Flow cytometry‐based enrichment for cell shape mutants identifies multiple genes that influence Helicobacter pylori morphology

Abstract

Summary The helical cell shape of Helicobacter pylori is highly conserved and contributes to its ability to swim through and colonize the viscous gastric mucus layer. A multi-faceted peptidoglycan (PG) modification program involving four recently characterized peptidases and two accessory proteins is essential for maintaining H. pylori's helicity. To expedite identification of additional shape-determining genes, we employed flow cytometry with fluorescence-activated cell sorting (FACS) to enrich a transposon library for bacterial cells with altered light scattering profiles that correlate with perturbed cell morphology. After a single round of sorting, 15% of our clones exhibited a stable cell shape defect, reflecting 37-fold enrichment. Sorted clones with straight rod morphology contained insertions in known PG peptidases, as well as an insertion in csd6, which we demonstrated has LD-carboxypeptidase activity and cleaves monomeric tetrapeptides in the PG sacculus, yielding tripeptides. Other mutants had only slight changes in helicity due to insertions in genes encoding MviN/MurJ, a protein possibly involved in initiating PG synthesis, and the hypothetical protein HPG27_782. Our findings demonstrate FACS robustly detects perturbations of bacterial cell shape and identify additional PG peptide modifications associated with helical cell shape in H. pylori.

Antimicrobial and Virulence-Modulating Effects of Clove Essential Oil on the Foodborne Pathogen Campylobacter jejuni

“…Some essential oils have been described as having modulating effects on the morphology of other bacteria: cumin EO on Klebsiella (58), lemon EO on E. coli (59), and oregano EO on Pseudomonas aeruginosa and Staphylococcus aureus (60). In line with these findings, our observations raise the possibility of one or some components of EOs having an effect on the structures determining cell shape, e.g., peptidoglycan (61) and certain other proteins, mostly those anchored in the cell membrane.…”

Helical Shape of Helicobacter pylori Requires an Atypical Glutamine as a Zinc Ligand in the Carboxypeptidase Csd4

“…8A) but has an increased population of straight cells that have side curvature values less than 4 compared with the strain with a single copy of the Q allele (Fig. 8B), as was reported previously (10). The perturbation of cell shape during overexpression was suggested to result from a requirement of precise asymmetric localization of Csd4 activity to generate proper helical curvature.…”

“…In turn, the tetrapeptidecleaving LD-carboxypeptidase Csd6 was recently shown to provide the tripeptide substrate for Csd4. Overexpression (as well as loss) of either Csd4 or Csd6 perturbs helical morphology; thus peptide hydrolysis by Csd4 to achieve a helical bacterial shape is likely to require precise spatial coordination within the PG layer (10).…”

Staying in Shape: the Impact of Cell Shape on Bacterial Survival in Diverse Environments

“…These bacteria do not possess an equivalent creS homolog, indicating that the vibrioid morphology is generated by an alternate mechanism. Interestingly, members of the Vibrionaceae family contain homologs of genes that promote helical shape in Helicobacter pylori and Campylobacter jejuni (described in the next section) (19,(44)(45)(46)(47). Identification of the genes governing curved shape in the Vibrionaceae would allow exploration of the functional significance of the vibrioid shape of these organisms in the aquatic environment, the mammalian host, or both.…”