An investigation of the molecular basis of the spontaneous occurrence of a catalase-negative phenotype in Helicobacter pylori.
BACKGROUND
The discovery of a highly active catalase in Helicobacter pylori that in some strains may lose its activity has generated strong scientific interest. We have characterized a spontaneous catalase-negative isolate of H. pylori (UNSW-RU1) and sequenced katA in the parent strain and the promoters of both phenotypes as a prelude to understanding the genetic processes leading to the failure to express catalase.


METHODS
Protein extracts from both phenotypes were examined for catalase on 2D-PAGE and analyzed by Western blot-based immuno-analysis. Presence of catalase mRNA was detected by Northern blot. Hi-Fidelity PCR was used to sequence the katA promoter while katA was sequenced using cycle-sequencing. The transcription start site was located by primer extension.


RESULTS
Catalase protein was absent in UNSW-RU1 (KatA-) by 2D-PAGE and Western blot, as was catalase mRNA by Northern blot, indicating that the cause of the KatA- phenotype was at the level of transcription. No mutations were found in the promoter region of the KatA- isolate. The transcription start site was identified 55 bp upstream of the ATG site and putative RNA polymerase binding sites were mapped at "-10" and "-35". A Fur box was identified 181 bp upstream of the transcription start site. The sequences of an 876 bp ORF and a 366 bp Escherichia coli phnA homologue were identified.


CONCLUSIONS
The UNSW-RU1 (KatA-) phenotype does not express KatA or transcribe katA. The absence of defects in its promoter and a large part of its ORF indicates that loss of activity may be due to a mutation in an accessory gene essential for catalase expression, or to the binding of a repressor preventing katA transcription.
Manos
J
J
School of Microbiology and Immunology, University of New South Wales, Sydney, Australia.
Kolesnikow
T
T
Hazell
S L
SL
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D002374:Catalase / Q000032:analysis* / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010641:Phenotype; D019105:Random Amplified Polymorphic DNA Technique; D017421:Sequence Analysis; D014158:Transcription, Genetic / Q000502:physiology
D001426:Bacterial Proteins; D002374:Catalase