Figure 3.

Complementation of C. burnetii proline and tyrosine auxotrophies for transformant selection. (A) Pathway of proline biosynthesis from glutamate. C. burnetii has lost the proA and proB genes that catalyze the first two steps of proline biosynthesis from glutamate while retaining proC, which catalyzes the final step in the proline biosynthesis pathway. (B) Schematic of the proBA operon cloned from Legionella and placed under the control of the CBU1169 promoter. The 1169^P -proBA operon was cloned into the pJB-CAT shuttle vector and the plasmid transformed into C. burnetii. (C) Complementation of the proline auxotrophy was tested using ACCM-D plus or minus proline. Expression of proBA in NMII (NMII proBA) complemented growth of the bacterium in the absence of proline, whereas the wild-type bacteria (NMII) could only grow in the presence of added proline. (D) Pathway of tyrosine biosynthesis from chorismate. C. burnetii has lost both genes (tyrA and tyrB) required for biosynthesis of tyrosine from chorismate. (E) Schematic of the tyrB gene cloned from E. coli and placed under the control of the CBU1169 promoter. This fragment was then cloned into pJB-CAT and the plasmid (pJB-CAT-tyrB) transformed into C. burnetii. (F) Complementation of the tyrosine auxotrophy was tested using ACCM-D with the addition of the TyrB substrate 4-hydroxyphenylpyruvic acid (4-HPP) and plus or minus tyrosine. Expression of tyrB in NMII (NMII tyrB) complemented growth of the bacterium in ACCM-D containing 4-HPP but minus tyrosine, whereas the wild-type bacteria (NMII) could only grow in the presence of added tyrosine and not in the presence of the TyrB substrate 4-HPP alone.