Rnaprotect bacteria reagent and rneasy mini kit

Geobacillus kaustophilus strains MK536 and MK536_up1 were cultured in MM medium at 60°C. Cells were collected at OD₆₀₀ = 1, and RNA was purified using an RNAprotect Bacteria Reagent and RNeasy Mini Kit (Qiagen, Venlo, Netherlands) with gDNA Eraser (Takara Bio). The pyrF transcript was detected using endpoint reverse transcription-polymerase chain reaction (RT-PCR). The RT reaction was performed using a PrimeScript RT reagent Kit (Takara Bio) with the PyrF_TR primer, whereas PCR was performed using Quick Taq HS DyeMix (Toyobo, Osaka, Japan) with two sets of primers: pyrF_0F and pyrF_600R (primer A) and is701_250F and pyrF_200R (primer B). Thermal cycles comprised 94°C for 2 min followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 2 min. The reaction without reverse transcriptase was used as negative control. The transcription of rpoB, which encodes for RNA polymerase β subunit, was detected as positive control using the primers rpoB_2800F and rpoB_3800R.

One hundred microliter of the frozen stock of each strain was inoculated into 100 ml of LB broth containing CTX and incubated at 37°C under constant shaking. The optical density at 600 nm (OD₆₀₀) of each culture was measured and a portion of the culture was collected for DNA and RNA extraction in early logarithmic phase (OD₆₀₀ = 0.5), middle logarithmic phase (OD₆₀₀ = 1.5), late logarithmic phase (OD₆₀₀ = 2.5), early stationary phase (OD₆₀₀ = 3.0), and late stationary phase (24 h). The genomic DNA was extracted as described above and total RNA was extracted using RNAprotect Bacteria Reagent and RNeasy Mini Kit (Qiagen, Venlo, Netherlands). mRNA was converted to cDNA using the PrimeScript RT reagent Kit (Takara Bio Inc., Shiga, Japan) with random hexamers provided with the kit. qPCR was performed as described earlier to determine the relative expression level of mRNA for bla_CMY−2. The parent strain, L-3553, was used to normalize the mRNA fold change. Because our preliminary data suggested that the nrfG expression was stable throughout different growth stages (data not shown), it was used as an internal control for mRNA quantification.