H. pylori cell shape loci and their associated mutant morphologies. A) Three-gene shape locus identified in visual screen. The insertion site of the curved rod shape mutant’s transposon encoding chloramphenicol acetyltransferase (cat) in reverse orientation occurred approximately 100 bp into the HPG27_1481 open reading frame. B–G) Phase contrast and transmission electron microscopy (TEM) images of wild-type and mutant cells. H) Single-gene shape locus containing csd3. I–L) TEM images of single csd3 mutant cells (I–J) and csd3 mutant cells that are elongated and preparing to divide (K–L, septa indicated by arrows). M–O) Phase contrast and TEM images (inset) of wild-type and mutant cells treated with the filamenting drug aztreonam. Strains used: LSH13, KGH8, KGH10, LSH112. See also Figures S1 and S2.

Conservation of Csd proteins in Epsilonproteobacteria and Gammaproteobacteria. A) Multisequence alignment of LytM domains in H. pylori, C. jejuni, V. cholerae, and T. crunogena Csd1 and Csd3 protein homologues listed by species and locus tag. Stars indicate positions of conserved LytM active site motifs HXXXD and HXH (Ragumani et al., 2008). The arrow shows histitidine residue mutated to alanine (H250A). B) Distribution of wild-type, csd1 null, and csd1^H250A point mutant populations. C) Distribution of csd1 null population compared to cross-species complementation of csd1 from C. jejuni (Cj) and V. cholerae (Vc). Semi-helical shapes rarely observed in cross-species complementation strains and never in the parent csd1 null are marked with green arrowheads. D) Smooth histograms showing the side curvature distribution for the above populations. All mutant and complement populations are significantly different from wild-type (bootstrapped K–S p-value < 0.00001), but indistinguishable from csd1 null (p > 0.9). Strains used: NSH57, LSH13, LSH153, NSH142, NSH144..See also Table S1 and Figure S3.

Phenotypes of non-helical mutants and complemented strains in the mouse stomach and related in vitro conditions. Complementation strains have the native locus disrupted by deletion and insertion of cat and the indicated gene expressed from the rdxA locus. A) Motility phenotype of mutant and complemented strains in soft agar (mean halo diameter inclusive of two or three independent experiments totaling 40–50 stabs per strain ± SD in 0.3% soft agar after four days). Stars indicate significant difference from wild-type (p < 0.05, ANOVA with Bonferroni correction). B–C) Survival in stomach stress conditions from two independent experiments, each with four replicates per strain and condition (mean survival ratio ± SEM). D–E) One week C57BL/6 mouse competition data. Each data point represents a single mouse. Data are plotted as a competitive index: [CFU/mL_MUT:CFU/mL_WT stomach output]/[CFU/mL_MUT:CFU/mL_WT inoculum]. Filled points indicate mice from which only one strain was recovered, causing the competitive index to be driven largely by the load of the infecting (wild-type) strain. Mean loads were 560 CFU/stomach, range 100–1050 (D) and 6600, range 2100–19100 (E). Black bars are the geometric mean. F) One week C57BL/6 single strain colonization data. Each data point represents the load (CFU/g) from a single mouse. Filled points indicate mice from which no bacteria were recovered and are plotted at the detection limit. Black bars are the geometric mean. Strains used: A, D, and F) LSH100, LSH113, LSH141, LSH142, LSH119, LSH126; B–C) NSH57, LSH13; E) NSH57, LSH11, LSH102. See also Figure S5.

Morphological characterization of single and multiple-gene deletion mutants. A, D, E) Scatter plots arraying wild-type and/or mutant populations by cell length (x-axis) and cell curvature (y-axis). A small proportion (~14–18%) of cells in mutant strains containing a csd3 deletion were so highly curved as to erroneously appear coccoid after image processing; these cells were manually removed from the analysis. B, C, F) Smooth histograms displaying population cell curvature (x-axis) as a density function (y-axis) and p-values from bootstrapped Kolmogorov–Smirnov statistical comparisons. Replicate csd1 mutant populations used to define the null distribution for statistical comparisons of curved rod strains are shown in panel B. Strains used: NSH57, LSH13, KGH8, KGH10, LSH112, LSH129, LSH130, LSH136, LSH143.

Transmission electron microscopy (TEM) images and schematic illustration of peptidoglycan (PG) sacculi. A–B) Schematic drawing (A) outlining the poorly visible uranyl acetate-stained wild-type H. pylori sacculus depicted in the image directly below (B). C) Negatively stained wild-type H. pylori sacculi. Morphological variability among the sacculi is a product of their two-dimensional flattening during fixation on the grid, but the apparent wave form of the sacculi approximates the helical morphology of intact cells as observed by TEM (Figure 1C). D–F). csd1 (D) and csd3 (E–F) mutant sacculi illustrating their slightly (csd1) or strongly (csd3) curved rod morphologies, as seen in the intact cells (Figure 1E and 1I). Panel E shows sacculi contrasted with uranyl acetate, others are negatively stained. The scale bar (0.5 μm) applies to all images. Strains used: LSH108, LSH115, LSH119. G) Schematic illustration of PG mesh structure that envelops the cell and is composed of glycan strands (green) interconnected by peptide cross-links (red). Glycan consists of repeating N-acetylglucosamine (NAG or GlcNAc)–N-acetylmuramic acid (NAM or MurNAc) disaccharides with a 1,6–anhydro ring (anh) on NAM sugars at strand termini. Peptide chains extend from each NAM. Shown is the tetra–penta-peptide muropeptide with the peptide cross-link highlighted in grey. m-Dap refers to meso-diaminopimelic acid. Peptide bonds marked with orange scissors may be hydrolyzed by DD-endopeptidases (cleaving DD-peptide bridges) or DD-carboxypeptidases (producing tetra-peptide), those marked with blue scissors may be hydrolyzed by LD-endopeptidases (producing tri-peptide), and those marked by green scissors may be hydrolyzed by DL-endopeptidases or DL-carboxypeptidases (producing di-peptide). Altogether, these modification activites give rise the other muropeptides depicted in Figure S4A.

Model of how H. pylori’s peptidoglycan sacculus forms helical curvature and twist. Glycan strands are green and peptide cross-links red. Blue, pink, and yellow highlights indicate possible zones where relaxation of peptide cross-linking may contribute to the indicated curved and twisting morphologies.