Excision of the kanamycin

resistance FRT cassette was con

Excision of the kanamycin

resistance FRT cassette was confirmed by PCR and sequencing to be correct. Southern blot using the FRT scar site region as a probe was also used to confirm that the final mutants were as intended. LPS serotype was confirmed by agglutination with anti-04 serotype antiserum using anti-09 antiserum as a negative control SAR405838 order (Remel Europe Ltd./Oxoid Ltd., Basingstoke UK). For complementation of SL1344 atp, lacking the entire atp operon, PCR was used to amplify the entire atp operon from SL1344 fused to a chloramphenicol resistance cassette, from pACYC184. This was inserted into the malXY pseudogene region on the Salmonella chromosome using ODM with selection on chloramphenicol. Insertion of the atp operon into malXY was confirmed by PCR and sequencing Veliparib mouse of the mutated malXY junction and by Southern blotting using atpG as the probe. In addition to the complemented strain, SL1344 atp (malXY atp operon+), a complementation control strain was also generated, SL1344 atp

(malXY CmR). For this control strain a chloramphenicol resistance cassette was inserted into the malXY pseudogene region of SL1344 atp to ensure the insertion into the pseudogene had no phenotypic effects. Cultures in 5 ml of LB broth were incubated overnight with shaking (180 rpm) at 37 °C. Cultures were diluted 1:100,000 into 100 ml of pre-warmed LB broth, and incubated with shaking at 37 °C. Growth was measured by viable count on LB agar plates. Exponential generation times were calculated from growth rates between 4 and 6 h. To assess the ability to utilise succinate as a sole carbon source wild type and the many various atp mutants were grown in M9 minimal medium supplemented

with 0.4% (w/v) of sodium succinate. Growth was assessed by OD595 after 24 and 48 h. Inocula were prepared from overnight cultures grown statically in LB broth at 37 °C. Cultures were centrifuged and bacteria were re-suspended in phosphate buffered saline (pH 7.4) to the required concentration. Seven to nine week-old female BALB/c mice (Harlan, Oxon, UK) were inoculated with 200 μl of bacteria suspension via intravenous injection, or they were lightly anaesthetised with halothane and inoculated by oral gavage. Doses of bacteria given were confirmed by viable counts in LB agar. Gene knock-out mice lacking gp91phox or IFNγR1 on a C57/BL6j background where originally purchased from Jackson Laboratory (Bar Marbour, ME) and maintained as homozygous matings at the Wellcome Trust Sanger Institute. C57/BL6j age- and sex-matched control mice were purchased from Harlan (Oxon, UK). At pre-determined time points postinfection animals were killed, spleens and livers removed and homogenised in 5 ml of sterile water in a Stomacher® 80 Lab System (Seward). Bacterial numbers were enumerated via serial dilutions and plating in LB agar. When required, blood was collected via cardiac puncture under terminal anaesthesia.

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