A c-di-AMP riboswitch controlling kdpFABC operon transcription regulates the potassium transporter system in Bacillus thuringiensis

Abstract

The intracellular K + level in bacteria is strictly controlled by K + uptake and efflux systems. Among these, KdpFABC is a high-affinity K + transporter system that is generally activated by the KdpDE two-component system in response to K + limitation stress. However, the regulatory mechanism remains obscure in bacteria lacking the kdpDE genes. Here we report that the transcription of a kdpFABC operon is distinctively regulated by a cyclic diadenylate monophosphate (c-di-AMP) riboswitch located at the 5′-untranslated region of kdp transcript, and binding of c-di-AMP to the riboswitch promotes its intrinsic termination that blocks the kdpFABC transcription. Further, the intracellular c-di-AMP concentration was found to decrease under the K + limitation stress, leading to transcriptional read-through over the terminator to allow kdpFABC expression. This regulatory element is found predominantly in the Bacillus cereus group and correlate well with the K + and c-di-AMP homeostasis that affects a variety of crucial cellular functions.

6S-1 RNA Contributes to Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis

“…Bacillus thuringiensis BMB171 and its derivative strains were cultured at 28°C in the GYS medium (g/L: glucose, 1.00; yeast extract, 2.00; K 2 HPO 4 •3H 2 O, 0.66; (NH 4 ) 2 SO 4 , 2.00; MgSO 4 •7H 2 O, 0.04; MnSO 4 •H 2 O, 0.04; and CaCl 2 , 0.08), with the medium autoclaved at 115°C for 30 min after pH value being adjusted to 7.8. When necessary, relevant antibiotics were added to the cultures with the following final concentration: 50 μg/ml kanamycin, 25 μg/ml erythromycin, 100 μg/ml ampicillin, 300 μg/ml spectinomycin, or 60 U polymyxin (Wang et al, 2019). For the decoyinineadded experiment, cells were grown to an OD 600 of 0.5 in GYS medium, followed by the addition of decoyinine to 0.4 mg/ml as previously described (Mitani et al, 1977;Ikehara et al, 1982;Cavanagh and Wassarman, 2013).…”

“…The cDNA was then PCR amplified using primers of Primer-8 and ssrSB-R as listed in Supplementary Table S1. The PCR products were then cloned to the pMD19-T vector (Takara, Japan) and sequenced (Wang et al, 2019).…”

“…The Bacillus cereus group is a taxonomic group comprising closely related species of the Bacillus genus that includes more than 20 different species (Miller et al, 2016;Liu et al, 2017). Among them, the insect pathogen Bacillus thuringiensis, the anthracnose pathogen Bacillus anthracis, and the food-borne opportunistic pathogen B. cereus (Wang et al, 2019), have attracted extensive attention. Different from B. subtilis that harbors two 6S RNA-encoding genes located in different regions, B. cereus group bacteria contain the 6S RNA-encoding genes arranged in tandem in their genomes (Wehner et al, 2014).…”

2‐Methylcitrate cycle: a well‐regulated controller of Bacillus sporulation

“…We first prepared total RNA from the wild-type strain BMB171 (cultured in GYS medium for 12 h). The TSS of prp operon was then determined by the 5 0 -RACE experiment based on the terminal deoxynucleotidyl transferase (TdT) as described previously (Ali et al, 2017;Wang et al, 2019).…”

“…BMB171 and its mutants containing the transcriptional fusion plasmid pHT1K-P prp -lacZ were grown at 28 C in an orbital shaker at 200 rpm in 200 ml GYS medium with 25 μg ml −1 erythromycin. Four millilitres of cultures were sampled at 2 h intervals and used for the determination of β-galactosidase activity as described previously (Wang et al, 2013c;Zheng et al, 2015;Tang et al, 2016;Wang et al, 2019).…”