Systematic Identification of Selective Essential Genes in Helicobacter pylori by Genome Prioritization and Allelic Replacement Mutagenesis

Abstract

A comparative genomic approach was used to identify Helicobacter pylori 26695 open reading frames (ORFs) which are conserved in H. pylori J99 but highly diverged in other eubacteria. A survey of selected pathways of central intermediary metabolism was also carried out, and genes with a potentially selective role in H. pylori were identified. Forty-five ORFs identified in these two analyses were screened using a rapid vector-free allelic replacement mutagenesis technique, and 33 were shown to be essential in vitro. Notably, 13 ORFs gave essentiality results which are unexpected in view of their known or proposed functions, and phylogenetic analysis was used to investigate the annotation of 7 such ORFs which are highly diverged. We propose that the products of a number of these H. pylori-specific essential genes may be suitable targets for novel anti-H. pylori therapies.

Alternative Spermidine Biosynthetic Route Is Critical for Growth of Campylobacter jejuni and Is the Dominant Polyamine Pathway in Human Gut Microbiota

“…However, H. pylori contains spermidine as its only polyamine (29), and as no orthologue of AdoMetDC is present, the H. pylori CANSDC must be considered a CASDC. There is no AdoMetDC orthologue in H. pylori; however, a divergent SpdSyn-like gene is present, which was shown by Chalker et al (28) to be inessential for colony growth.…”

“…A gene described as a CANSDC was deleted in the gastric pathogen H. pylori 26695 as part of a systematic screen for genes essential for growth in this strain (28). The CANSDC orthologue was essential for growth as determined by a colony growth assay.…”

Characterization of Unique Modification of Flagellar Rod Protein FlgG by Campylobacter jejuni Lipid A Phosphoethanolamine Transferase, Linking Bacterial Locomotion and Antimicrobial Peptide Resistance

“…Construction of Select Deletion Mutants in C. jejuni-Deletion mutants of flgF and flgG gene operon (CJJ81176_720 and _721) were constructed as described previously (20). Briefly, an upstream (primers 1 and 3) and downstream (primers 2 and 6) region flanking the flgFG operon (Ϯ 1,000 bp) was PCR-amplified from C. jejuni strain 81-176 genomic DNA.…”

Helicobacter pylori Adhesion to Carbohydrates

“…In our experience, all selected transformants contain the desired mutant allele in place of the original wild-type allele, unless the gene in question is essential for viability (Chalker et al, 2001;Tan and Berg, 2004;Tan et al, 2005). …”

“…In one application, this "crossover PCR" was used to map multiple sites of transposon insertion in a gene (Krishan et al, 1991). Chalker et al (2001), adapted this principle to the high throughput construction of deletion alleles of 42 H. pylori genes of to them, essentially as diagrammed in Fig. 4.…”