1 Supplemental methods 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 21 22 23 Quantitative reverse-transcription PCR. Transcript levels of flgs, flgr, flia and flha were monitored by quantitative reverse-transcription polymerase chain reaction (qrt-pcr) as described in Materials and Methods. Construction of rpon mutant. A disruption in rpon was constructed in H. pylori B128 by allelic exchange. The suicide vector used to generate the rpon mutant was created by amplifying rpon from H. pylori 26695 genomic DNA and cloning the resulting amplicon into the NdeI and HindIII sites of pet28a. A unique EcoRI site was introduced ~37 bp downstream of the start codon of rpon as described (1) and a cat cassette was inserted into the EcoRI site. This plasmid was introduced into H pylori B128 by natural transformation. The rpon mutation in H. pylori was confirmed by amplifying the region surrounding rpon and sequencing of the resulting amplicon. Western blotting. For detection of H. pylori RpoN, antibodies directed against MBP-RpoN were affinity-purified using the AminoLink Plus Immobilization Kit (Thermo Scientific) as follows. MBP-RpoN was purified as previously described (1) and immobilized to the AminoLink Plus Resin following the manufacturer s protocol. Two milliliters of antiserum was buffer-exchanged into phosphate-buffered saline (PBS) by diluting with 13 ml PBS, reducing volume to.5 ml using an Amicon Ultra-15 centrifugal filter two times. The buffer-exchanged antiserum was incubated with the AminoLink Plus Resin in the column to allow for binding to MBP-RpoN. The column was washed with 1 ml PBS before eluting the antibody with 8 ml of.1m glycine, ph 2.5. The eluted antibody was dialyzed against 4 L citric acid-phosphate buffer (55 mm citric acid, 5 mm K 2 HPO 4, ph 5.5) for 18 hours, followed by 4 L Tris-buffered saline 1
24 25 26 27 28 29 3 31 32 33 34 35 36 37 38 39 41 42 43 44 45 (5 mm Tris, 15 mm NaCl, ph 7.6) two times for 3 hours each and one time for 18 hours. The dialyzed antiserum was stored at - C. H. pylori cytoplasmic fractions were prepared for western blot analysis as follows. Cells were grown on agar medium for 24 hours before resuspending into 3 ml PBS. Cells were lysed with three passages through a French press at 1, kpa. Cellular debris was removed by centrifugation for 15 min at 6 x g. Membranes were separated from cytoplasmic proteins by centrifugation for 6 min at 1, x g. The supernatant containing the cytoplasmic proteins was concentrated by trichloroacetic acid precipitation as described previously (2). Protein concentrations were determined using the bicinchoninic acid protein assay (Thermo Scientific) following the manufacturer s instructions. µg protein was analyzed by western blotting using the affinity-purified RpoN antibody. Western blots were analyzed using the secondary antibody and imaged as described in Materials and Methods. Motility enrichment. Semisolid agar medium was inoculated by the pre-enrichment flio stock as described in Materials and Methods. After growth for seven days, cells from the edge of the halo were used to inoculate a fresh semisolid agar plate and to make a frozen glycerol stock. This motility enrichment process was repeated three times in the subsequent weeks to obtain the post-enrichment stock. After all stocks were made, the level of motility from each stock was determined on semisolid agar plates. Electron microscopy. Cells from the pre-motility enrichment stock and from the post-motility enrichment stock were analyzed by electron microscopy as described in Materials and Methods. Statistical significance between the pre-motility enrichment and post-motility enrichment samples was analyzed using the Mann-Whitney U test and the Fisher exact test. 46 2
46 47 48 49 5 51 52 References 1. Pereira L, Hoover TR. 5. Stable accumulation of sigma54 in Helicobacter pylori requires the novel protein HP958. J Bacteriol 187:4463-4469. 2. Smith TG, Pereira L, Hoover TR. 9. Helicobacter pylori FlhB processing-deficient variants affect flagellar assembly but not flagellar gene expression. Microbiology 155:117-118. 53 54 3
54 Table S1. Oligonucleotides used for this study. 55 56 Primer B128 flio forward B128 flio reverse cat forward cat reverse flio upstream forward flio upstream reverse flio downstream forward flio downstream reverse PfliN forward PfliN reverse flio forward flio reverse flioδ N reverse flioδ N forward flioδ C reverse flioδ C forward flioδ C 2 reverse Sequence GAT TCT AAC GCC ATT GTG AA TCC TGT TAG AAT GAT CCA CA GAT ATA GAT TGA AAA GTG GAT TTA TCA GTG CGA CAA ACT GGG GTC AGA AAC AGA AGC TAA TAA ATC CAC TTT TCA ATC TAT ATC TCT TCA GCC AGT AAC ATA AAA CCC AGT TTG TCG CAC TGA TAA TAC GGC CTG CTT TTA GAC A AGA AAG CTG GTT AGG GTC AA CTC GAG ACT CAG CGA TCT GTT TAA AGA AGA TAA TAT ACA ATG GCG TTA GTT TCT TTT TCG GCT ACT TTT AA CTA ACG CCA TTG TAT ATT ATC T ATT TTT CCC TAA CTT AGC AGG TTT ATC ATC ATC ATC TTT ATA ATC GTC ATC GTT TAA AGA TAT TT GAT TAT AAA GAT GAT GAT GAT AAA AAC ACT CTA GAG TTA AAA C TTT ATC ATC ATC ATC TTT ATA ATC AGA CCC TTG TGT GGG GAT TAT AAA GAT GAT GAT GAT AAA AAA TTA GGA AAT GGA TCC TTA AGA CCT TTG TGT GGG 4
56 57 58 Table S2. qrt-pcr oligonucleotide primers use for this study. Primer Sequence gyra qpcr forward GCT AGG ATC GTG GGT GAT GT gyra qpcr reverse TGG CTT CAG TGT AAC GCA TC flaa qpcr forward GGC AAG CGT TAT TGT CTG GT flaa qpcr reverse CGA TAC GAA CCT GAC CGA TT flge qpcr forward TGC GAA CGT GAA TAC CAC TG flge qpcr reverse GTC ATT CTG CCC TGC TAA CC flab qpcr forward ATC GCC GCT TTA ACT TCT CA flab qpcr reverse CGC CAT CCC ACT AGA ATC AT flgs qpcr forward AGC CCT TTA TGA GTG GGT CA flgs qpcr reverse TTG AAC ACC TCA TGG CTT TG flgr qpcr forward GCC ATT GAT TCC ATT CGT TT flgr qpcr reverse GCC TGT CGT TTG CTC TCT TC flia qpcr forward GCC TAT GAT GAG CAG CAA CA flia qpcr reverse GAA TCG TTT AAC GCG CTC TC flha qpcr forward GCG ATA TTA TCA CGG CGT TT flha qpcr reverse CGC GCC CTA ACT TCA GTA AC 5
Early% Middle% Late% FlgE% FlaB% FlaA% OM% L% P% rod% Flgs,%FlgR% IM% MS% C% EA% RpoD3dependent%(σ 8 ) % C%ring%(C)% %flin,%flim,%flig,%fliy% Export%apparatus%(EA)% %flha,%flhb,%flio,%flip,% %fliq,%flir,%flii,%flih,% %flij% Rod% Ms%ring%(MS)% L%ring%(L)% P%ring%(P)% RpoN3dependent%(σ 54 )% Rod% Hook%(flgE)% Hook%length%control% protein%(flik)% Minor%flagellin%(flaB)% FliA3dependent%(σ 28 )% Major%flagellin%(flaA)% Figure S1: A transcriptional hierarchy for expression of flagellar genes. Only proteins and genes mentioned in this study are included in this figure.
Diameter of growth (mm) 5 3 1 Week 1 Week 2 Week 3 Week 4 WT ΔfliO Figure S2: Motility enrichment of wild type and ΔfliO. Motility from each enrichment stock was analyzed on semisolid agar medium. Plates were incubated for seven days before halo diameters were measured.
A WT preenrichment B WT postenrichment Percentage 1 8 6 1 2 3 4 5 6 Percentage 1 8 6 1 2 3 4 5 6 Number of flagella Number of flagella C ΔfliO preenrichment D ΔfliO postenrichment Percentage 1 8 6 1 2 3 4 5 6 Percentage 1 8 6 1 2 3 4 5 6 Number of flagella Number of flagella Figure S3: Effects of motility enrichment on flagellar numbers. The number of flagella per cell was analyzed by electron microscopy after negative staining of cells. For each strain, 125 cells were visualized. Bars represent the percentage of cells possessing the same number of flagella. Pre- and post-enrichment populations were not significantly different from one another (WT p-value=.27, ΔfliO p-value =.). In the wild type, there was no significant difference in the number of cells with -3 flagella versus 4-6 flagella (p-value =.57) and in the ΔfliO mutant, there was no significant difference in the number of aflagellated versus flagellated cells (p-value =.9) pre- and post-enrichment.
WT mem WT ΔflhA ΔfliO WT / pflio ΔfliO / pflio ΔfliO / pflio ΔN ΔfliO / pflio ΔC KatA Figure S4: Detection of KatA in cytoplasmic fractions. 4 µg of each fraction was loaded into a 1% SDSPAGE gel. Lane one contains WT membrane and is a positive control for the western blot. All subsequent lanes contain the soluble fraction.