Escherichia coli strains were grown in Luria–Bertani (LB) medium. Listeria monocytogenes was grown in brain heart infusion (BHI) broth. Erythromycin (Ery) and chloramphenicol (Cm) were made up as concentrated stocks, and then added to media at the required levels. Where solid media was required, agar was added at 1.5%. Zinc limiting media was achieved via the addition of 0.5 M EDTA at the required concentration

followed by agitated overnight incubation to allow complete chelation. Where metals were AZD8055 used, they were prepared as 1 M stocks according to the manufacturer’s instructions and were added to media at the required concentrations. Gel extractions were performed using the Qiagen gel extraction kit (Qiagen). Plasmid DNA isolation was achieved utilizing the Qiagen QIAprep spin miniprep kit (Qiagen). Ligations were carried out using T4 DNA Ligase from Roche selleck screening library Diagnostics GmbH (Mannheim, Germany) according to the manufacturer’s instructions. Restriction enzymes were purchased

from New England Biolabs and were used according to the manufacturer’s recommendations. The splicing by overlap extension (SOEing) PCR procedure described by Horton et al. (1990) was used to create an isogenic nonpolar deletion mutant of zurR. Primers SOE AB and SOE CD were used to amplify regions of similar size flanking the sequence to be deleted. The resulting products were mixed in a 1 : 1 ration and were amplified using SOE A and D primers. The resulting product was digested with the appropriate restriction enzymes (XbaI and EcoRI), cloned into the temperature sensitive shuttle vector pKSV7, and transformed into E. coli DH5α.

The plasmid was subsequently transformed into L. monocytogenes EGDe. Chromosomal integration of the plasmid at 42 °C was selected by serial passage of a single colony in prewarmed BHI/Cm broth and streaking onto prewarmed BHI/Cm agar. Plasmid excision and curing was brought about by continuous passaging in BHI at 30 °C followed by spread plating onto BHI agar at 30 °C. Replica plating onto BHI and BHI/Cm was used to select for loss of plasmid and appropriate deletion events. The mutation was L-gulonolactone oxidase confirmed using primers upstream of SOE A (SOE X) and downstream of SOE D (SOE Y) (listed in Table 1). The pORI19 insertional mutant was constructed as outlined previously (Law et al., 1995; Rea et al., 2004). Briefly, a central portion of the zurR gene was amplified by PCR and cloned into the multiple cloning site of pORI19 (RepA−) and maintained in E. coli EC101. The plasmid was transformed by electroporation into L. monocytogenes EGDe containing the temperature sensitive RepA+ plasmid pVE6007. Loss of pVE6007 and integration of pORI19 to create zurR::pORI19 was carried out as described previously (Rea et al., 2004). No obvious specific ribosome-binding site was determined within zurM and zurR, which suggests a coupled translation mechanism (Dalet et al., 1999).