Figure 6. Growth analysis of R. sphaeroides ΔbluE and ΔbluE ΔcobB strains.

Growth analysis of R. sphaeroides bluE⁺, ΔbluE, and ΔcobB ΔbluE strains harboring a plasmid expressing R. sphaeroides bluE⁺ or carrying the empty pBBR1MCS-2 plasmid (vector). Cells were grown overnight in Sistrom’s medium and cultures were prepared as described in Materials and Methods. Cell growth was monitored normoxically at 30°C in Sistrom’s medium with acetate (30 mM), kanamycin (0.01 mg mL⁻¹), and when noted, Cby (15 nM) or Cbi (15 nM). Growth experiments were performed in triplicate in three independent experiments. Error bars represent the standard error of the mean. Pathway represents a simplified schematic of the roles of CobB (hydrogenobyrinate a,c-diamide synthase) and BluE (L-Thr kinase) in the synthesis of cobalamin in R. sphaeroides. CobD, AdoCbi-P synthase; CobC, L-Thr-P decarboxylase; CobP, AdoCbi kinase / AdoCbi-P guanylyltransferase; BluE, L-Thr kinase; Cby, cobyric acid; Cbi, cobinamide, AP-P, (R)-1-aminopropan-2-ol O-phosphate. Figure key: Panel A: cobB⁺ bluE⁺/vector (●), ΔbluE/pRsBluE (○), ΔbluE/vector (✲), Panel B: ΔcobB ΔbluE/pRsBluE (□), ΔcobB ΔbluE/vector (▲), cobB⁺ bluE⁺/pRsBluE (●), Panel C: cobB⁺ bluE⁺/vector (●), ΔcobB ΔbluE/vector (▲), ΔcobB/vector (□), ΔbluE/vector (✲).