5 μM was incubated with 100 ng DNA at

room temperature for 15 min, then separated on a non-denaturing 5% polyacrylamide gel by electrophoresis at 40V for 16 hours at 4°C. When Ler was present, essentially all of the DNA was bound in a nucleoprotein complex which was not disrupted by zinc acetate at any concentration up to 100 μM, and only partially at 1000 μM (the highest concentration tested). The upper and lower arrows mark the locations of bound and unbound DNA, respectively. Under normal physiological conditions, it is estimated that the concentration of free zinc within E. coli is in the femtomolar range, less then one zinc atom per cell [18], whereas the zinc quotient of the cell– that complexed with amino acids, ribosomal proteins and enzymes– reaches Milciclib mouse micromolar concentrations. Because millimolar RGFP966 molecular weight concentrations of zinc acetate see more were necessary for disrupting Ler binding to LEE4 (Figure 1) and no putative zinc binding domains are found within Ler (data not shown), we concluded that alterations of LEE gene expression by zinc did not involve direct interaction of

zinc with the regulatory protein Ler. LEE gene expression is reduced by zinc in K-12 laboratory strains To further our understanding of zinc alteration of LEE gene expression we transformed plasmids containing LEE1-lacZ and LEE4-lacZ fusions (pJLM164 and pJLM165; Table 1) into the prototypical EPEC strain E2348/69, EPEC strain LRT9, strain JPN15 lacking the EAF virulence plasmid, and the K-12 strain MC4100. Strains were grown

in DMEM medium in the presence and absence of 0.5 mM zinc acetate, and assayed for β-galactosidase activity. β-galactosidase activity derived from the LEE4 operon was significantly diminished in the presence of zinc in all four strains (Figures 2A-D). Similarly, β-galactosidase activity derived from the LEE1-lacZ, multi-copy fusion was also diminished by the presence of 0.5 mM zinc acetate in the four strains tested (Figures 2E-H). Table 1 Bacterial strains and plasmids used for in this study Strain or plasmid Genotype or description Source or reference Strains        E2348/69 Prototype EPEC strain (serotype O127:H6) [19]        JPN15 EAF plasmid-cured derivative of E2348/69 [20]        MC4100 araD139 Δ(argF−lac)U169 rpsL150 relA1 flbB5301 deoC1 ptsF25 rbsR [21]        JLM164 MC4100 ΦLEE 1−lacZ [14]        JLM165 MC4100 ΦLEE 4−lacZ [14]        SIP812 MC4100 zur::Spc r /Str r [22]        TB742 MC4100 ΔzntR [23]        CT32 MC4100 rpoE−lacZ [24]        MCamp MC4100 bla−lacZ [25]        CVD452 E2348/69 ΔescN::aphT [26]        LRT-9 EPEC O111:abH2 [27] Plasmids        pRS551 Promoterless lacZ reporter fusion vector [28]        pVSAPR bla−lacZ [25]        pJLM164 LEE 1−lacZ [14]        pJLM165 LEE 4−lacZ [14] Figure 2 Effect of zinc acetate on LEE gene expression.