The large number of membrane-associated proteins with an altered expression in the HP0256 mutant highlighted another aspect of the mutant phenotype: the alteration of the cell envelope architecture, likely responsible for the weak adhesion to, and the low inflammatory response induced in, host cells. We conclude that HP0256 is required for full motility of H. pylori, possibly through its involvement with the switch components, but that it also modulates directly or indirectly the normal H 89 ic50 expression of membrane proteins essential in pathogenesis. Methods Bacterial strains, media and growth conditions Bacterial strains used in this study are listed in Table 3. H. pylori strain P79 [46], a streptomycin

mutant

of the P1 selleck chemicals wild-type strain, was generously provided by Dr. R. Haas. H. pylori strains were cultured as previously described [26]. Two H. pylori mutants Tofacitinib lacking the HP0256 gene (one in CCUG17874 and one in P79) were generated as described below in Materials and Methods. Transformants were selected on CBA (Columbia agar base) plates supplemented with 10 μg/ml chloramphenicol (Sigma) and/or 50 μg/ml kanamycin (Sigma). One Shot TOP10 chemically competent E. coli cells (Invitrogen, CA, USA) were propagated on Luria-Bertani (LB) agar plates or LB broth at 37°C supplemented with antibiotics: 50 μg/ml kanamycin (Sigma), 100 μg/ml ampicillin (Merck, Germany) and 10 μg/ml chloramphenicol (Sigma). Table 3 Strains and plasmids used in this study. Strains or plasmids Relevant characteristics Reference or source Strains     H. pylori     Glutamate dehydrogenase CCUG17874 wild-type strain CCUG, Sweden hp0256 KO CCUG17874 Δhp0256::Cmr This study P1 wild-type strain [57] P79 P1 Strr [58] P79-hp0256KO P79 Δhp0256::Cmr This study P79-0256/pIR203K04 P79 Δhp0256::Cmr with pIR203K04 (Kanr) This study P79-0256/pIR0601 P79 Δhp0256::Cmr

with pIR0610 (Kanr) This study S. typhimurium     SJW1103 wild-type strain [59] MKM40 SJW1103 ΔfliJ [59] MKM40-pQE60 SJW1103 ΔfliJ with empty pQE-60 This study MKM40-pQE60-0256 SJW1103 ΔfliJ with pQE-60-0256 This study E. coli     One Shot TOP10 F- mcrA Δ(mrr-hsdRMS-mcrBC) ф80lacZ ΔM15 ΔlacX74 recA1 araΔ139 Δ(ara-leu)7697 galU galK rpsL (Strr) endA1 nupG Invitrogen, USA Plasmids     pIR203K04 kanamycin resistance cassette (Kanr) [51] pIR0601 pIR203K04 with hp0256 gene under the control of hp0601 promoter This study   C-term His-tagged expression vector (Ampr)   pQE-60 pQE-60 with hp0256 gene Qiagen, Germany pQE-60-0256 This study   Cm, chloramphenicol, Kan, kanamycin; Str, streptomycin Bioinformatics PSI-BLAST was performed using bacterial sequences from the NCBI non-redundant protein databank at NCBI-BLAST. Three to four iterations were run and false-positives were edited from the output. Searching with Salmonella or other FliJ sequences did not result in significant hits with any HP0256 homologues.