The amplified 5′ fragments of phoP and axyR were digested with BamHI and EcoRI and cloned into the thermosensitive plasmid pMAD using the corresponding restriction sites. Using the EcoRI and NcoI sites in the plasmids and fragments, the resulting constructs were used to clone the amplified 3′ fragments of phoP and axyR downstream of the 5′ fragments of the appropriate genes,

yielding constructs pMADΔphoP and pMADΔaxyR, {Selleck Anti-diabetic Compound Library|Selleck Antidiabetic Compound Library|Selleck Anti-diabetic Compound Library|Selleck Antidiabetic Compound Library|Selleckchem Anti-diabetic Compound Library|Selleckchem Antidiabetic Compound Library|Selleckchem Anti-diabetic Compound Library|Selleckchem Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|Anti-diabetic Compound Library|Antidiabetic Compound Library|buy Anti-diabetic Compound Library|Anti-diabetic Compound Library ic50|Anti-diabetic Compound Library price|Anti-diabetic Compound Library cost|Anti-diabetic Compound Library solubility dmso|Anti-diabetic Compound Library purchase|Anti-diabetic Compound Library manufacturer|Anti-diabetic Compound Library research buy|Anti-diabetic Compound Library order|Anti-diabetic Compound Library mouse|Anti-diabetic Compound Library chemical structure|Anti-diabetic Compound Library mw|Anti-diabetic Compound Library molecular weight|Anti-diabetic Compound Library datasheet|Anti-diabetic Compound Library supplier|Anti-diabetic Compound Library in vitro|Anti-diabetic Compound Library cell line|Anti-diabetic Compound Library concentration|Anti-diabetic Compound Library nmr|Anti-diabetic Compound Library in vivo|Anti-diabetic Compound Library clinical trial|Anti-diabetic Compound Library cell assay|Anti-diabetic Compound Library screening|Anti-diabetic Compound Library high throughput|buy Antidiabetic Compound Library|Antidiabetic Compound Library ic50|Antidiabetic Compound Library price|Antidiabetic Compound Library cost|Antidiabetic Compound Library solubility dmso|Antidiabetic Compound Library purchase|Antidiabetic Compound Library manufacturer|Antidiabetic Compound Library research buy|Antidiabetic Compound Library order|Antidiabetic Compound Library chemical structure|Antidiabetic Compound Library datasheet|Antidiabetic Compound Library supplier|Antidiabetic Compound Library in vitro|Antidiabetic Compound Library cell line|Antidiabetic Compound Library concentration|Antidiabetic Compound Library clinical trial|Antidiabetic Compound Library cell assay|Antidiabetic Compound Library screening|Antidiabetic Compound Library high throughput|Anti-diabetic Compound high throughput screening| respectively. These plasmids were introduced into L. monocytogenes EGD by electroporation and gene replacement was performed as described previously [36]. Erythromycin-sensitive clones were screened for the presence of the phoP and axyR deletion by PCR with primers phoP-1 and phoP-4, and primers axyR-1 and axyR-4, respectively. The shorter PCR products amplified from these strains were sequenced buy BV-6 to verify that they carried the desired deletions. Antibiotic susceptibility GANT61 tests The susceptibility of L. monocytogenes strains to different

antibiotics was examined using a microdilution test. The antimicrobial agents were obtained as powders (Sigma-Aldrich, St. Louis, USA) and stock solutions were prepared immediately before use. The microdilution method was performed according to guidelines of the Clinical and Laboratory Standards Institute [37]. Briefly, an overnight culture of each strain was serially diluted in BHI broth to a cell density of 105 CFU/ml and 100 μl aliquots were added to the wells of 96-well microdilution plates containing 100 μl of two-fold dilutions of the different antimicrobial agents in BHI broth. These plates were then incubated at 37°C for 18–22 h before the MIC endpoints were read. The MIC was determined as the lowest antibiotic concentration

that resulted in the absence of apparent growth of the bacteria. MIC determinations were carried out in triplicate. For quality control of performance and reliability of the results of MIC determination, standard Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 strains were used in parallel tests. The growth of L. monocytogenes strains Diflunisal in the presence of a sublethal level of penicillin G was examined by plotting growth curves. Overnight cultures were diluted (1:100) into BHI broth supplemented with 0.09 μg/ml penicillin G and incubated with shaking at 37°C. Cell growth was monitored spectrophotometrically by measuring the OD600. The presented results are the average of three independent experiments, each carried out in triplicate. The tolerance of L. monocytogenes strains to penicillin G was tested as described previously [12], except that cultures in the exponential rather than the stationary phase of growth were used for the assays. Briefly, cultures in mid-exponential phase (OD600 0.4) were diluted (5 × 107 CFU/ml) into BHI broth supplemented with 32 μg/ml penicillin G and incubated with shaking at 37°C.