DMSO was used as a control at the same buy BIBW2992 concentration as present in INP0403-treated samples (0.1% v/v). A nalidixic acid (Nal)-resistant derivative of S. Typhimurium strain 4/74 (Morgan et al., 2004), a bovine diarrhoea isolate that is the parent of the genome-sequenced hisG derivative
SL1344, was used unless otherwise stated. SL1344 derivatives were used to study the effect of inhibitor on transcription of single-copy gfp+ transcriptional fusions to the T3SS-1 gene prgH (prgH′-gfp+; JH3010), the T3SS-2 gene ssaG (ssaG′-gfp+; JH3009), the housekeeping gene rpsM (rpsM′-gfp+; JH3016) and a promoterless gfp+ (JH3008) (Hautefort et al., 2003). In studies to investigate whether inhibition of Salmonella T3SS-1 was dependent on ferric uptake regulator (Fur) regulation
of SPI-1, S. Typhimurium SL1344 wild-type and fur deletion mutant (SL1344 Δfur) strains were used (Karavolos et al., 2008). Bacteria were cultured in Luria–Bertani (LB) media at 37 °C with shaking unless otherwise stated and supplemented with nalidixic acid at 20 μg mL−1 where appropriate. For experiments requiring induction of T3SS-1, bacteria were grown in LB media overnight with shaking at 25 °C, diluted 1 : 10 into fresh LB media and then incubated at 37 °C for 4 h. This temperature-shift method results in elevated secretion of proteins via T3SS-1 into the culture supernatant (Wood et al., 1996). We have previously reported that INP0403 does not affect bacterial viability or growth
during selleck chemical culture MG-132 ic50 in LB medium over this time course (Hudson et al., 2007). Ten millilitres of LB broth supplemented with nalidixic acid was inoculated with fresh single colonies of S. Typhimurium 4/74 NalR and incubated overnight with shaking at 25 °C. Bacteria were collected by centrifugation, resuspended in 10 mL fresh LB, diluted 1 : 10 into LB containing 100 μM INP0403 or 0.1% v/v DMSO and cultured at 37 °C shaking for 90 min. 2.0 OD600 nm units of each culture were incubated in one-fifth culture volume 5% v/v phenol pH 4.3/95% v/v ethanol solution for 30 min on ice to stabilize RNA. RNA was extracted using the SV Total RNA purification kit (Promega, Southampton, UK). Purified total RNA (10 μg) was labelled with Cy5-dCTP (Amersham Biosciences, Little Chalfont, UK). All hybridizations were performed as indirect comparison experiments, using Cy3-dCTP-labelled S. Typhimurium SL1344 as the common reference as described (Yang & Speed, 2002). SALSA microarrays covering 92% of the genes common between S. Typhimurium LT2 and SL1344 strains were used (Nagy et al., 2006). Fluorescence intensities of scanned microarrays were quantified using genepix pro software, version 6.0 (Axon Instruments Inc., Foster City, CA). Data were filtered and spots showing a reference signal lower than background+2 SDs were discarded.