BioProject

In this study targets of the RNA pyrophosphohydrolase RppH in H. pylori 26695 (HP1228) were identified. More...

In this study targets of the RNA pyrophosphohydrolase RppH in H. pylori 26695 (HP1228) were identified. Overall design: To investigate the global role of HpRppH (HP1228) in converting 5’ triphosphates to monophosphates in H. pylori, we used a variant of differential RNA-seq (dRNA-seq) to compare the transcriptome of isogenic strains containing or lacking the rppH gene. For this purpose, we constructed two derivatives of the wild-type H. pylori strain 26695: an rppH deletion mutant (ΔrppH) and an rppH complementation strain (CrppH) bearing an ectopic copy of the rppH gene. The ΔrppH strain was generated by replacing the rppH gene of wild-type cells (WT) with a kanamycin-resistance cassette. The CrppH strain was then constructed by complementing this deletion with an ectopic copy of the H. pylori rppH gene under the control of its own promoter, which was introduced at an unrelated locus (rdxA) previously used as a site for integrating genes into the H. pylori chromosome. These isogenic H. pylori strains were grown to log phase, and total RNA isolated from each was used to generate three libraries specific for transcripts bearing (1) a 5'-triphosphate, (2) a 5'-monophosphate, or (3) either a 5'-triphosphate or a 5'-monophosphate. This was accomplished by differential treatment of cellular RNA with Terminator 5‘-phosphate-dependent exonuclease (TEX) and Tobacco Acid Pyrophosphatase (TAP). The 5’ exonuclease activity of TEX digests 5‘-monophosphorylated RNAs but leaves triphosphorylated transcripts intact. Subsequent treatment of the latter set of transcripts with TAP generates monophosphorylated 5’ ends to which an RNA oligonucleotide can be ligated, thereby enabling cDNA synthesis. By contrast, treatment with TAP alone enables cDNA synthesis from both triphosphorylated and monophosphorylated RNAs, whereas treatment with neither enzyme allows cDNA synthesis only from cellular RNAs that are already monophosphorylated. Therefore, to identify RNAs in each category, we generated cDNA libraries specific for transcripts with a 5'-triphosphate (+TEX/+TAP), a 5'-monophosphate (-TEX/-TAP), or both (-TEX/+TAP) from all three strains (Figure 5A) and subjected them to Illumina sequencing. Less...