Acid-induced gene expression in Helicobacter pylori: study in genomic scale by microarray

Abstract

To understand the RNA expression in response to acid stress of Helicobacter pylori in genomic scale, a microarray membrane containing 1,534 open reading frames (ORFs) from strain 26695 was used. Total RNAs of H. pylori under growth conditions of pH 7.2 and 5.5 were extracted, reverse transcribed into cDNA, and labeled with biotin. Each microarray membrane was hybridized with cDNA probe from the same strain under two different pH conditions and developed by a catalyzed reporter deposition method. Gene expression of all ORFs was measured by densitometry. Among the 1,534 ORFs, 53 ORFs were highly expressed (> or = 30% of rRNA control in densitometry ratios). There were 445 ORFs which were stably expressed (<30% of rRNA in densitometry) under both pH conditions without significant variation. A total of 80 ORFs had significantly increased expression levels at low pH, while expressions of 4 ORFs were suppressed under acidic condition. The remaining 952 ORFs were not detectable under either pH condition. These data were highly reproducible and comparable to those obtained by the RNA slot blot method. Our results suggest that microarray can be used in monitoring prokaryotic gene expression in genomic scale.

FIG. 1

Colorimetric detection of H. pylori total RNA expression on a microarray. Comparison of acidic stress responses by culture at pH 7.2 (A) and pH 5.5 (B). The expression level of 23S rRNA at pH 5.5 was slightly higher than those at pH 7.2; however, each mRNA expression was standardized by using 23S rRNA as an internal control. 23S: additional 23S rRNA gene. The figures are amplified from 0.4 by 0.4 in. to 4 by 4 in.

FIG. 2

Slot blot hybridization of RNA isolated from H. pylori cells grown at pH 7.2 and 5.5. Probes were either the cloned 23S H. pylori rRNA gene or the pCRII insert (H. pylori ORFs). The amount of RNA used for all conditions was standardized at 10 μg per slot. The expressions of these ORFs were much greater at acidic pHs than at pH 7.2. 23S, H. pylori 23S rRNA, 5.5, total RNA isolated from growth condition at pH 5.5, 7.2, total RNA isolated from growth condition at pH 7.2; ORF, probes that were used for hybridization.

FIG. 3

Semiquantification of HP1037 ORF mRNA by PCR. Total RNAs were isolated at pH 7.2 (A) and pH 5.5 (B) separately. One microgram from each was reverse transcribed with antisense primer HP1037-r. Tenfold serial dilutions of cDNA were made, and the end point was determined by negativity of PCR. (−), RNA templates not subjected to the RT reaction served as negative control to exclude the possibility of contamination of genomic DNA; 1×, 1 μg of total RNA used as template; 10×, 100×, and 1000×, sample was at a 10-, 100-, or 1,000-fold serial dilution, respectively.

FIG. 4

Determination of detection limit of CARD method. To determine the detection limit of the CARD method, spotted 23S rRNA genes in the array membrane were hybridized with in an vitro-transcribed 23S rRNA fragment. Biotin-cDNA probe was synthesized during the RT reaction. Tenfold dilutions of cDNA probes from 1 μg of rRNA were made according to CARD protocols. The limit of CARD was determined to be 50 pg, which corresponds to approximately 10⁸ copies of 23S rRNA.