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

Laura K. Sycuro, Chelsea S. Rule, Timothy W. Petersen, Timna J. Wyckoff, Tate Sessler, Dilip B. Nagarkar, Fakhra Khalid, Zachary Pincus, Jacoby Biboy, Waldemar Vollmer, Nina R. Salama

Research output: Contribution to journalArticlepeer-review

63 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)869-883
Number of pages15
JournalMolecular Microbiology
Volume90
Issue number4
DOIs
StatePublished - Nov 2013

Fingerprint

Dive into the research topics of 'Flow cytometry-based enrichment for cell shape mutants identifies multiple genes that influence Helicobacter pylori morphology'. Together they form a unique fingerprint.

Cite this