacteria has been well documented (20, 44, 51), and the elevated rate should result in the production of large quantities of reactive oxygen intermediates (ROIs) unless the organism successfully eliminates all but trace amounts of O2. In contrast to the metabolism in nitrogen-fixing conditions, the metabolism of encysted A. vinelandii is very low (40).

From these observations it can be seen that A. vinelandii experiences two metabolic extremes. At one extreme the ROI load is potentially very high, but active metabolism allows the rapid production and maintenance of protective enzymes. At the other extreme, the ROI load is low, but the quiescent state of the cell is not amenable to enzyme synthesis. Since the cystlike structures remain viable for years (31, 41), one can postulate that the quiescent organism either actively produces enzymes to deal with toxic oxygen products or synthesizes very stable enzymes which require infrequent replacement.

The major ROIs produced during bacterial metabolism are superoxide (·O2–) and hydrogen peroxide (H2O2), although hydroxyl radicals can be generated if ·O2– or H2O2 undergoes the Fenton reaction with iron. Superoxide dismutases protect the cell by converting superoxide to hydrogen peroxide, and the expression of two such enzymes has been described for A. vinelandii (37). Hydrogen peroxide is then removed from the cell by either dismutation by a catalase (equation 1) or reduction by a peroxidase (equation 2):

Formula 1(1)

Formula 2(2)
where E is the enzyme, AH2 is a hydrogen donor, and ·AH is the radical form of the donor. Thus, hydrogen peroxide degradation by catalatic activity is independent of the metabolic state of the bacterium, whereas peroxidatic activity is metabolically dependent since the electron donor must be regenerated.

Several classes of catalases have been described. The monofunctional heme catalases tend to have very high catalatic rates but exhibit no peroxidatic activity. This group is further divided into the small- and large-subunit categories. The small-subunit catalases tend to be more heat resistant and, in the case of Escherichia coli KatE (HPIIEc), also protease resistant (6). The nonheme catalases have a manganese cofactor rather than an iron cofactor at the active site. These enzymes tend to be heat stable and, with one notable exception (1), have lower catalatic activities than the heme-based monofunctional catalases (21). Finally, the bifunctional catalases, frequently referred to as HPI or KatG, exhibit both strong catalatic activity and moderate peroxidatic activity.

The bacterial cytochrome c peroxidases (BCCPs) constitute another class of antioxidant enzymes. First identified in Pseudomonas fluorescens (24), BCCPs reduce hydrogen peroxide to water peroxidatically using small protein donors, such as cytochrome c, azurin, or pseudoazurin. Members of this class of proteins contain two type c heme groups with different redox potentials. Each heme group plays a distinct role in peroxide reduction (35, 39), requiring communication of electrons from one heme site to the other. Because type c heme groups are covalently bound to the protein, they can be distinguished from type b and d heme groups, such as those found in the hemic catalases described to date, which are bound noncovalently.

It was shown previously that A. vinelandii produces two catalase proteins when it is grown on the minimal medium Burk's buffer salts supplemented with 55 mM glucose, 15 mM ammonium acetate, and 18 µM ferric citrate (BBGN) (41). One of these is expressed during exponential growth, whereas the other catalase is expressed in an RpoS-dependent manner during periods of nutrient limitation and in the stationary phase (41). Here we identified the catalases by liquid chromatography-tandem mass spectroscopy (LC-MS/MS) following protease digestion. The exponentially expressed protein is a KatG homologue. The RpoS-dependent enzyme is a novel cytochrome c catalase (CCC) that belongs to a diheme protein family which has received little attention to date. It is the first protein of this family for which a definite enzymatic function has been assigned.


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MATERIALS AND METHODS
 
Bacterial strains and growth conditions. A. vinelandii strains UW (= ATCC 13705), UWD (= ATCC 53799), and UWDS (41) were grown in the minimal medium BBGN (33) or BBG (41). To obtain optimal aeration, 200-ml cultures were grown in 500-ml flasks with rapid shaking (250 rpm) at 28°C. Escherichia coli DH5{alpha} was grown in liquid LB medium with shaking at 37°C and on solid LB medium with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and 40 µg/ml 5-bromo-4-chloro-3-β-D-galactopyranoside (X-Gal). The antibiotics kanamycin, gentamicin, and ampicillin were used in solid media at concentrations of 10 µg ml–1, 500 ng ml–1, and 100 µg ml–1, respectively.

Preparation of cell lysates. Cell lysates were prepared by the bead beating method described by Benov and Al-Ibraheem (5) in 50 mM potassium phosphate buffer (pH 7.7) amended with 1 µM Pepstatin A and 1 mM phenylmethylsulfonyl fluoride (41). Lysates were clarified by 5 min of centrifugation at 5,000 x g (SS-34 rotor; Sorvall). Protein concentrations were determined by the method of Lowry et al. (28).

In situ catalase activity staining. Protein samples were separated electrophoretically (Hoefer Scientific SE500) on 7.5% nondenaturing polyacrylamide gels by standard methods and were analyzed zymographically for catalases by the method of Clare et al. (8). Protein quantities are indicated in Results. To assess residual catalase activities following heat treatment, a protein sample was incubated for 30 min in a preheated thermocycler (Mastercycler; Eppendorf) and then immediately chilled on ice. Samples were added to nondenaturing loading dye and immediately loaded onto the gel without further heating.

Identification of catalases by trypsin digestion and LC-MS/MS. Duplicate samples containing 80 µg of cell lysate were resolved electrophoretically on 7.5% nondenaturing polyacrylamide gels. One half of each gel was stained for catalase activity, and the other half was stained using Coomassie blue R-250 (Sigma). The Coomassie blue-stained protein bands corresponding to the activity bands were excised, destained, reduced with 37.1 mM dithiothreitol, alkylated with 32 mM iodoacetamide, and digested with sequencing grade modified trypsin (Promega) overnight. The peptides were extracted from the gel, and LC-MS/MS was performed using a CapLC high-performance liquid chromatograph (Waters) coupled to a Q-TOF-2 mass spectrometer (Waters). Data were analyzed using Mascot (Matrix Science).

Kat1 purification. Cell lysates were heat treated for 15 min at 75°C, chilled on ice, and then centrifuged at 5,000 x g (SS-34 rotor; Sorvall) to remove heat-labile protein complexes that had precipitated out of solution. The supernatant was then concentrated by ultrafiltration (YM100; Millipore). The retentate was washed with 5 volumes of ice-cold 50 mM Tris-HCl (pH 7.0) to facilitate removal of low-molecular-weight proteins and peptides. The resultant solution was fractionated by fast protein liquid chromatography (AKTA Explorer 100A; Amersham) using a Q-Sepharose column (Pharmacia). Graduated elution was performed using 50 mM Tris-HCl (pH 7.0) containing 1 M NaCl. Active fractions were quantitatively assayed for total antioxidant activity (catalatic or peroxidatic) by monitoring the degradation of hydrogen peroxide spectrophotometrically at A240 in 50 mM potassium phosphate buffer (pH 7.0) (3). Fractions containing 33% of the peak activity were pooled and concentrated by ultrafiltration (Amicon Ultra-15; Millipore). The solution was prefiltered through a 0.8-µm filter under a vacuum (type AA; Millipore) and then separated by size exclusion chromatography (Sephacryl S-300; Pharmacia). The antioxidant activity of each fraction was assessed as described above.

De novo identification of the stationary-phase catalase following proteinase K digestion. De novo identification was performed by the proteinase K method of Wu et al. (53). Briefly, an equal volume of 200 mM Na2CO3 (pH 11) was added to an aliquot of purified protein. The sample was incubated on ice for 1 h, and urea was added to a final concentration of 8 M. The sample was reduced with 25 mM dithiothreitol and alkylated with 30 mM iodoacetamide. Digestion with proteinase K (Sigma) was performed for either 3 or 18 h with continuous mixing and was stopped by addition of 5% formic acid. LC-MS/MS analysis was performed using a CapLC high-performance liquid chromatograph (Waters) coupled to a Q-TOF Global Ultima mass spectrometer (Micromass). Data were processed using the automated peptide function of MassLynx 4.0 (Micromass) and were analyzed using both Mascot (Matrix Science) and PEAKS online (Bioinformatics Solutions).

N-terminal sequence determination. Purified catalase multimer was dissociated in denaturing load dye for 3 min at 95°C and separated on 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gels that had been aged for at least 24 h. The protein was transferred to a polyvinylidene difluoride membrane (Immun-Blot; Bio-Rad) in 10 mM 3-cyclohexamino-1-propanesolfonic acid-5% methanol (pH 11.0) buffer in a wet cell device (TE Transfor; Hoefer Scientific) for 16 h at 100 mA at 4°C. The membrane was washed twice with 50% methanol and stained using Coomassie blue R-250 (Bio-Rad). The band containing the catalase monomer (51 kDa) was excised and analyzed following Edman degradation with a Procise protein sequencing system (Perkin-Elmer).

Heme staining. Protein was added to loading dye containing β-mercaptoethanol (βME) and SDS at final concentrations of 10% (vol/vol) and 4% (wt/vol), respectively. Samples were separated on 10% SDS-polyacrylamide gels that had been aged for 24 h to ensure that residual peroxides had dissipated. The gels were then stained for heme groups by the method of Goodhew et al. (12); each gel was soaked in methanol-250 mM sodium acetate (30:70; pH 5.0) for 30 min and stained with 1.25 mM 3,3',5,5,'-tetramethylbenzidine (TMBZ) (Sigma) in the same buffer for 30 min. Colorimetric development (cyan) was initiated by addition of 20 mM H2O2.

Interruption of the cccA gene. The cccA gene was amplified with Taq polymerase using forward primer WJP352 (5'-GGCTTTCGCTTCCTGGTCCTTTC-3') and reverse primer WJP353 (5'-GTACTGCAACTGATCCTCGCCCG-3'). An additional 10-min extension step was included for preferential addition of a single adenosine residue at the 3' end. The 1.1-kb product was separated electrophoretically on a 0.8% agarose gel (OmniPur; EM Science) and was extracted using a QIAquick kit (Qiagen). The product was ligated into pGEM-T Easy (Promega), producing plasmid pCCC. Sequence analysis with the M13 forward primer (5'-GTAAAACGACGGCCAGT-3') confirmed insertion of the cccA gene. pCCC was digested with StuI, and the linearized plasmid was purified from a 0.8% agarose gel. The gentamicin antibiotic cassette (Gmr) was excised from plasmid p34S-Gm (9) by SmaI digestion, and the host vector was destroyed by cleavage with NdeI. The Gmr cassette was treated with shrimp alkaline phosphatase (Roche), purified from an agarose gel, and ligated into StuI-linearized pCCC using a rapid ligation kit (Fermentas). Transformants were selected on LB plates containing gentamicin, and plasmid purification was performed using cultures inoculated with isolated colonies. The orientation of the Gmr cassette was determined by restriction analysis and was confirmed by sequencing. Plasmids containing the interrupted cccA gene were transformed into A. vinelandii strains UWD and UWDS by natural transformation techniques (32). Mutants were selected on BBGN plates containing gentamicin.

Western analysis of RpoS. Western analysis of cell lysates was performed as described in the accompanying paper (41). Cell protein was separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immun-Blot; Bio-Rad) by wet cell transfer (TE Transfor; Hoefer Scientific) in 20% methanol-27 mM Tris base-192 mM glycine (pH 8.1) transfer buffer at 100 mA for 16 h at 4°C. Membranes were first stained with the primary antibody (His-RpoS rabbit antibody [41]) and then with a fluorescently conjugated secondary antibody (IRDye800-conjugated donkey anti-rabbit antibody; Rockland Immunochemicals). Detection was performed using the Odyssey infrared imaging system (Li-Cor) at a resolution of 84 µm.

Survival in the presence of hydrogen peroxide. A. vinelandii cultures were grown in BBGN to either exponential or stationary phase. Cultures were pelleted by centrifugation at 2,500 rpm for 5 min and resuspended in an appropriate volume to obtain ca. 1.8 x 108 CFU ml–1. To maintain the growth state of the cultures, exponentially growing cells were resuspended in sterile BBG, whereas stationary-phase cells were resuspended in sterile Burk's buffer salts. The cultures were divided into smaller aliquots and grown at 28°C with agitation. Hydrogen peroxide was added to a final concentration of 20, 40, or 60 mM. After 20 min of exposure to hydrogen peroxide, samples were serially diluted and enumerated on BBGN agar plates.


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RESULTS
 
Stationary-phase catalase is thermostable and monofunctional and exponential-phase catalase is heat labile and bifunctional. Expression of two catalases by A. vinelandii is described in the accompanying paper (41). These enzymes were designated strictly on the basis of their relative electrophoretic mobilities. In this study whole-cell extracts of mid-exponential-phase A. vinelandii cultures were heated for 30 min at various temperatures and then rapidly cooled on ice. The lysates were separated by 7.5% nondenaturing PAGE and subsequently stained for catalase activity (Fig. 1). The lower catalase, previously designated Kat2, was heat labile at temperatures above 46°C, whereas Kat1 remained functional following heat treatment at temperatures as high as 90°C. Increasing the temperature shifted the migration pattern of Kat2 to a previously identified form, Kat3 (41). Kat2 and Kat3 were found to have peroxidase activity when they were stained with the donor 3,3'-diaminobenzidine in the absence of horseradish peroxidase (data not shown), indicating that they are capable of bifunctional catalase-peroxidase activities. Kat1 was considered a monofunctional catalase as it exhibited no peroxidase activity in the presence of 3,3'-diaminobenzidine.


Figure 1
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  		FIG. 1. Zymographic analysis of residual catalase activity following 30-min heat treatments of cell lysates. Following heat treatment, 50-µl samples were separated by 7.5% nondenaturing PAGE and stained for the presence of catalases by the method of Clare et al. (8). The temperatures used are indicated above the lanes. The positions of the electrophoretically discernible catalase species Kat1, Kat2, and Kat3 are indicated. The image quality was improved by color inversion (Adobe Photoshop v5.5).

Identification of the exponential-phase catalase, a KatG homologue. To determine the identities of the enzymes responsible for each of the distinct catalase bands, 80 µg of mid-exponential-phase cell lysate was separated in duplicate on a nondenaturing polyacrylamide gel; one half of the gel was stained for total protein with Coomassie blue, while the other half was stained for catalase activity. Coomassie blue-stained protein bands corresponding to the catalase activity bands were excised, digested with trypsin, and subsequently analyzed by LC-MS/MS. The A. vinelandii KatG protein (accession number EAM04829) was detected in both the Kat2 and Kat3 bands. Cumulatively, 16.4% peptide coverage of KatGAv was obtained (see Fig. S1 in the supplemental material). KatGAv was found to share 65% protein identity with E. coli KatG (accession number AAC76924) and 74% protein identity with Pseudomonas syringae KatG (accession number NP_794283).

No catalase homologues were identified in the trypsin-treated Kat1 band, although a single peptide of a putative antioxidant protein (accession number EAM04846) similar to the BCCP/MauG family of proteins (COG 1858; pfam 03150) was detected. This identification was suspect since the fragment only gave 3.7% coverage of the protein and because no BCCP has ever been reported to have catalase activity despite decades of scrutiny. Further analysis demonstrated that the enzyme responsible for the Kat1 band was protease resistant (including the proteases pronase E, AspN, and trypsin) (data not shown), a feature shared with HPIIEc (6) and KatA of P. aeruginosa (13). While an evaluation of the A. vinelandii genome (http://www.azotobacter.org) did not reveal an HPII catalase, three other putative heme catalases were present in the genome. A hypothetical Mn catalase, conserved in the Pseudomonas aeruginosa PAO1 genome, was also present.

Purification of the stationary-phase catalase. Since the stationary-phase catalase was heat stable (Fig. 1), cell extract was heated at 75°C for 15 min, chilled on ice for 10 min, and clarified by centrifugation. Low-molecular-weight proteins and peptides were removed using a 100-kDa-cutoff filter (YM100; Millipore). During fractionation on a Q-Sepharose column (Pharmacia), the approximate protein concentrations of the fractions were monitored at 215 nm, the presence of heme was monitored at 407 nm, and the relative enzyme activities were determined as described in Materials and Methods (Fig. 2A). Because the enzyme assay method followed only the degradation of hydrogen peroxide, the assay could potentially measure both catalatic and peroxidatic activities. However, significant oxygen liberation was observed during the assays, which suggested that there was a dominant catalase function. Three peaks absorbed strongly at 407 nm, and one of them corresponded to the fractions exhibiting the strongest enzymatic activity. Maximal elution of the catalase occurred between 252 and 265 mM NaCl. The most active fractions were pooled and fractionated by size exclusion chromatography (S-300), and the protein concentration, heme content, and relative activity were again monitored. The fractions with the highest activity absorbed strongly at 407 nm (Fig. 2B), which suggested that the enzyme was a heme-type catalase.


Figure 2
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  		FIG. 2. Elution profiles of A. vinelandii whole-cell lysates fractionated by Q-Sepharose (A) and S-300 (B) fast protein liquid chromatography. For each fraction the approximate relative protein concentration was determined at 215 nm (dashed line), the heme content was measured at 407 nm (solid line), and the catalase/peroxidase activity (expressed as a percentage) was determined (circles). The dashed and dotted line indicates the relative NaCl concentration during elution of the Q-Sepharose column in panel A. Protein concentration estimates were scaled down 30- and 12-fold in panels A and B to allow use of a single axis (in milliabsorbance units [mAU]). The graphs were prepared using Origin 5.0. Elution profiles of the void volume were not included for the sake of clarity.

Several fractions were separated by 10% denaturing SDS-PAGE and stained with Coomassie blue (Fig. 3A) to determine protein purity. The fractions with the highest activities had a dominant protein band at a molecular mass of approximately 215 kDa and three minor contaminants with molecular masses of 58, 38, and 37 kDa (Fig. 3A). Fractions with at least 20% of the peak activity were pooled. Duplicate samples of the pooled S-300 solution were separated by 10% denaturing SDS-PAGE; one half of the gel was stained for total protein with Coomassie blue, and the other half was stained for catalase activity. The zymographically detected catalase band corresponded to the dominant protein band at 215 kDa (Fig. 3B).


Figure 3
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  		FIG. 3. (A) Assessment of the purity of selected fractions that exhibited strong catalase activity following S-300 chromatography by electrophoretic separation by 10% SDS-PAGE and subsequent staining with Coomassie blue. The positions of the molecular weight standards (BroadRange; Bio-Rad) are indicated on the left. The most active fractions had a major band at 215 kDa and three minor contaminants with molecular masses of 68, 38, and 37 kDa. The samples were pooled as described in the text. (B) Duplicate lanes containing 2.3 µg of the pooled protein were separated by 10% SDS-PAGE and stained for total protein (lane 1) with Coomassie blue or for catalase activity (lane 2) by the method of Clare et al. (8). The mobility of the catalase activity band (Kat1) corresponded to that of the 215-kDa protein complex.

Identification of Kat1, a protein similar to Snr-1 of P. aeruginosa. Before fractions were pooled, S-300 fraction 27 (ca. 57 to 58 ml) (Fig. 2B) was selected for further analysis. The proteins in this fraction identified by trypsin fragmentation and LC-MS/MS analysis included the 60-kDa GroEL chaperonin (accession number AAL25964.1), with 10.99% peptide coverage, and the 36.6-kDa acetohydroxy acid isomeroreductase (accession number EAM03644.1), with 9.76% peptide coverage. The identity of the 38-kDa protein could not be determined using fraction 27. Two additional fragments of the previously identified BCCP/MauG homologue (accession number EAM04846) were also observed.

Analysis of the peptides liberated by proteinase K digestion of fraction 27 resulted in an additional 21.6% coverage of the theoretical open reading frame (ORF) of the BCCP-like catalase (Fig. 4), which provided composite coverage of 27.7% with the trypsin fragments. The level of similarity between the catalase and the BCCPs was low; there was only 35% shared protein identity across 51 residues with the BCCP of P. aeruginosa (accession number NP_253277). Instead, the enzyme shared the greatest degree of similarity with two novel diheme proteins: 78% protein identity with the Snr-1 protein of P. aeruginosa PAO1 (Snr-1Pa) (accession number NP_251722) and 72% protein identity with the Hsc protein of Desulfonmonile tiedjei DCB-1 (HscDt) (accession number AAB66558).


Figure 4
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  		FIG. 4. Multiple alignment of CCCAv (AvCCC) (accession number EAM04846) with its closest homologues, the inducible cytochrome c protein of D. tiedjei DCB-1 (DtHsc) (accession number AAB66558) and the Snr-1 protein of P. aeruginosa PAO1 (PaSnr) (accession number NP_251722), performed by using ClustalW (15). Sequences detected by LC-MS/MS are indicated as follows: italics and underlining with a solid line, sequences identified following trypsin digestion; bold type and underlining, sequences identified following proteinase K digestion. The single Trp residue (W) is indicated by a triangle, two heme binding sites adhering to the CXXCH motif are enclosed in solid boxes, and the putative Ca2+ site is enclosed in a dashed box. Leader peptide sequences predicted by SignalP 3.0 (4) are indicated by italics and underlining with a dashed line. The N-terminal extension unique to CCCAv is indicated by lowercase letters and is discussed in the text. An asterisk indicates complete residue conservation, a colon indicates strong group conservation, a period indicates weak group conservation, and a blank space indicates no conservation of residues.

Sequence analysis. Two c-type heme binding sites adhering to the motif CXXCH were observed in the primary amino acid sequence at the C-terminal end of the catalase (Fig. 4), in agreement with findings for the diheme proteins HscDt and Snr-1Pa. This motif distribution is not similar to that of the BCCP proteins, in which only one of the heme sites occurs in the C-terminal end. All of the Cys residues found in the protein mapped to the two heme motifs, a surprising observation considering the thermal stability of the protein (Fig. 1). Because they are expected to be covalently bound to heme groups, it is unlikely that any of the Cys residues are involved in intrapeptide cross-linkage or that they act as axial ligands to either heme.

Analysis of the amino acid content of the protein indicated a paucity of aromatic amino acids. In particular, only a single Trp residue was observed. This residue was localized near the N terminus of the protein (W102), as previously described for the BCCPs (11).

Calcium has been shown to be critical for the activation and multimerization of several BCCPs (10, 36, 47). A sequence bearing some resemblance to the consensus Ca2+ binding site of the BCCPs (TXPYXHXG) was observed at the C-terminal end of the protein (423-SPPYLHDG-430) (Fig. 4).

Sequence analysis using the publicly available signal peptide prediction software SignalP3.0 (4) indicated that there is a high-probability (0.993) signal peptide starting at residue M124 of the CCCAv ORF. Revision of the N-terminal ORF boundary so that it started at residue M124 rather than at V1 and subsequent cleavage of the 26-amino-acid leader sequence (Fig. 4) gave a theoretical monomeric molecular mass of 50.6 kDa (including two 616-Da heme groups) rather than 66.3 kDa (PeptideMass) (50). Furthermore, the adjusted conceptual protein had a theoretical pI of 6.20 (PeptideMass) (50). It is unlikely that a protein with the original ORF boundaries and the corresponding theoretical pI (pI 9.60) would have bound Q-Sepharose in the buffer used in this study (50 mM Tris-HCl, pH 7.0). To further elucidate the ORF boundaries of CCCAv, N-terminal sequencing by Edman degradation (Procise; Perkin-Elmer) was attempted. Little useful data was obtained, likely due to natural modification at the N terminus. However a third-position codon bias plot (Artemis; Sanger Institute), which can be particularly useful for defining coding regions in G+C-rich genomes (17), supported the revised N-terminal boundary (Fig. 5B).


Figure 5
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  		FIG. 5. (A) Gene map of cccA and the surrounding ORFs shown to scale. The N-terminal extension predicted by automated annotation is indicated by a dashed box, and the adjusted ORF is indicated by the lightly shaded arrow (see text). The positions of the putative heme sites within the cccA ORF are indicated by cross-hatched boxes. The Gmr antibiotic cassette was inserted at the StuI site, and the orientations of the cassette in the cccA1 and cccA2 mutants are indicated. (B) Third-position G+C bias plot aligned with panel A and to scale. The three different reading frames for the top strand are indicated by the solid, dotted, and dashed lines. The G+C bias was plotted in Artemis (Sanger Institute).

A. vinelandii Kat1 is a novel CCC. In order to determine the validity of the predicted heme binding sites, purified protein was subjected to denaturation by several methods, separated by denaturing PAGE, and then stained with TMBZ for detection of heme groups (Fig. 6). Low-temperature denaturation (37°C, 15 min) in the presence of SDS and βME did not cause dissociation of the catalase complex, which migrated at about 215 kDa and stained poorly. Higher-temperature treatment (100°C) in the presence or absence of SDS and βME (Fig. 6, lanes 4 and 5) dissociated the active complex to various degrees. The resolution of a 51-kDa monomer corresponded to the hypothetical ORF mass (with hemes), 50.6 kDa. Strong staining of the monomer by TMBZ indicated the presence of covalently bound heme groups. As a result, we designated this protein the A. vinelandii cytochrome c catalase (CCCAv). Treatment with the chelating agent EGTA resulted in some dissociation, suggesting that Ca2+ has a role in enzyme stabilization (Fig. 6, lane 6).


Figure 6
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  		FIG. 6. Heme stained proteins separated by 10% SDS-PAGE. Lane 1 contained 154 µg of whole-cell lysate, lane 2 contained 9 µg of Q-Sepharose pooled protein, and lanes 3 through 6 contained 3.4 µg of S-300 pooled protein. Prior to loading, the samples were treated as follows: lanes 1 to 3 were treated with loading dye containing βME and SDS for 15 min at 37°C; lane 4 was treated with loading dye for 3 min at 100°C; lane 5 was treated for 30 min at 100°C in aqueous buffer, and then loading dye was added at room temperature; and lane 6 was treated with 1 mM EGTA and loading dye for 15 min at 37°C. The proteins were separated electrophoretically and stained by the method of Goodhew et al. (12). Band A, which corresponded to the catalase multimer, stained poorly. The monomeric form of the enzyme, band B, stained strongly and had a molecular mass of approximately 51 kDa. The positions of the molecular mass standards (BroadRange; Bio-Rad) are indicated on the left.

Effects of the cccA::Gm interruption on expression of the stationary-phase catalase. The cccA gene was amplified by PCR and cloned into pGEM-T Easy (Promega), resulting in plasmid pCCC. This plasmid was cleaved with StuI, leaving blunt ends into which a SmaI-liberated Gmr cassette from p34S-Gm was ligated. Plasmids pCCC::Gm1 and pCCC::Gm2 were produced with the gentamicin cassette in the forward and reverse orientations, respectively. Each of these plasmids was transformed into A. vinelandii strain UWD (Fig. 5A) and the rpoS mutant strain UWDS. Whereas strain UWD produced significant quantities of CCCAv during early stationary phase (Fig. 7A), mutant strains UWD cccA1 and UWD cccA2 produced none. Consistent with previous observations, neither the rpoS strain (41) nor the rpoS cccA1 double mutant (Fig. 7A) produced measurable levels of the catalase. To ensure that the effect of the mutation was direct and not due to the abolition of RpoS expression, a Western analysis of RpoS levels was performed using the same cell lysates (Fig. 7B). RpoS expression was not eliminated by the cccA::Gmr mutation, as similar levels were observed in strains UWD, UWD cccA1, and UWD cccA2. Expression of the sigma factor was completely abolished in the rpoS mutant strains.


Figure 7
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  		FIG. 7. Assessment of the A. vinelandii cccA mutants during stationary phase. Cell lysates of strains UWD (lane 1), UWD cccA1 (lane 2), UWD cccA2 (lane 3), UWD rpoS (lane 4), and UWD cccA1 rpoS (lane 5) were separated by 10% SDS-PAGE. (A) Zymographic analysis for catalase activity performed with 50 µg of cell extract per lane. (B) Western analysis of RpoS performed with 25 µg of cell extract.

Survival of the cccA mutant in the presence of hydrogen peroxide. The survival of strains UWD and UWD cccA1 following oxidative stress was assessed by adding H2O2 to a final concentration of 20, 40, or 60 mM during exponential- or stationary-phase growth (Table 1). Strain UWD was very sensitive to hydrogen peroxide treatment during the exponential phase, with only 0.00096% survival following addition of 20 mM H2O2 and no detectable CFU following treatment with 40 mM peroxide. Survival of strain UWD was 2,700-fold greater during stationary phase, with 0.01% survival following the addition of 20 mM H2O2 (Table 1). The opposite profile was observed for UWD cccA1 since survival was 34-fold lower in stationary phase than in exponential phase after treatment with 20 mM H2O2. In general, the mutant resisted peroxide exposure poorly and did not exhibit measurable survival following the addition of 40 mM H2O2.


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  		TABLE 1. Survival of A. vinelandii strains UWD and UWD cccA1 following 20 min of exposure to 20, 40, or 60 mM hydrogen peroxide during exponential or stationary phase growth in liquid media


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DISCUSSION
 
One of the dangers inherent in oxygen-based metabolism is the generation of toxic coproducts, such as oxygen radicals, hydroxyl radicals, and hydrogen peroxide, which can damage virtually any biomolecule in the cell. This is particularly important for organisms that live in hostile environments (43), organisms that have high respiration rates (16), or bacteria that enter quiescent states measured in years (30, 49) and so are unable to repair damage in a timely manner. We report here that A. vinelandii, which has the highest respiration rate of any known bacterium (20, 44, 51), synthesizes two catalases under specific physiological conditions. The first enzyme, a bifunctional KatG homologue, shares a high degree of similarity with previously described catalases in this class. Previous work suggested that the stationary-phase form of this enzyme exhibits different migratory patterns when it is separated by nondenaturing PAGE (41). A similar shift in migration was observed following heat treatment of the enzyme. The enzyme was found to be somewhat more heat labile than KatGEc, losing detectable activity at temperatures above 46°C. KatGAv expression was strong during exponential-phase growth, so this enzyme is considered the "housekeeping" catalase (41). Weak or no KatGAv expression was observed in stationary phase unless A. vinelandii was grown under high-aeration conditions (Fig. 7), supporting the suggestion that KatGAv may be oxygen or ROI inducible (41).

The second catalase, a stationary-phase inducible enzyme, was found to be both thermostable and protease resistant. While some denaturation of the catalase was observed following boiling in the presence of βME and SDS, a small but detectable amount of the enzyme complex remained in the multimeric form (Fig. 6, lane 4). Despite its thermal stability, the CCCAv protein had a low Cys content. It was determined that only four Cys residues were present in the protein and that all of them were confined to the heme binding motifs and so were not available for the formation of intrapeptide disulfide bonds. Although the vegetative form of A. vinelandii UWD does not grow at temperatures above 37°C and the encysted form of the organism is not considered heat resistant (45), the heat tolerance of the enzyme was equivalent to that of the catalase of thermotolerant Bacillus terminalis spores (23). A similar degree of heat resistance has been described for HPII/KatE of the mesophile E. coli (29). It has been suggested that either HPII developed protease resistance due to its role as the stationary-phase catalase and that heat resistance arose coincidentally (7) or that katE was transferred horizontally from a thermophilic organism (46). Whether CCCAv developed within A. vinelandii or the gene arrived by horizontal transfer, the presence of a protease-resistant enzyme is advantageous for the stationary and quiescent phases of A. vinelandii, since the frequency with which the enzyme needs to be manufactured is decreased. Additionally, whereas peroxidatic activity is to some degree metabolically dependent due to donor regeneration, catalatic protection would have the added advantage of being "metabolically independent" during a period of nutrient limitation.

Unlike the BCCPs, which in most cases are paradoxically expressed under anoxic or microaerophilic conditions (26, 48), expression of the stationary-phase catalase seemed to be insensitive to the aerobic state of the culture (41). Initially recognized by the automated annotation software as a BCCP/MauG homologue (COG1858; pfam 03150), the novel diheme catalase shares limited similarity with other members of this protein family. The only motifs shared by CCCAv and the BCCPs appear to be the putative Ca2+ binding site at the C terminus and two CXXCH heme binding sites. Treatment of the protein with EGTA resulted in partial dissociation of the protein, so either Ca2+ is less critical for multimer stability than it is in the BCCPs or binding at this site is stronger than it is at the equivalent BCCP site. It should be noted that the heme binding sites had a very unusual distribution in CCCAv, where the two sites were both relegated to the C-terminal half of the protein rather than equally distributed between the N and C termini. It is not known what the sixth axial ligands, if any, are for each of the heme groups, as typically spaced methionine and histidine candidates were not observed.

The catalase was also unique in that it contained only one tryptophan residue (W102). From the crystal structure of P. aeruginosa cytochrome c peroxidase it is known that a highly conserved N-terminal Trp residue is localized in three-dimensional space between the two heme groups and is believed to mediate the transfer of electrons from the high-spin accepting heme to the low-spin peroxidatic heme (11). The primary sequence context of this residue is highly conserved in the BCCPs (92-QFWDGRA-98, CCPPa numbering) and is similar in the MauG family. However, this conserved sequence is not found around the Trp of CCCAv (100-TNWDDLG-106), which could indicate that W102 is not involved in electron transfer. One hypothesis is that the absence of the conserved Trp precludes intraheme communication. Such an insulated state could give rise to two independent catalytic centers. Biochemical and structural studies are being performed to test the validity of this assertion.

CCCAv exhibits the greatest levels of similarity to the less-studied diheme cytochrome c proteins Snr-1Pa and HscDt. It is not certain that these proteins have significant catalase activity since definitive enzymatic and physiological functions have yet to be elucidated (J. J. Rowe, personal communication; W. W. Mohn, personal communication). However, CCCAv and HscDt share many physiochemical properties, including a similar monomer mass (around 51 kDa), a multimer mass just greater than 200 kDa (and thus an apparent tetrameric organization), and the presence of covalently bound heme groups (27).

In agreement with the studies described in the accompanying paper (41), A. vinelandii resistance to hydrogen peroxide stress during exponential phase, when KatGAv was the dominant catalase, was quite low. Although the survival during stationary phase was 2,700-fold higher than the survival during exponential phase, the resistance to this stress was much poorer than that of other soil pseudomonads (14, 19). This may seem paradoxical considering the high metabolic rate of A. vinelandii, but mounting evidence indicates that endogenously produced H2O2 is largely removed by peroxidases (34). In the case of a free-living soil organism like A. vinelandii, the exposure to exogenous peroxides from plants and other bacteria may be relatively minor, requiring less antioxidant protection. Interruption of the cccA gene had little effect on peroxide survival during exponential phase but resulted in a survival rate that was nearly 8,700-fold lower than that of the wild-type strain in stationary phase. So unlike many of the BCCPs, which have only assumed physiological roles at this time (43), a clear role has been determined for CCCAv.

Despite their energetic potential to function in a catalatic manner, neither the yeast cytochrome c peroxidase (42) nor the BCCPs exhibit measurable catalase activities. It could be argued that CCCAv does not normally have catalase activities but became damaged during the heating stage of the purification process, resulting in release of new enzymatic capabilities. Such "functional conversion" is observed when certain cytochrome c proteins are treated with proteases, resulting in activation of previously unobserved peroxidase and catalase functions (18). However, CCCAv also exhibited strong catalatic activities when it was liberated by gentle cell lysis methods in the absence of heat (Fig. 1) (41). As a result, we report here the first BCCP-like protein, and possibly the first naturally occurring cytochrome c-type protein, to demonstrate significant catalase activity. If the closely related proteins HscDt and Snr-1Pa have significant catalatic functions, these enzymes would constitute the first new class of catalases since the description of the manganese (nonheme) catalases (22).


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ACKNOWLEDGMENTS
 
We thank Amanda L. Doherty-Kirby of the Siebens-Drake Research Institute (University of Western Ontario), who performed the proteinase K LC-MS/MS identifications. Her willingness to try the method and her invaluable conversations were greatly appreciated. We also thank Lorne Burke (Institute for Biomolecular Design, University of Alberta) for his assistance with trypsin LC-MS/MS identifications. N-terminal sequencing was done at the Synthesis and Sequencing Facility at the John Hopkins University School of Medicine.

Financial support for this work was provided by the Natural Sciences and Engineering Research Council (NSERC) of Canada.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G2E9. Phone: (780) 492-4758. Fax: (780) 492-7033. E-mail: bill.page{at}ualberta.ca Back

{triangledown} Published ahead of print on 30 November 2007. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


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Journal of Bacteriology, February 2008, p. 954-962, Vol. 190, No. 3
0021-9193/08/$08.00+0     doi:10.1128/JB.01572-06
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