Ml Erlenmeyer flask containing ml of LB broth for h at on a rotary shaker at rpm. Exactly l of your cultures have been then transferred into ml Erlenmeyer flasks containing ml of LB broth and grown inside a temperature controlled rotary shaker at rpm (LSIR, Labtech Shaking Incubator, Indonesia). Optical density (OD) was measured until reaching the preferred OD for treatment with HO ( corresponding to an incubation time of about. h). These circumstances closely resemble these employed within a earlier study with E. coli, 
exactly where ml of culture grown at rpm in a shaking incubator at to an OD of. exhibited a pO of. Strong media contained agar ( g l), and plates have been incubated at. When important, growth media was supplemented together with the suitable antibiotics.Microarray alysisOvernight cultures of strains s and arcA have been diluted (:) and cells were grown to OD. as described. At this point, HO ( mM) was added and cells had been grown for min. Manage cells received no therapy. Experiments had been performed in triplicate on distinctive days. Right after exposure towards the toxic compound, ml of ice cold (vv) phenol pH. (vv) ethanol was added to ml of culture and left on ice for min. Subsequently, ml of this remedy have been centrifuged for min at rpm, the supertant was removed along with the bacterial pellet was resuspended with l of mM Tris Cl (pH.) that included l of lysozyme PubMed ID:http://jpet.aspetjournals.org/content/110/1/93 ( mgml). The reaction was incubated for min at, and total R was extracted employing the Higher Pure R Isolation kit (Roche) following the RN-1734 site manufacturer’s instructions. R was eluted in l of water and treated with DseI (Roche) at for min. Total R was recovered employing the Qiagen CAY10505 web RNeasy kit (Qiagen), following the manufacturer’s directions. R was eluted in l and subjected to a second round of DseI treatment (Ambion Turbo Dfree kit) at for min, purified, recovered applying the Qiagen RNeasy kit (Qiagen) following the manufacturer’s instructions and eluted in l of water.Morales et al. BMC Genomics, : biomedcentral.comPage ofExactly g of total R were used for labeling with Cy or Cy. Briefly, the R volume was adjusted to l, l of random hexamers N (Sigma, gl) were added plus the mixture was incubated for min at. Subsequently, cD waenerated working with Superscript II (Invitrogen) following the manufacturer’s guidelines. Fil nucleotide concentrations in the reaction have been. mM dATP, dTTP, dGTP and. mM dCTP. Immediately after addition of the master mix, l of mM dye labeled dCTP (Cy or Cy) were added to the reaction plus the mixture was incubated at for min. Right after this time, l of Superscript II have been added along with the reaction was incubated at for an additiol min. The reaction was stopped by adding l of M OH and incubating at for min. The pH was neutralized by adding l of M HCl. The labeled cD was purified utilizing the Qiagen PCR purification kit following the manufacturer’s guidelines. The purified labeled cD ( g) was hybridized to a. mer NimbleGen microarray (Roche NimbleGen), tiling the S. Typhimurium enome at overlapping intervals of about bases on each strands, as previously described.Data acquisition and alysisArrays have been scanned using a GenePix B laser scanner (Molecular Devices, Sunnyvale, California) at m resolution. Sigl intensities were quantified making use of NimbleScan application v. (Roche NimbleGen). Intensity values were background subtracted, normalized inside (median) and amongst (quantile) the arrays utilizing WebarrayDB, and converted to log values. For each and every array, the background was calculated as follows: log median intensity worth for negative handle probes + (.Ml Erlenmeyer flask containing ml of LB broth for h at on a rotary shaker at rpm. Precisely l in the cultures had been then transferred into ml Erlenmeyer flasks containing ml of LB broth and grown within a temperature controlled rotary shaker at rpm (LSIR, Labtech Shaking Incubator, Indonesia). Optical density (OD) was measured until reaching the desired OD for remedy with HO ( corresponding to an incubation time of about. h). These conditions closely resemble those utilized inside a prior study with E. coli, where ml of culture grown at rpm within a shaking incubator at to an OD of. exhibited a pO of. Solid media contained agar ( g l), and plates have been incubated at. When needed, growth media was supplemented using the proper antibiotics.Microarray alysisOvernight cultures of strains s and arcA were diluted (:) and cells had been grown to OD. as described. At this point, HO ( mM) was added and cells had been grown for min. Manage cells received no treatment. Experiments had been performed in triplicate on distinctive days. Just after exposure towards the toxic compound, ml of ice cold (vv) phenol pH. (vv) ethanol was added to ml of culture and left on ice for min. Subsequently, ml of this resolution have been centrifuged for min at rpm, the supertant was removed as well as the bacterial pellet was resuspended with l of mM Tris Cl (pH.) that incorporated l of lysozyme PubMed ID:http://jpet.aspetjournals.org/content/110/1/93 ( mgml). The reaction was incubated for min at, and total R was extracted making use of the Higher Pure R Isolation kit (Roche) following the manufacturer’s instructions. R was eluted in l of water and treated with DseI (Roche) at for min. Total R was recovered working with the Qiagen RNeasy kit (Qiagen), following the manufacturer’s instructions. R was eluted in l and subjected to a second round of DseI therapy (Ambion Turbo Dfree kit) at for min, purified, recovered utilizing the Qiagen RNeasy kit (Qiagen) following the manufacturer’s guidelines and eluted in l of water.Morales et al. BMC Genomics, : biomedcentral.comPage ofExactly g of total R had been applied for labeling with Cy or Cy. Briefly, the R volume was adjusted to l, l of random hexamers N (Sigma, gl) had been added along with the mixture was incubated for min at. Subsequently, cD waenerated working with Superscript II (Invitrogen) following the manufacturer’s guidelines. Fil nucleotide concentrations of your reaction 
were. mM dATP, dTTP, dGTP and. mM dCTP. Soon after addition with the master mix, l of mM dye labeled dCTP (Cy or Cy) have been added for the reaction and also the mixture was incubated at for min. Following this time, l of Superscript II have been added plus the reaction was incubated at for an additiol min. The reaction was stopped by adding l of M OH and incubating at for min. The pH was neutralized by adding l of M HCl. The labeled cD was purified making use of the Qiagen PCR purification kit following the manufacturer’s directions. The purified labeled cD ( g) was hybridized to a. mer NimbleGen microarray (Roche NimbleGen), tiling the S. Typhimurium enome at overlapping intervals of about bases on each strands, as previously described.Data acquisition and alysisArrays have been scanned employing a GenePix B laser scanner (Molecular Devices, Sunnyvale, California) at m resolution. Sigl intensities had been quantified making use of NimbleScan software program v. (Roche NimbleGen). Intensity values had been background subtracted, normalized within (median) and in between (quantile) the arrays employing WebarrayDB, and converted to log values. For every single array, the background was calculated as follows: log median intensity value for unfavorable manage probes + (.