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 programme 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. © 2013 John Wiley & Sons Ltd.
CITATION STYLE
Sycuro, L. K., Rule, C. S., Petersen, T. W., Wyckoff, T. J., Sessler, T., Nagarkar, D. B., … Salama, N. R. (2013). Flow cytometry-based enrichment for cell shape mutants identifies multiple genes that influence Helicobacter pylori morphology. Molecular Microbiology, 90(4), 869–883. https://doi.org/10.1111/mmi.12405
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