Flanking regions of the mlr gene cluster were amplified by PCR walking. Two groups of primers for this purpose were designed based on mlrC and mlrB* sequences of THN1 (Table 1). General amplifications, purification and sequencing of the PCR products were performed as described previously (Lin et al., 2010), except that the annealing temperature was adjusted according BKM120 to the Tm values of different primers. A Genome Walking Kit (Takara, Japan) was utilized for PCR walking according to procedures provided by the manufacturer. All amplifications

were conducted in an MJ mini personal thermal cycler (Bio-Rad). Sequences were compared with known mlr genes in GenBank using blastn. A single colony of the bacterium was inoculated into 20 mL R2A medium and cultivated overnight. One milliliter of the culture was centrifuged at 3000 g for 1 min. The pellet was resuspended in 20 mL fresh medium within a conical flask and cultivated to an OD600 nm=0.6 http://www.selleckchem.com/products/azd-1208.html at 28 °C. Nine flasks of this kind were

divided into three groups for independent experiments. Within each group, three parallel cultures were prepared. Then, microcystin LR was added to a final concentrations of 0.4 and 2.0 mg L−1, respectively, and sterile water with no microcystin was used as a control. Two milliliters of culture were taken from the flasks 10, 20, 30, 45, 60, 90 and 120 min after inoculation, and centrifuged (12 000 g, 1 min) at 4 °C. The supernatant was decanted and the bacterial pellet was resuspended in 1 mL Trizol reagent (Invitrogen). Total RNA extraction, reverse transcription and Real-time PCR were performed as described previously (Shao et al., 2009), except that a MyiQ mini Real-time system (Bio-Rad) was used in our study. not Two pairs of specific primers, qmlrAF/qmlrAR and q16SF/q16SR (Table 1), were used for quantification of mlrA and the 16S rRNA gene, respectively. The mRNA copy number was determined using the Ct value. The induction ratio was calculated by where

ΔΔCt=(Ct, target gene−Ct, 16S rrn)stress−(Ct, target gene−Ct, 16S rrn)control according to the handbook for the Bio-Rad Real-time PCR system. Significant differences between treatments and control at different times were determined by independent-samples t-test with spss 13.0 for Windows, and differences were considered to be significant at P<0.05. The RNA and cDNA samples were obtained from bacterial cultures containing 2.0 mg L−1 microcystin LR as described in the above section and were used in this section. Before reverse transcription, total RNA extracts were digested by DNase to eliminate genomic DNA contamination. Total cDNA of pure RNA extracts were used for detecting mlrB* using primer sets mlrB-84 and mlrB-203 (Table 1). Positive and negative controls were performed using THN1 cells and pure RNA extracts as templates, respectively. Amplification of the mlrA gene was also performed using primer sets qmlrAF and qmlrAR to ensure template quality.