The PCR product was cut with BamHI and HindIII and cloned into the plasmid pSP72 (Promega, Madison, WI) which had been cut with the same enzymes, transformed into DH5α, and selected for bright blue

colonies on LB-amp plates containing 40 μg/ml GSK3326595 in vivo X-gal. The plasmid was subsequently transformed to the restriction minus methylation plus strain YS501 before transforming other Salmonella strains. β-gal assays were performed according to the instructions for the Galacto-Star™ chemiluminescent reporter gene assay system (Applied Biosystems, Bedford, Massachusetts). Briefly, 1 ml of bacterial culture expressing β-gal from pSP72lacZ was pelleted at 13,000 × g for 5 min. Supernatants were filtered through a 0.2 μm syringe filter and then assayed immediately or frozen at -80°C until assayed with no further processing. Cell pellets were quickly freeze-thawed and suspended in 50 μl or 200 μl B-PER™ bacterial cell lysis reagent (Pierce click here Chemical) containing Selleck XL184 10 mg/ml lysozyme (Sigma). Bacteria were allowed to lyse for 10–20 min. at room temperature and were then placed on ice. All reagents and samples were allowed to adjust to room temperature before use. Filtered supernatants and bacterial lysates were diluted as needed in Galacto-Star™ Lysis Solution or assayed directly.

β-gal standard curves were made by preparing recombinant β-gal (Sigma, 600 units/mg) to 4.3 mg/ml stock concentration in 1× PBS. The stock was diluted in Lysis Solution to

prepare a standard curve of 100 ng/ml- 0.05 ng/ml in doubling dilutions. 20 μl of standard or sample was added to each well of a 96-well tissue culture plate. 100 μl of Galacto-Star™ Subtrate, diluted 1:50 in Reaction Buffer Diluent, was added to each well and the plate rotated gently to mix. The plate was incubated for 90 minutes at 25°C in the dark and then read for 1 second/well in an L-max™ plate luminometer (Molecular Devices). Sample light units/ml were compared to the standard curve and values converted to units β-gal/ml. Percent release of β-gal was determined by dividing units/ml supernatant by total units/ml (units/ml supernatant + units/ml pellet). All samples were assayed in triplicate. Acknowledgements We wish to thank the reviewers Sulfite dehydrogenase for helpful suggestions, and Diana Downs and Eugenio I. Vivas (University of Wisconsin, Madison) for expeditiously providing gnd mutants. This work was supported by Vion Pharmaceuticals, New Haven, CT. SRM was supported by NIH Grant 1SC2 GM084860-01. DB thanks Caroline Clairmont for informing him of the plating results at the NCI. References 1. Nikaido H: Outer membrane. Escherichia coli and Salmonella: Cellular and molecular biology (Edited by: Neidhardt F, Curtiss R, Ingraham J, Lin ECC, Low KB, Magasanik B, Reznikoff M, Riley M, Schaechter M, Umbager HE). Washington D.C.: ASM Press 1996, 29–47. 2.