The F0 subunit of the ATPase is a hydrophobic membrane-embedded proton channel encoded by genes atpBEF. The F1 subunit constitutes the catalytic ATPase, encoded by atpHAGDC [19] and [21]. The first gene in the operon, atpI, has no defined function and does not appear to form part of the F0F1 ATPase complex [22]. This genetic organisation is conserved between E. coli and S. Typhimurium. A comprehensive identification of genes required for S. Typhimurium infection of mice by our laboratory identified mutation of atpA as an attenuating lesion [23]. A defined atpA deletion mutant was subsequently confirmed to be attenuated for growth in vivo and furthermore was found to offer significant protection against

subsequent SB203580 price challenge [23]. Here we present a full analysis of the role of the F0F1 ATPase in S. Typhimurium infection and the potential use of mutants in the atp operon as live attenuated vaccines. The bacterial strains and plasmids used in this study are shown in Table 1. Bacteria were grown at 37 °C in Luria–Bertani (LB) broth or on LB agar. Media were supplemented Onalespib cost with antibiotics

where stated, at the following concentrations, kanamycin 50 μg/ml, ampicillin 100 μg/ml and chloramphenicol 25 μg/ml. Minimal medium (used to determine carbon source utilisation) consisted of M9 salts (Sigma Dorset UK) supplemented with 0.1 mM CaCl2, 1 mM MgSO4, 4 μg/ml histidine and the stated carbon source at 0.4% (final w/v). Oligo-directed mutagenesis (ODM), an adaptation of ET-cloning, was used to replace the target genes on the Salmonella chromosome with a kanamycin resistance cassette flanked with FRT regions from pBADkanFRT [24] and [25]. PCR was used to amplify the kanamycin resistance FRT cassette with 5′ and 3′ 60 bp arms homologous to DNA flanking the target genes (see Table 2 for primer sequences). S. Typhimurium LB5010 containing pBADλred was grown in

LB broth supplemented with ampicillin to an OD595 of 0.25. Arabinose was added to 0.2% (final almost w/v) to induce red gene expression. Cultures were grown to OD595 0.5 and electroporated with the purified ODM PCR product described above. Mutant colonies were selected on LB agar plates supplemented with 50 μg/ml kanamycin. The desired allelic replacement of the target genes was confirmed by PCR (see Table 2 for primer sequences). Mutations in S. Typhimurium LB5010 were transduced into SL1344 by bacteriophage P22 as described previously [26] with selection on LB agar plus kanamycin and gene deletions were confirmed to be correct by PCR and sequencing. The kanamycin resistance FRT cassette was then excised to leave only a 128 bp FRT scar site. Briefly, electrocompetent mutants of SL1344 were transformed with pCP20 [24] grown at 30 °C and then plated onto LB agar containing 100 μg/ml ampicillin. Single colonies were grown in LB at 39 °C (to prevent replication of pCP20) for 6 h then diluted and plated onto LB agar and incubated overnight at 39 °C.