Manos J, Kolesnikow T, Hazell S L
School of Microbiology and Immunology, University of New South Wales, Sydney, Australia.
Helicobacter. 1998 Mar;3(1):28-38. doi: 10.1046/j.1523-5378.1998.08030.x.
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.
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.
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.
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.
幽门螺杆菌中发现了一种高活性过氧化氢酶，在某些菌株中该酶可能会失去活性，这引起了强烈的科学兴趣。我们对一株自发过氧化氢酶阴性的幽门螺杆菌分离株（新南威尔士大学-RU1）进行了特性分析，并对亲本菌株中的katA以及两种表型的启动子进行了测序，以此作为理解导致过氧化氢酶表达失败的遗传过程的前奏。
通过二维聚丙烯酰胺凝胶电泳（2D-PAGE）检测两种表型的蛋白质提取物中的过氧化氢酶，并通过基于蛋白质印迹的免疫分析进行分析。通过Northern印迹检测过氧化氢酶mRNA的存在。使用高保真PCR对katA启动子进行测序，同时使用循环测序法对katA进行测序。通过引物延伸确定转录起始位点。
二维聚丙烯酰胺凝胶电泳和蛋白质印迹显示新南威尔士大学-RU1（KatA-）中不存在过氧化氢酶蛋白，Northern印迹显示也不存在过氧化氢酶mRNA，这表明KatA-表型的原因在于转录水平。在KatA-分离株的启动子区域未发现突变。转录起始位点位于ATG位点上游55 bp处，推定的RNA聚合酶结合位点定位于“-10”和“-35”。在转录起始位点上游181 bp处鉴定出一个铁摄取调节蛋白（Fur）盒。鉴定出一个876 bp开放阅读框（ORF）和一个366 bp大肠杆菌phnA同源物的序列。
新南威尔士大学-RU1（KatA-）表型不表达KatA或转录katA。其启动子和大部分开放阅读框没有缺陷，这表明活性丧失可能是由于过氧化氢酶表达所必需的辅助基因发生突变，或者是由于阻遏物结合阻止了katA转录。
