An Investigation of the Molecular Basis of the Spontaneous Occurrence of a Catalase‐Negative Phenotype in Helicobacter pylori

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Abstract
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. Materials and 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.