Cloning, characterization and phenotypic expression in Escherichia coli of catF, which encodes the catalytic subunit of catalase isozyme CatF of Pseudomonas syringae.
The phytophathogenic, gram-negative bacterium Pseudomonas syringae pv. syringae 61 contains three isozymes of catalase (EC 1.11.1.6), which have been proposed to play a role in the bacterium's responses to various environmental stresses. To study the role of individual isozymes, the gene coding for the catalytic subunit of one catalase isozyme was cloned from a cosmid library hosted in Escherichia coli DH5 alpha by using a designed catalase-specific DNA probe for the screening. One out of four clones with a catalase-positive genotype was subcloned and a pUC19-based 2.7 x 10(3)-base (2.7-kb) insert subclone, pMK3E5, was used to transform catalase-deficient E. coli strain UM255 (HPI-, HPII-). The transformants contained a single isozyme of catalase that had electrophoretic and enzymic properties similar to catalase isozyme CatF from P. syringae pv. syringae 61. Analysis of the sequenced 2.7-kb insert DNA revealed six putative open-reading frames (ORF). The 1542-base-pair DNA sequence of ORF2, called catF, encodes a peptide of 513 amino acid residues with a calculated molecular mass of 66.6 kDa. The amino acid sequence deduced from catF had homology to the primary structure of true catalases from mammals, plants, yeasts and bacteria. The activity of the recombinant catalase was inhibited by 3-amino-1,2,4-triazole and azide and stimulated by chloramphenicol. The N terminus contained a signal sequence of 26 amino acids necessary for secretion into the periplasm, a so-far unique property of Pseudomonas catalases.
Klotz
M G
MG
Biology Department, Utah State University, Logan 84322-5305, USA.
Kim
Y C
YC
Katsuwon
J
J
Anderson
A J
AJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002374:Catalase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010641:Phenotype; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis
D007527:Isoenzymes; D011994:Recombinant Proteins; D002374:Catalase