Departamento Patología Animal I, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain1
Centro de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain2
Centre National de Référence de Toxémies à Staphylocoques, EA 1655, Faculté de Médecine, rue Guillaume Paradin, 69372 Lyon cedex 08, France3
Author for correspondence: Ricardo de la Fuente. Tel: +34 1 3943703. Fax: +34 1 3943908. e-mail: rifuente{at}eucmax.sim.ucm.es
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: catalase, catalase gene,, Staphylococcus aureus subsp. anaerobius
Abbreviations: BLC, bovine liver catalase
The GenBank accession numbers for the sequences reported in this paper are AJ000472 (S. aureus ATCC 12600 katA) and AJ000471 (S. aureus subsp. anaerobius MVF 213 katB).
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Catalase is a haem-containing enzyme involved in dismutation of hydrogen peroxide generated during cellular metabolism to water and molecular oxygen. Most of the catalases characterized can be classified into one of two types based on their enzymological properties: monofunctional or typical catalases, and bifunctional catalase-peroxidases (Loewen, 1992 ). In many bacteria, both types of catalase are present and each enzyme is encoded by a different gene (e.g. in Escherichia coli, katE and katG code for monofunctional and bifunctional catalases, respectively). Monofunctional catalases have been described as proteins with molecular masses of approximately 220350 kDa and are normally formed by four identical subunits, each containing one (proto-)haem group (Haas & Brehm, 1993
). Their active centre and NADPH-binding site have also been described in detail (Fita & Rossmann, 1985a
, b
). Comparison of the deduced amino acid sequences of these enzymes indicates that typical catalases share regions that are highly conserved among microbial, plant and mammalian enzymes (Switala et al., 1990
; Von Ossowski et al., 1993
).
In S. aureus, a typical catalase with high levels of enzymic activity and formed by four identical subunits of approximately 60 kDa has been described (Rupprecht & Schleifer, 1979 ; Ruiz Santa Quiteria et al., 1992
). However, the gene encoding the apoenzyme of staphylococcal catalase has not yet been studied. Previous studies on catalase deficiency of S. aureus subsp. anaerobius have shown that this bacterium synthesizes haem (De la Fuente et al., 1987
), the prosthetic group of catalase and cytochromes, but that no protein reacting with purified immunoglobulins against S. aureus catalase is detected in crude and partially purified cellular extracts (Ruiz Santa Quiteria et al., 1992
). These findings suggest that the catalase deficiency of S. aureus subsp. anaerobius could be due to mutations in the structural gene of the apoenzyme, or to alterations in the sequences that control its expression. The aim of this work was to establish the molecular basis of the catalase deficiency of S. aureus subsp. anaerobius by means of a genetic approach. Cloning, sequencing and characterization of the catalase gene from S. aureus was a prerequisite to analysing the corresponding gene from S. aureus subsp. anaerobius.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Southern blot analysis.
DNA was digested with different restriction enzymes (HindIII, HindIII/SacI, HindIII/SalI) in accordance with the manufacturers suggestions (New England Biolabs), fractionated on 1% agarose gels and transferred to nylon membranes (N+, Amersham) by the capillary blot technique. DNA probe was radiolabelled with [32P]dCTP by using the Multiprimer DNA labelling system (Amersham Life Science). Prehybridization reactions were carried out at 56 °C for 1 h in a solution containing 7% (w/v) SDS, 1 mM EDTA and 0·5 M sodium phosphate (pH 7·2). Hybridization was performed for 24 h at 56 °C. The nylon membranes were washed at 45 °C in 5x SSC (1x SSC is 0·15 M NaCl plus 0·015 M sodium citrate)/0·1% SDS for 30 min and exposed to X-ray films at -70 °C for 124 h.
Northern blot analysis.
Total RNA was separated by electrophoresis on 1% agarose gels containing 2·2 M formaldehyde. Nucleic acids were vacuum-transferred to nylon membranes (N+, Amersham) and cross-linked by UV light. RNA probe was synthesized by in vitro T3 polymerase transcription of pKat1 with digoxigenin-labelled UTP (Boehringer Mannheim) as substrate. Hybridizations were performed at 68 °C in 5x SSC plus formamide 50% (v/v). Detection was performed with the DIG kit (Boehringer Mannheim) according to the manufacturers instructions and signals were detected by chemiluminescence. Staphylococcal 16S and 23S rRNAs were used as size markers.
PCR amplification.
Oligonucleotide primers (Table 2) were constructed by Isogen Bioscience. PCR was carried out in a 50 µl volume containing 50 ng genomic DNA with reagents and protocols supplied by the manufacturer (Perkin Elmer). Thermocycler reaction conditions were 1 min at 94 °C, 1 min at 52 °C and 1 or 1·5 min at 72 °C for 30 cycles. All PCR amplifications included preliminary denaturation at 94 °C for 10 min and a final incubation at 72 °C for 10 min. Amplified PCR products were analysed by electrophoresis on 1% agarose gels.
Cloning of catalase.
Digested total DNA from S. aureus ATCC 12600 or S. aureus subsp. anaerobius MVF 213 was recovered from agarose gels with the Qiaquick gel extraction kit (Qiagen) and ligated with the T4 ligase (Amersham Life Science) into pUC18. Calcium chloride competent E. coli CC118 cells were transformed as described by Sambrook et al. (1989) . Transformants were selected on LB agar containing ampicillin (100 µg ml-1). Colonies were screened by colony hybridization and by their oxygen formation when they were overlaid with 3% H2O2. kat gene fragments amplified by PCR from S. aureus or S. aureus subsp. anaerobius genomic DNA were cloned in E. coli by using the pMOSBlue T-vector kit (Amersham).
Construction of kat hybrid genes.
Recombinant hybrid genes were constructed by the method of gene SOEing or splicing by overlap extension (Higuchi, 1989 ). Oligonucleotides cat3 and cat4 (Table 2
) are complementary and represent nucleotides from 992 to 1011 of the katA and katB genes. In separate reactions (30 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min and extension at 72 °C for 1 min), oligonucleotides cat1 and cat3 and oligonucleotides cat2 and cat4 were used to amplify, from the katA and katB genes, PCR products representing sequences from each end of these genes. Samples of the products of each reaction were combined and subjected to a subsequent reaction with oligonucleotides cat1 and cat2 to amplify a full-length recombinant product representing the 5' end of katB with the 3' end of katA (katBA) and the 5' end of katA with the 3' end of katB (katAB). Amplified products were cloned into E. coli by using the pMOSBlue T-vector kit (Amersham) (Fig. 1
). Recombinant hybrids thus generated were confirmed by automatic nucleotide sequencing.
|
Data research and computer analysis.
Genetic data were analysed using the University of Wisconsin Genetics Computer Group program library (GCG) (Devereux et al., 1984 ). CLUSTALX (1·5b) was used for comparison of analysed sequences. Phylogenetic relationships were inferred from a multiple amino acid sequence alignment and a consensus tree was constructed with the program TreeView 1.5.
Preparation of cell lysates.
Bacterial lysates were prepared by resuspending cell pellets in 3 ml extraction buffer A (50 mM Tris/HCl, pH 8·0; 0·5 mM EDTA; 50 µg sodium azide ml-1; 0·25 mg lysozyme ml-1). S. aureus and S. aureus subsp. anaerobius cells were lysed with 0·2 mg lysostaphin ml-1. The suspensions were incubated at 37 °C for 15 min. Following incubation, 0·3 ml extraction buffer B was added (1·5 M NaCl, 0·1 M CaCl2, 0·1 M MgCl2, 0·02 mg DNase I ml-1; 0·05 mg ovomucoid protease inhibitor ml-1). After incubation at room temperature for 5 min, the suspensions were centrifuged at 10000 g for 1 h. The supernatant was retained and stored in an ice bath or frozen at -20 °C until needed. Protein concentration was determined by the Bradford method using bovine serum albumin as the standard.
Polyacrylamide gel electrophoresis and immunoblotting.
Protein extracts were separated on 10% (w/v) polyacrylamide gels under native and denaturing conditions. Denaturing gels were stained with Coomassie blue and nondenaturing gels were stained for enzyme activity as described below. The purified anti-catalase immunoglobulins for Western blot analysis were obtained as described by Ruiz Santa Quiteria et al. (1992) .
Determination of enzymic activity.
Quantitative determination of catalase activity present in extracts was performed by the colorimetric assay of Sinha (1972) . Catalase activity present in extracts was also determined by staining nondenaturing polyacrylamide gels according to the method of Clare et al. (1984)
using 5 mM H2O2.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Analysis of the catalase nucleotide sequence
The nucleotide sequence of an internal 1756 bp fragment carried by pKatA was determined on both strands. A 1518 bp single ORF initiating at ATG (base-pair 166) and terminating at TAA (base-pair 1683) was identified within this sequence. The ORF encodes a protein of 505 amino acids with a calculated molecular mass of 58347 Da. A -10 region identical to the E. coli consensus sequence was detected upstream of the start codon [TATAAT in S. aureus ATCC 12600 and E. coli (Horwitz & Loeb, 1990 )]. A putative -35 region similar to the corresponding region in E. coli [TTGTAA in S. aureus ATCC 12600 and TTGAAG in E. coli (Horwitz & Loeb, 1990
)] was also observed. Finally, a potential ribosome-binding site sequence was found at positions -12 to -8 upstream of the ATG initiation codon (GGAGG). The codon usage was similar to that detected in previously described S. aureus genes (Wada et al., 1992
). The 65·5 mol% A+T content of katA is consistent with the high percentage A+T of base-pairs found in other S. aureus genes.
In the case of S. aureus subsp. anaerobius, sequencing and analysis of a 1747 bp fragment revealed a single ORF of 1368 bp (nucleotides 1661533). This ORF encodes a protein of 455 amino acids with a predicted molecular mass of 52584 Da. Potential -10 and -35 promoter sequences and the putative ribosome-binding site detected in this sequence were identical to those described for S. aureus.
Comparison of amino acid sequences
The predicted amino acid sequences obtained for KatA and KatB were compared to other catalase proteins in the GenBank-EMBL/SWISS-PROT databases. The search revealed significant similarities between the analysed catalases and large regions of numerous eukaryotic and prokaryotic catalases. Considering both identical and conservative replacements of amino acids, KatA and KatB showed more than 50% identity with Bacteroides fragilis catalase KatB, Streptomyces coelicolor catalase CatA, Haemophilus influenzae catalase HktE, Bordetella pertussis catalase KatA, Pseudomonas fluorescens catalase and Neisseria gonorrhoeae catalase (Fig. 4). The degree of similarity was greater in the core of the deduced amino acid sequences than in the N-terminal and C-terminal regions.
|
In the case of the S. aureus subsp. anaerobius catalase, all the residues involved in the active site, haem-binding site and NADPH-binding site are identical to those described for the S. aureus catalase KatA, except the residue Pro-317, involved in the proximal haem-binding site, which is replaced by a Ser residue.
Transcription of katA and katB
Northern blot assays with a katA-specific RNA probe revealed the presence of a transcript of approximately 1·8 kb in samples from early-exponential-phase cultures of S. aureus strains ATCC 12600, MN 42 and MN 73 (Fig. 5). Total RNA isolated from exponentially growing cells of S. aureus subsp. anaerobius hybridized with the same katA-specific RNA probe, showing the presence of a kat-related transcript larger than that observed in S. aureus (approx. 3·7 kb) (Fig. 5
). In addition, other smaller positive hybridization signals were detected; these bands are probably degraded katB-transcript products. When samples were collected from stationary-phase cultures of S. aureus subsp. anaerobius, no hybridization was detected in the tested strains.
|
|
|
Comparison of the katA and katB nucleotide sequences
The nucleotide sequence of S. aureus subsp. anaerobius katB showed 98·7% identity to that of S. aureus katA. Comparative analysis of the two sequences revealed the presence of several mutations in the S. aureus subsp. anaerobius katB gene compared with S. aureus katA. Firstly, a single base-pair deletion located at 1338 bp from the initiation codon was found. This deletion would originate a shift of the nucleotide reading frame and, as a consequence, a termination codon (TAA) is present at position 1368. In addition, 14 single base substitutions were found in katB, eight of which were silent mutations while six were mis-sense. Four of the mis-sense mutations detected in katB lead to a non-conservative replacement of the corresponding amino acid (Arg108Ser, Asp238Gly, Pro317Ser and Asp440Gly) (Fig. 8), while those located at codons 215 and 313 produce a conservative replacement in the KatB amino acid sequence. The most relevant substitution was that located at the first nucleotide of codon 317 (CCA in katA
TCA in katB). As result of this mutation, the non-polar amino acid Pro-317, which is involved in the proximal haem-binding site, was replaced by the polar amino acid Ser-317 (Fig. 8
).
|
Construction and characterization of kat hybrid genes
The katAB construction comprises the 5' end of katA (of approximately 1033 bp) and the 3' end of katB (of approximately 570 bp). In this construction all of the mutations described above have been repaired except the non-conservative substitution at position 1320 (GAC in katAGGC in katB) and the deletion of the nucleotide T-1338. The katBA construction includes the 5' end of katB (of approximately 1033 bp) and the 3' end of katA (of approximately 570 bp). In katBA the deletion of the T-1338 nucleotide as well as the non-conservative substitution located at the second nucleotide of the codon 440 (GAC in katA
GGC in katB) have been repaired. Recombinant hybrids thus generated were confirmed by automatic nucleotide sequencing. Constructions were cloned into pMOSBlue in E. coli, yielding plasmids pKatAB and pKatBA, respectively.
As shown in Fig. 6, E. coli MOSBlue(pKatAB) and E. coli MOSBlue(pKatBA) showed the same pattern of catalase activity as that of the wild-type E. coli strain. In addition, levels of catalase activity showed by E. coli harbouring katAB (185 U mg-1) or katBA (190 U mg-1) constructions were similar to that of the E. coli control (200 U mg-1). The protein band pattern of cell lysates from the E. coli parent strain in SDS-polyacrylamide gels was similar to that of the E. coli strains harbouring pKatAB or pKatBA (Fig. 7a
). When cell extracts from E. coli harbouring pKatAB or pKatBA were analysed by Western blotting, no protein reacting with the purified immunoglobulins against S. aureus catalase was detected (Fig. 7b
).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of katA in E. coli and nucleotide sequence analysis indicated that the cloned DNA encodes the catalase of S. aureus. Multiple amino acid sequence alignment showed a strong relationship between KatA and the prokaryotic catalases from Bacteroides fragilis (65% identity), Streptomyces coelicolor (64% identity), Proteus mirabilis (62% identity) and Haemophilus influenzae (61% identity). However, the KatA sequence showed less than 50% amino acid identity with eukaryotic catalases; this is in agreement with data reported by Von Ossowski et al. (1993) and Klotz et al. (1997)
, who established that animal, plant and fungal catalases in general form a homogeneous group in terms of evolution whereas bacterial catalases appear to have a different phylogenetic origin.
The changes detected in KatA in comparison with BLC in both the proximal haem site (Val-73 in BLCMet-55 in KatA) and the NADPH-binding site (Asp-212
His-194, Lys-236
Arg-218 and Tyr-214
Phe-196) have also been described in the Proteus mirabilis catalase (Gouet et al., 1995
) and, according to biochemical results and molecular modelling, they do not appear to influence either the specific activity of catalase or the interaction of the enzyme with the NADPH molecule (Gouet et al., 1995
).
Northern blotting showed the presence of a kat-specific transcript in samples from exponential-phase cultures of all the S. aureus subsp. anaerobius strains tested, although katB is probably transcribed with other gene(s) since the observed transcript in S. aureus subsp. anaerobius is larger than that of S. aureus controls. The fact that all of the S. aureus subsp. anaerobius strains showed a kat mRNA transcript but no catalase activity could be due to a post-transcriptional defect. Western blot assays favour this hypothesis, as none of these strains showed a catalase-specific band with antisera against the purified S. aureus catalase. Similar results have been reported in several human-pathogenic isolates of coagulase-negative S. aureus, which showed a coa RNA transcript but no detectable coagulase activity (Vandenesch et al., 1994 ). Whether the lack of a kat-related transcript in samples from stationary-phase cultures of S. aureus subsp. anaerobius is related to regulation of catalase gene expression was not investigated; this requires further analysis.
Comparison of the katA and katB nucleotide sequences revealed significant identity (96·5% similarity) between the two catalase genes, but there are some mutations in the S. aureus subsp. anaerobius catalase gene. Firstly, a single base-pair deletion at position 1338 originates a shift of the nucleotide reading frame and would be responsible for the presence of an early termination codon at 1368 bp from the initiation codon. As a consequence of this deletion, the last 50 amino acids of the C-terminal region of KatB would be absent (Fig. 8). As shown by Rocha & Smith (1995)
, The C-terminal region of catalase seems to be essential for enzymic activity. These authors, studying the Bacteroides fragilis catalase gene, found that catalase activity was lost when the last 21 codons of the gene were deleted. Therefore, the loss of the last 50 amino acids from the C terminus in KatB could account for the catalase deficiency of S. aureus subsp. anaerobius. Based upon the three-dimensional structure of the Proteus mirabilis catalase (Gouet et al., 1995
) and a high degree of similarity between the amino acid sequences of P. mirabilis and S. aureus subsp. anaerobius catalases, a hypothesis about the role of this deletion in the lack of catalase activity in S. aureus subsp. anaerobius could be established. Gouet et al. (1995)
determined the structure of the P. mirabilis catalase (484 amino acids) and established that each subunit of this enzyme is composed of four domains. The fourth domain is a bundle of four
-helices (
10 to
13) comprising residues 417 to 484. Residues in this region are present at the protein surface, exhibit a high temperature factor and could play a role in the protein stability (Fita & Rossmann, 1985b
; Gouet et al., 1995
). In addition, these
-helices appear to form a pocket lined by several hydrophobic residues where NADPH is bound (Melik-Adamyan et al., 1986
), and they contribute to forming the hydrophobic channel leading to the haem group (Murthy et al., 1981
). In KatB, as a consequence of the deletion described above, the
12 and
13 helices and part of the
11 helix are not present. The lack of these
-helices would produce an alteration of the NADPH peripheral site and could change the entrance of substrate to the active centre. Moreover, the loss of the
11,
12 and
13 helices in the C-terminal region would sensitize the polypeptide chain to the action of proteases and other degrading enzymes due to the presence of structural changes in the protein surface.
In addition, 14 base substitutions were found in katB, six of which were mis-sense mutations. Four of the mis-sense substitutions in katB would lead to a non-conservative amino acid replacement (Fig. 8), the most significant being that located at position 951 (CCA in katA
TCA in katB) because the affected amino acid is involved in the proximal haem-binding site. As a result of this mutation, the non-polar amino acid proline is replaced by the polar amino acid serine. Fita & Rossmann (1985b)
described the active centre of BLC and proposed that the residue Pro-335 (Pro-317 in KatA) provides a non-polar face which hinders the movement of Tyr-357 (Tyr-339 in KatA). The phenolic hydroxyl group of Tyr-357 interacts with the haem iron and forms hydrogen bonds with Arg-353 (Arg-335 in KatA). Therefore, Pro-335 is involved in the binding of catalase to the haem group and influences the tertiary structure of catalase. The polar serine could thus modify the position of Tyr-339. The Pro
Ser replacement could influence binding of catalase to the haem group and could modify the tertiary structure of the protein, thus altering the formation of hydrogen bonds among other amino acids in the polypeptide chain.
With regard to the other non-conservative substitutions described for katB, it seems that these mutations are not involved in the active site or in the binding of catalase to the haem group and NADPH cofactor. Nevertheless, it cannot be ruled out that some of these amino acid replacements cause a conformational change of the protein such that biological activity of catalase is decreased or nullified.
All of the mutations described in the katB gene isolated from S. aureus subsp. anaerobius MVF 213 were also detected in three other strains of this bacterium isolated from outbreaks of abscess disease which occurred in different Spanish regions from 1981 to 1992. These results suggest that the mutations may be widely present in S. aureus subsp. anaerobius strains, and that the strains tested could have a common origin.
Two chimeras (katAB and katBA) were constructed for establishing whether the two main mutations described above are involved in the lack of catalase activity in S. aureus subsp. anaerobius. Both catalase activity and Western blot analysis of the recombinant hybrids were similar to those found in an E. coli wild-type strain. Consequently, each of the two main alterations found in katB (the deletion and the substitution in the 317 codon) seems to contribute to the lack of catalase activity in S. aureus subsp. anaerobius. Nevertheless, to clarify the individual role of each of the mutations detected in KatB in enzymic activity and in the tertiary structure of the protein, it would be necessary to replace the corresponding residue in S. aureus catalase by site-directed mutagenesis and to analyse the biological activity of each variant enzyme.
In conclusion, a 1368 bp gene isolated from S. aureus subsp. anaerobius with high homology to the S. aureus katA gene, described in this report, has been identified and designated as katB. Northern blotting has showed that katB is transcribed to mRNA and, therefore, the lack of catalase activity in this bacterium is probably due to a post-transcriptional defect. According to data described in this report and taking into account that transcription and translation occur almost simultaneously in bacteria, a protein similar to but smaller than S. aureus catalase is probably synthesized. It is possible that this protein is degraded after synthesis due to the alterations present in the structure and/or in the active site. That could provide an explanation for the lack of reaction of anti-catalase immunoglobulins with crude extracts from S. aureus subsp. anaerobius or E. coli harbouring the katB gene or the kat hybrid genes.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was partially supported by DGICYT (grants PB91-0865 and AGF98-0743).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Clare, D. A., Duong, M. N., Darr, D., Archibald, F. & Fridovich, I. (1984). Effects of molecular oxygen on detection of superoxide radical with nitroblue tetrazolium and on activity stains for catalase.Anal Biochem 140, 532-537.[Medline]
De la Fuente, R. & Suarez, G. (1985). Respiratory deficient Staphylococcus aureus as the aetiological agent of abscess disease.Zentbl Vet Med B 32, 397-406.
De la Fuente, R., Suarez, G. & Schleifer, K. H. (1985). Staphylococcus aureus subsp. anaerobius subsp. nov., the causal agent of abscess disease of sheep.Int J Syst Bacteriol 35, 99-102.
De la Fuente, R., Götz, F. & Shleifer, K. H. (1987). Comparative biochemical studies on aerobic mutants of Staphylococcus aureus subsp. anaerobius.Syst Appl Microbiol 9, 29-33.
De la Fuente, R., Ruiz Santa Quiteria, J. A., Cid, D., Domingo, M. & Suarez, G. (1993). Experimental intramammary infection of ewes with Staphylococcus aureus subsp anaerobius.Res Vet Sci 54, 221-226.[Medline]
Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX.Nucleic Acids Res 12, 387-395.[Abstract]
Fita, I. & Rossmann, M. G.(1985a). Tha NADPH binding site on beef liver catalase.Proc Natl Acad Sci USA 82, 1604-1608.[Abstract]
Fita, I. & Rossmann, M. G. (1985b). The active center of catalase.J Mol Biol 185, 21-37.[Medline]
Gouet, P., Jouve, H. M. & Dideberg, O. (1995). Crystal structure of Proteus mirabilis PR catalase with and without bound NADPH.J Mol Biol 249, 933-954.[Medline]
Haas, A. & Brehm, K. (1993). Superoxide dismutases and catalases: biochemistry, molecular biology and some biomedical aspects.Genet Eng Biotechnol 13, 243-269.
Higuchi, R. (1989). Using PCR to engineer DNA. In PCR Technology. Principles and Applications for DNA Amplification, pp. 61-70. Edited by H. A. Erlich. New York: Macmillan.
Horwitz, M. S. Z. & Loeb, L. A. (1990). Structurefunction relationship in Escherichia coli promoter DNA.Prog Nucleic Acid Res Mol Biol 38, 137-164.[Medline]
Kanafani, H. & Martin, S. E. (1985). Catalase and superoxide dismutase activities in virulent and nonvirulent Staphylococcus aureus isolates.J Clin Microbiol 21, 607-610.[Medline]
Klotz, M. G., Klassen, G. R. & Loewen, P. C. (1997). Phylogenetic relationships among prokaryotic and eukaryotic catalases.Mol Biol Evol 14, 951-958.[Abstract]
Kornblum, J. S., Projan, S. J., Moghazeh, S. L. & Novick, R. P. (1988). A rapid method to quantitate nonlabeled RNA species in bacterial cells.Gene 63, 75-85.[Medline]
Loewen, P. C. (1992). Regulation of bacterial catalase synthesis. In Molecular Biology of Free Radical Scavenging Systems, pp. 96-116. Edited by J. Scandalios. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Mandell, G. L. (1975). Catalase, superoxide dismutase, and virulence of Staphylococcus aureus. In vivo and in vitro studies with emphasis on staphylococcalleucocyte interaction.J Clin Invest 55, 561-566.[Medline]
Melik-Adamyan, W. R., Barynin, V. V., Vagin, A. A., Borisov, V. V., Vainshtein, B. K., Fita, I., Murthy, M. R. N. & Rossmann, M. G. (1986). Comparison of beef liver and Penicillium vitale catalases.J Mol Biol 188, 63-72.[Medline]
Murthy, M. R. N., Reid, T. J.III, Sicignano, A., Tanaka, N. & Rossmann, M. G. (1981). Structure of beef liver catalase.J Mol Biol 152, 465-499.[Medline]
Rocha, E. R. & Smith, C. J. (1995). Biochemical and genetic analyses of a catalase from the anaerobic bacterium Bacteroides fragilis.J Bacteriol 177, 3111-3119.[Abstract]
Ruiz Santa Quiteria, J. A., Cid, D., Bellahsene, R., Suarez, G. & De la Fuente, R. (1992). Polyclonal antibodies against Staphylococcus aureus ATCC 12600 catalase do not recognize any protein in cellular extracts from S. aureus subsp. anaerobius.FEMS Microbiol Lett 72, 173-176.[Medline]
Rupprecht, M. & Schleifer, K. H. (1979). A comparative immunological study of catalases from coagulase-positive staphylococci.Arch Microbiol 120, 53-56.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors.Proc Natl Acad Sci USA 74, 5463-5467.[Abstract]
Sinha, A. K. (1972). Colorimetric assay of catalase.Anal Biochem 47, 389-394.[Medline]
Switala, J., Triggs-Raine, B. L. & Loewen, P. C. (1990). Homology among bacterial catalase genes.Can J Microbiol 36, 728-731.[Medline]
Timoney, J. F., Gillespie, J. H., Scott, F. W. & Baslough, J. E. (1988). The staphylococci. In Hagan and Bruners Microbiology and Infectious Diseases of Domestic Animals, 8th edn, pp. 171180. Ithaca & London: Comstock Publishing.
Vandenesch, F., Lebeau, C., Bes, M., McDevitt, D., Greenland, T., Novick, R. P. & Etienne, J. (1994). Coagulase deficiency in clinical isolates of Staphylococcus aureus involves both transcriptional and post-transcriptional defects.J Med Microbiol 40, 344-349.[Abstract]
Von Ossowski, I., Hausner, G. & Loewen, P. C. (1993). Molecular evolutionary analysis based on the amino acid sequence of catalase.J Mol Evol 37, 71-76.[Medline]
Wada, K., Wada, Y., Ishibashi, F., Gojobori, T. & Ikemura, T. (1992). Codon usage tabulated from the GenBank genetic sequence data.Nucleic Acids Res 20, 2111-2118.[Medline]
Watson, D. L. (1988). Vaccination againts experimental staphylococcal mastitis in ewes.Res Vet Sci 45, 16-21.[Medline]
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13, mp18 and pUC19 vectors.Gene 33, 103-119.[Medline]
Received 11 October 1999;
accepted 5 November 1999.