FIG 3.

C. crescentus cells are starved for B₁₂ in laboratory growth medium. (A) Fraction of ribosome footprints for the most highly translated mRNAs in M2G. B₁₂-related genes are colored. (B) Pathway of methionine biosynthesis, MetX1 (CCNA_03309), MetX2 (CCNA_00559), MetZ (CCNA_02321), MetE (CCNA_00515), MetH (CCNA_02221), and CCNA_02222. Pathway for B₁₂ utilization, BtuB (CCNA_01826) and BtuR (CCNA_02321). Pathway for tRNA modification, MtaB (CCNA_03798). Pathway for nucleotide reduction, NrdJ (CCNA_01966), NrdE (CCNA_03607), and NrdB (CCNA_00261). Pathway for succinyl-CoA biosynthesis, MeaA (CCNA_03177) and MutB (CCNA_02459). Square boxes next to each enzyme contain an orange heat map which represents the fraction of ribosome footprints. ADO (adenosyl) and CN (cyano) refer to the B₁₂ upper ligand. (C) Negative regulation by B₁₂ riboswitches on the btuB and metE genes. Translation reporters for the btuB and metE genes fused to mCherry assayed in M2G with the indicated concentrations of B₁₂. Error bars represent standard deviation for mCherry fluorescence in three biological replicates of the B₁₂ dilution series (n = 3). Solid blue and green horizontal lines indicate the mChy fluorescence without vitamin B₁₂ for btuB and metE, respectively, and dashed lines indicate the standard deviation. (D) Cartoon of B₁₂-regulated pathways in C. crescentus. btuB and metE genes contain negative regulatory B₁₂ riboswitches. BtuB and BtuR are part of the B₁₂ import and utilization pathway. MetH, NrdE, MeaA, MutB, and MtaB are B₁₂-dependent enzymes for methionine biosynthesis.