Bacterial Strains and Cell Growth

R.sphaeroides 2.4.1 or 2.4.1（pUIIA2）was used where indicated in this study.Strain 2.4.1（pUIIA2）contains plasmid pUIIA2 which has an R.sphaeroides promoter and translational regulatory regions fused to the lac operon described by Casadaban et al.［12］which is contained in the broad host range plasmid pUI108［13］.This strain was employed for the studies described，because measurement of p-galactosidase activity and lac-specific mRNA represented reliable internal controls.Theβ-galactosidase activity was measured as described previously［12］.Control experiments（some of which are contained in this paper）have shown no differences in the expression of the puf operon in this strain relative to that observed for R.sphaeroides 2.4.1.The cells were grown on Sistrom's medium A containing 0.4%butyrate and 0.1%NaHCO3 as the carbon source unless otherwise stated.For experiments employing R.sphaeroides（pUI1A2）this medium was supplemented with streptomycin at 25 μg/ml.Photoheterotrophically grown cells were cultured to a cell density of 5×108 cells/ml at 30℃under different light intensities：10 watts/m2 was designated high intensity light（HL）and 3 watts/m2 was designated low intensity light（LL），as previously described［10］.The generation times of R.sphaeroides 2.4.1（pUI1A2）are 5.5 and 12 h in the presence of high and low light，respectively.Dark treatment was performed by wrapping the growth vessels from high light-grown cells with aluminum foil for 1 h at 30℃.Aerobic growth（5-h generation time）for strain 2.4.1（pUI1A2）was maintained by sparging with a gas mixture of O2∶N2∶CO2（25∶74∶1）.

In experiments determining the decay of puf-specific mRNA，cells were grown on succinate（0.4%）containing medium sparged with a gas mixture of N2∶CO2（98∶2）to a cell density of 2×109 cells/ml at high intensity light.Where indicated，transcription initiation was inhibited by the addition of rifampicin（200 μg/ml），and cells were harvested for RNA isolation at the times indicated.The decay of the puf-specific mRNA was also measured following either a shift of photoheterotrophically grown cells to dark conditions or by changing the gas mixture from N2∶CO2（98∶2）to O2∶N2∶CO2（25∶74∶1）.Cells were withdrawn for RNA isolation at various time intervals as indicated in the text.Zero time samples were those withdrawn from the culture just prior to imposition of the new growth conditions.(https://www.daowen.com)