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.
Highlights
Manual visual screens of transposon mutant libraries for rare bacterial cell shape mutants, though tedious, have been fruitful in uncovering novel machinery that generates cell shape
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
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
Summary
Manual visual screens of transposon mutant libraries for rare bacterial cell shape mutants, though tedious, have been fruitful in uncovering novel machinery that generates cell shape. CreS, the Caulobacter crescentus cytoskeletal protein required for that organism’s curved rod shape, was first discovered in such a screen (Ausmees et al, 2003), as were Csd and Csd, the peptidoglycan (PG) peptidases we previously reported to be essential for Helicobacter pylori’s helical morphology (Sycuro et al, 2010; 2012). One advantage of these types of screens is their indiscriminate nature; any and all types of morphological aberrations may be observed and isolated for study. According to current models of PG assembly, high-molecular-weight penicillinbinding proteins (H. pylori has one class A PBP, PBP1, and two class B PBPs, PBP2 and PBP3) carry out glycosyl-
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