Catalase deficiency in Staphylococcus aureus subsp. anaerobius is associated with natural loss-of-function mutations within the structural gene.
Degenerate oligonucleotide primers based on internal peptide sequences obtained by HPLC from purified Staphylococcus aureus catalase were used to locate the S. aureus and S. aureus subsp. anaerobius kat regions by PCR. Southern hybridization analysis with a probe derived from a 1.1 kb PCR-amplified fragment showed that a single copy of the putative catalase gene was present in the S. aureus and S. aureus subsp. anaerobius chromosome. The nucleotide sequence of S. aureus katA revealed a 1518 bp open reading frame for a protein with 505 amino acids and a predicted molecular mass of 58347 Da, whereas S. aureus subsp. anaerobius katB is 1368 nt long and encodes a polypeptide of 455 amino acids with a predicted molecular mass of 52 584 Da. These catalases are highly homologous to typical monofunctional catalases from prokaryotes. The active-site residues, proximal and distal haem-binding ligands and NADPH-binding residues of the bovine liver catalase-type enzyme were highly conserved in S. aureus KatA. Escherichia coli cells carrying cloned katA had a catalase activity approximately 1000 times that of untransformed E. coli, but no detectable increase in catalase activity was observed with E. coli carrying cloned katB. Northern blotting showed the presence of a kat-specific transcript in S. aureus subsp. anaerobius, suggesting that the lack of catalase activity in this bacterium is due to a post-transcriptional alteration. Compared to the nucleotide sequence of katA, katB showed a single base-pair deletion and six mis-sense mutations, and these alterations were present in three other S. aureus subsp. anaerobius strains analysed. The deletion, located at 1338 bp from the initiation codon, originates a shift of the nucleotide reading frame and is responsible for the premature translation termination at 1368 bp, generating a KatB polypeptide 50 amino acid residues shorter than KatA. Moreover, four of the mis-sense mutations present in katB lead to non-conservative amino acid replacements, the most significant being that located at residue 317 (Pro in KatA-->Ser in KatB) because the affected amino acid is involved in determining the proximal haem-binding site. Both the main alterations found in KatB (the deletion and the substitution in residue 317) seem to contribute to the lack of catalase activity in S. aureus subsp. anaerobius, as deduced from results obtained with chimeric catalase constructs.
Sanz
Rosario
R
Departamento Patologı́a Animal I, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain1.
Marı N
Irma
I
Centro de Biologı́a Molecular, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain2.
Ruiz-Santa-Quiteria
Jose A
JA
Departamento Patologı́a Animal I, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain1.
Orden
Jose A
JA
Departamento Patologı́a Animal I, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain1.
Cid
Dolores
D
Departamento Patologı́a Animal I, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain1.
Diez
Rosa M
RM
Departamento Patologı́a Animal I, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain1.
Silhadi
K Souad
KS
Centre National de Référence de Toxémies à Staphylocoques, EA 1655, Faculté de Médecine, rue Guillaume Paradin, 69372 Lyon cedex 08, France3.
Amils
Ricardo
R
Centro de Biologı́a Molecular, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain2.
de la Fuente
Ricardo
R
Departamento Patologı́a Animal I, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain1.
D000595:Amino Acid Sequence; D000818:Animals; D002374:Catalase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013211:Staphylococcus aureus / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic
D011994:Recombinant Proteins; D002374:Catalase