The lacZ fusion in pKSK001
was recombined onto the chromosome (KSK003) using the transducing λ phage system, λRS45 , via a double recombination event and was verified as previously described . Strain ΔymdB was constructed by eliminating the kanamycin cassette (ymdB::km R ) from Keio-ΔymdB as described previously . Verification of Keio-ΔymdB, ΔymdB (KSK002), Keio-ΔrpoS, or rnc14∙Keio-ΔrpoS (KSK005) was carried out by colony PCR using primer pairs ymdB-F/-R or rpoS-F/-R and Emerald PCR premix (Takara) (Additional file 1: Figure S1), and the PCR products were read by DNA sequencing analysis using the same primers (data not shown). Verification of RNase III mutation was confirmed by Western blot analysis using antibodies against RNase III (Additional file 1: Figure S1). Bacteria were grown in Luria-Bertani (LB) broth or on LB plates at 37°C #see more randurls[1|1|,|CHEM1|]# throughout this study. Antibiotics were Akt inhibitor used at the following concentrations: kanamycin, 50 μg/mL; tetracycline, 10 μg/mL; and chloramphenicol,
30 μg/mL. Microarray analysis Total RNA was extracted from IPTG (0.1 mM final concentration)-induced E. coli BW25113 cells (at an OD600 of 1.0) containing either pCA24N (−gfp) or ASKA-ymdB (−) using an RNeasy® Kit (Qiagen) with two additional DNase treatments. The integrity of the bacterial total RNA was checked by an Agilent 2100 Bioanalyzer. The cDNA probes were prepared by reverse transcription with random priming of total RNA (25 μg) in the presence of aminoallyl-dUTP for 3 h, followed by coupling of probes with Cy3 dye (for the reference) or Cy5 dye (for the test sample) (AP Biotech). The Cy3- or Cy5-labeled cDNA probes were purified, dried, and resuspended in hybridization buffer containing 30% formamide, 5× SSC, 0.1% SDS,
and 0.1 mg/mL salmon sperm DNA. The cDNA probes were mixed together and hybridized to customized microarray slides (E. coli K12 3 × 15 K microarray; http://www.Mycroarray.com). The image of the slide was scanned with a GenePix 4000B (Axon Instruments, USA) and analyzed by GenePix Pro 3.0 software (Axon Instruments) to obtain the gene expression ratios (reference vs. test sample). Microarray data analysis was performed using Genowiz 4.0™ (Ocimum Biosolutions). Global lowess (Locally weighted scatter plot smoothing) method was used for data selleck compound normalization. The cut-off values for up- or down-regulated genes in duplicate hybridizations were 1.5- or 0.6-fold, respectively. RT-qPCR analysis The E. coli strains listed in Additional file 1: Table S1 were grown in LB medium to an OD600 of 1.0, and the total RNA was extracted using an RNeasy Mini Kit (Qiagen). Reverse transcription and qPCR (RT-qPCR) analyses were performed using CFX96 (Bio-Rad) with IQ™ SYBR® Green Supermix (Bio-Rad), as described previously  and gene specific primers designed by PrimerQuest (http://www.idtdna.com; Additional file 1: Table S2).