CBBR, A LYSR-TYPE TRANSCRIPTIONAL ACTIVATOR, IS REQUIRED FOR EXPRESSION OF THE AUTOTROPHIC CO2 FIXATION ENZYMES OF XANTHOBACTER-FLAVUS

Xanthobacter flavus is able to grow autotrophically with the enzymes o f the Calvin cycle for the fixation of CO2, which are specified by the cbbLSXFP gene cluster. Previously, the 5' end of an open reading fram e (cbbR), displaying a high sequence similarity to the LysR family of regulatory proteins and transcribed divergently from cbbLSXFP, was ide ntified (W. G. Meijer, A. C. Arnberg, H. G. Enequist, P. Terpstra, M. E. Lidstrom, and L. Dijkhuizen, Mol. Gen. Genet. 225:320-330, 1991). T his paper reports the complete nucleotide sequence of cbbR and a funct ional characterization of the gene. The cbbR gene of X. flavus specifi es a 333-amino-acid polypeptide, with a molecular weight of 35,971. Do wnstream from cbbR, the 3' end of an open reading frame displaying a h igh similarity to ORF60K from Pseudomonas putida and ORF261 from Bacil lus subtilis was identified. ORF60K and ORF261 are located at the repl ication origin of the bacterial chromosome. Inactivation of cbbR, via the insertion of an antibiotic resistance gene, rendered X. flavus una ble to grow autotrophically. This was caused not by an inability to ox idize autotrophic substrates (e.g., formate) but by a complete lack of expression of the cbb genes. The expression of the CbbR protein in Es cherichia coli was achieved by placing cbbR behind a strong promoter a nd optimization of the translational signals of cbbR. CbbR binds speci fically to two binding sites in the cbbR-cbbL intergenic region.