Transcriptional analysis of the divergent cagAB genes encoded by the pathogenicity island of Helicobacter pylori

Abstract

SummaryHelicobacter pylori strains isolated from most patients with peptic ulcer disease and adenocarcinoma express the vacuolating toxin VacA and contain a pathogenicity island named cag. The cag pathogenicity island codes for more than 40 putative proteins with features similar to bacterial secretion systems. One of these proteins, CagA, is an immunodominant antigen with unknown function encoded by the cagA gene. In the present study, we have analysed the functional promoter elements of the H. pylori cagA gene as well as of the divergently transcribed cagB gene. Primer extension analyses identified a single 5Ј end of the cagA mRNA, while two initiation sites were mapped in the case of the cagB mRNA. The promoters deduced upstream of these start points of transcription contained conserved ¹10 regions but no ¹35 regions with respect to the Escherichia coli 70 consensus sequence. Nevertheless, they could be activated in E. coli and in vitro by purified E. coli RNA polymerase. Deletion analyses indicated that the cagA and cagB genes are transcribed by overlapping promoters and that full activation requires sequences up to ¹70 and ¹96 respectively. Instead, basal transcription is likely to be mediated by ¹10 extended promoter-like sequences. RNA polymerase is able to bind the ¹40 to ¹60 region of the cagA promoter, and its binding is mediated by the ␣-subunit. This region resembles the UP elements of prokaryotic promoters in location, sequence and mechanism of interaction with the RNA polymerase. We discuss the features of these promoters and propose that they could represent a class of minimum promoters, which ensures a basic level of transcription, while full activation requires regulatory elements or a defined promoter context.

Growth Phase-Dependent Regulation of Target Gene Promoters for Binding of the Essential Orphan Response Regulator HP1043 of Helicobacter pylori

“…Total H. pylori RNA was extracted as described previously (45). Briefly, 25 ml of H. pylori grown in modified brucella broth at 37°C was harvested at an OD 590 between 0.6 and 1.0 (or otherwise where indicated).…”

“…Progressing through stationary to late stationary phase (represented in lanes 6 to 9), the transcription from both promoters is further coordinately reduced. As a control of this experiment, we also monitored transcription from P cagA , a promoter whose expression has been studied in detail (45). The P cagA promoter is expressed throughout the time course experiment (Fig.…”

The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in Neisseria meningitidis

“…After 15 min, the cultures were added to an equal volume of equivalent frozen medium to bring the temperature immediately to 4°C and then centrifuged at 3,000 rpm in a benchtop centrifuge at 4°C. RNA was extracted from the pelleted cells as previously described (31) or for use in microarray experiments using an RNeasy minikit (Qiagen).…”

Promoters of the CATG-Specific Methyltransferase Gene hpyIM Differ between iceA1 and iceA2 Helicobacter pylori Strains

“…The hpyIM promoters contain Ϫ10 hexamers with homology to the E. coli Ϫ10 consensus sequence; however, except for the promoter P I , which exhibits a Ϫ35 hexamer with similarity to the E. coli Ϫ35 consensus sequence, no obvious Ϫ35 box was found in the hpyIM promoters. Promoter differences between H. pylori and other bacteria, including E. coli, have been reported (13,21,24). New motifs, TTAAGC or TTTTAA, have been proposed to be combined with the Ϫ10 hexamer in H. pylori promoters (24).…”