Biomedical subjects

B L Triggs-Raine

Publications and source records attributed to B L Triggs-Raine.

23 records · Page 2Linked to original sources

PubMed1988

Cloning and physical characterization of katE and katF required for catalase HPII expression in Escherichia coli.

Two genes, katE and katF, affecting the synthesis of catalase HPII in Escherichia coli, have been cloned. The multistep cloning protocol involved: screening for the tet gene in a transposon interrupting the genes, selecting DNA adjacent to the transposon, and using it to probe a library of wild-type DNA to select clones from which katE and katF were subcloned into pAT153. The clones were physically characterized and the presence of the genes confirmed by complementation of their respective mutations. The location of the transposon insertions in the two genes was determined by Southern blotting of genomic digests to further confirm the identity of the cloned genes. A 93-kDa protein, the same size as the subunit of HPII, was encoded by the katE plasmid, indicating that katE was the structural gene for HPII. A 44-kDa protein was encoded by the katF plasmid.

Bacteriophage lambda↗

PubMed1988

Nucleotide sequence of katG, encoding catalase HPI of Escherichia coli.

The gene katG, encoding catalase HPI of Escherichia coli, was sequenced, predicting a 726-amino-acid protein. The sequence was confirmed by identification of potential regulatory elements and amino acid sequencing of peptides. HPI shows no homology to other catalases. The distances between katG, metF, and ppc were defined.

Amino Acid Sequence↗

PubMed1987

Physical characterization of katG, encoding catalase HPI of Escherichia coli.

The gene encoding the bifunctional catalase-peroxidase HPI from Escherichia coli was located on a 3.8-kb HindIII fragment of the Clarke and Carbon plasmid pLC36-19 using transposon Tn5 insertions. This fragment was subcloned into the HindIII site of pAT153 to create pBT22. The size of the insert was reduced by BAL 31 digestion of one end to an apparent minimum size for catalase expression of approx. 2.5 kb as determined by complementation and expression in maxicell strains. Further reduction in size or digestion from the opposite end inactivated the gene. The location and orientation of the promoter at the 0 kb end of the insert in pBT22 was confirmed by cloning a 320-bp BglII fragment into the promoter-cloning vector pKK232-8. Differences in the Southern blots of genomic DNA from a wild-type strain and a katG17::Tn10 mutant digested with HincII and probed with pBT22 confirmed that the transposon previously mapped in katG was located in the 2.5-kb coding region for HPI.

Catalase↗

PubMed1985

Catalases HPI and HPII in Escherichia coli are induced independently.

Three strains of Escherichia coli differing only in the catalase locus mutated by transposon Tn10 were constructed. These strains produced only catalase HPI (katE::Tn10 and katF::Tn10 strains) or catalase HPII (katG::Tn10). HPI levels increased gradually about twofold during logarithmic growth but did not increase during growth into stationary phase in rich medium. HPII levels, which were initially threefold lower than HPI levels, did not change during logarithmic growth but did increase tenfold during growth into stationary phase. HPI levels increased in response to ascorbate or H2O2 being added to the medium but HPII levels did not. In minimal medium, any carbon source derived from the tricarboxylic acid cycle caused five- to tenfold higher HPII levels during logarithmic growth but had very little effect on HPI levels. Active electron transport did not affect either HPI or HPII levels.

Ascorbic Acid↗

PubMedDate not supplied

Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli.

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, less than 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to beta-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.

Base Sequence↗