Experimental analysis of Helicobacter pylori transcriptional terminators suggests this microbe uses both intrinsic and factor‐dependent termination

Abstract

SummaryIn this study, we report experimental analysis of transcriptional terminators in the human pathogen Helicobacter pylori. Previous bioinformatics approaches came to differing conclusions regarding transcriptional termination in this bacterium. We used a reporter construct, the tnpR-encoded resolvase, to assess terminators. In our first experiments, we found that a subset of previously predicted intrinsic terminators for H. pylori are functional. In our second experiments, we used an unbiased screen to identify putative terminators and then characterized 18 of these. We found that these putative terminators overlap genomic regions that are either intergenic or intragenic. Using reverse transcription PCR, we showed that an intergenic terminator and an intragenic antisense terminator function at their endogenous loci. Additionally, we found that putative terminators contain features of both intrinsic and Rho-dependent termination, but that intrinsic terminators define the majority. We were unable to delete rho, however, in H. pylori, suggesting that it is essential and likely important. Finally, we carried out a mutational analysis of one of our randomly identified terminators that has both intrinsic and Rho-dependent features, and found that they are both functional. In conclusion, we found that H. pylori possesses numerous Rho-dependent and intrinsic terminators including some found in intragenic regions.

Functional Analysis of theHelicobacter pyloriFlagellar Switch Proteins

“…In each case, the majority of the open reading frame was deleted in frame and replaced with a cat gene (that lacked its transcriptional terminator), as detailed in Table 1, to create null mutants. Previous work from our lab has shown that this cat allele is not polar (8,24,45), and we verified that genes downstream of fliN, fliM, and fliY were unaffected using reverse transcription-PCR (see Fig. S2 in the supplemental material), suggesting that these mutations are nonpolar.…”

The Complete Genome Sequence ofHelicobacter pyloriStrain G27

“…In addition, it efficiently delivers the translocated effector protein CagA to cells in culture, facilitating the cell biological analysis of this important virulence factor (2,8,9,12,13). The strain has also been subjected to multiple experimental adaptations to new environments, including growth on canine kidney epithelial cells (2), serial passage through the mouse stomach (3,5), and adaptation to in vitro growth in the presence or absence of a functional natural transformation system (4). Determination of the complete genome sequence of G27 will facilitate research with this strain and provide a foundation for molecular evolution studies of the genetic basis for its adaptation to new environments.…”

Recombination-Based In Vivo Expression Technology IdentifiesHelicobacter pyloriGenes Important for Host Colonization

“…H. pylori strain mG27 is a mouse-adapted descendant of clinical isolate G27 (19,23). mG27 was generated by serially passaging the G27 H. pylori strain in mice (19). All H. pylori strains were cultured on Columbia horse blood agar (CHBA) or in brucella broth supplemented with 10% fetal bovine serum (BB10) and were grown at 37°C under microaerobic conditions with a gas mixture containing 5 to 10% O 2 , 10% CO 2 , and 80 to 85% N 2 .…”

“…H. pylori strain mG27 is a mouse-adapted descendant of clinical isolate G27 (19,23). mG27 was generated by serially passaging the G27 H. pylori strain in mice (19).…”

“…This plasmid contains the Campylobacter coli gene for Cm resistance (Cm r ) (cat), a strong E. coli transcriptional terminator (rrnBT 1 T 2 ) (62), and a promoterless tnpR gene, all of which are flanked by sequences of the H. pylori rdxA gene. cat is transcribed from its own promoter, and the strong E. coli terminator rrnBT 1 T 2 , prevents read-through transcription into tnpR (19). The pcat-T-tnpR plasmid directs recombination into the middle of the rdxA locus on the H. pylori chromosome (80).…”