Fig. 3. Leaderless expression of cvkR genes.

a Deduced amino acid sequence alignment of 53 MerR-family CvkR homologs. The sequences of the MerR-family CvkR homologs for the multiple sequence alignment were recovered from public databases and are available in Supplementary Data 1. The sequences were aligned using MAFFT⁸⁷ and visualized with UGENE⁸⁸. b Differential RNA sequencing data²⁸ indicate that the TSSs of cvkR and cas12k were located internal to the NCBI-annotated coding sequences under nitrogen repletion (dark green) and 8 h of nitrogen depletion (light green). Yellow boxes indicate the corresponding sections not used in translation. Data shown as primary read coverage mapped to the corresponding genomic region. Each TSS is represented by an arrow. c Distances between the cas12k effector protein and regulator genes are plotted according to the respective type of regulator, CvkR (n = 51 examined independent CAST systems), Arc_1 (n = 21) and Arc_2 (n = 11). The box plots are colored according to the respective DNA-binding domain (HTH: blue; RHH: red) and structured as center: median; bounds of box: upper and lower quartiles; whiskers: 1.5× interquartile range; dots: individual samples. d qRT–PCR analyses verify that cvkR is transcribed in ΔcvkRCom strains. The amounts of cvkR transcripts were normalized to those of rnpB as an internal standard. Data are presented as mean values ± SD (n = 3 biologically independent experiments). e Western blot analyses of different ΔcvkR complementation strains show expression of differently sized CvkR proteins. Upper panel, Western blot against the C-terminal 3xFLAG tag; Lower panel, ponceau S staining shows that equal amounts of protein were loaded (100 μg). The calculated molecular masses for CvkR-S and CvkR-L were 20.03 kDa and 22.41 kDa, respectively. Two independent experiments were performed, which showed consistent results. The ΔcvkRCom-1, 2, and 3 strains are detailed in Supplementary Fig. 4c. Source data are provided as a Source Data file.