For the iodine staining, patches of bacteria or diluted samples were grown overnight on LB plates, stored at 4°C for 24 h and then flooded with iodine. The intensity of the brown colour varies according to glycogen concentration in the cell and indirectly reveals the

level of RpoS [17, 18]. rpoS + strains stain brown to dark brown. Western-blot of RpoS Western-blot analyses were performed essentially as described [47]. Briefly, 2 × 109 bacteria grown overnight in LB-broth were resuspended in 200 μl application buffer Ferrostatin-1 clinical trial (0.5 M Tris/HCl, 2% SDS, 5% 2-mercaptoethanol, 10%, v/v, glycerol and 0.01% bromophenol blue) and boiled for 5 min. Proteins were resolved in a 12.5% denaturing polyacrylamide gel and transferred to a nitrocelullose membrane (GE HealthCare) by capillary action. Following blocking with 5% skim milk, the membrane was incubated with 2, 000-fold diluted monoclonal anti-RpoS antibodies (Santa Cruz) and 20, 000-fold diluted peroxidase conjugated anti-mouse IgG (Pierce). The Super Signal West Pico kit (Pierce) was used to detect the RpoS bands as recommended by the manufacturer and the membrane was exposed to X-ray films. Knock-out of rssB A KmR cassete was inserted into rssB ORF by homologous recombination using the λ-Red system as described [48]. The rssB gene was PCR BAY 11-7082 concentration amplified from E.

coli chromosome with primers rssB94F (5′-CGCACCAACATTTGACCAG) and rssB1368R (5′-GTATCGCATCCCAGTATATCAG)

and ligated into pGEM T-easy (Promega), resulting in plasmid pBS23. The KmR gene was excised from pUC4K by digesting with EcoRI and ligated into the MunI site of rssB in pBS23. The resulting plasmid (pBS25) was used as a template for the PCR amplification of the rssB-KmR fragment. The PCR product was resolved by electrophoresis, extracted Sclareol from the gel and purified using the Wizard SV gel and PCR clean-up system (Promega). The linear DNA carrying rssB-KmR was electrotransformed into strain KM32 and plated on Km plates. One out of three colonies was KmR and AmpS, suggesting that the resistance to Km was due to insertion of KmR into the chromosome and not due to transfomation of pBS25 leftovers. The KmR insertion in rssB was verified by PCR. The rssB::KmR mutation was transferred to strain MC4100BS by P1 transduction [46]. Cloning of rssAB A DNA fragment containing the entire rssAB operon was obtained by PCR amplification with primers rssA231F (5′-CCATCAATTCGGCACGTAAC) and rssB1368R (5′-GTATCGCATCCCAGTATATCAG) and cloned in pGEM T-easy (Promega) following the manufacturer instructions. The resulting plasmid was then digested with EcoRI and the rssAB fragment was ligated to the low-copy vector CAL-101 mouse pWKS130 [44] previously linearised with EcoRI, resulting in plasmid pBS28. Strain DH10B was used as a recipient for DNA transformation.