EXPRESSION OF THE CBBLCBBS AND CBBM GENES AND DISTINCT ORGANIZATION OF THE CBB CALVIN CYCLE STRUCTURAL GENES OF RHODOBACTER-CAPSULATUS

Rhodobacter capsulatus fixes CO2 via the Calvin reductive pentose phos phate pathway and, like some other nonsulfur purple bacteria, is known to synthesize two distinct structural forms of ribulose 1,5-bisphosph ate carboxylase/oxygenase (RubisCO). Cosmid clones that hybridized to form I(cbbLcbbS) and form II (cbbM) RubisCO gene probes were isolated from a genomic library of R, capsulatus strain SB1003. Southern blotti ng and hybridization analysis with gene-specific probes derived from R hodobacter sphaeroides revealed that R, capsulatus cbbM is clustered w ith genes encoding other enzymes of the Calvin cycle, including fructo se 1,6/sedoheptulose 1,7-bisphosphatase (cbbF), phosphoribulokinase (c bbP), transketolase (cbbT), glyceraldehyde-3-phosphate dehydrogenase ( cbbG), and fructose 1,6-bisphosphate aldolase (cbbA), as well as a gen e (cbbR) encoding a divergently transcribed LysR-type regulatory prote in. Surprisingly, a cosmid clone containing the R, capsulatus form I R ubisCO genes (cbbL and cbbS) failed to hybridize to the other cbb stru ctural gene probes, unlike the situation with the closely related orga nism R. sphaeroides. The form I and form II RubisCO genes were cloned into pUC-derived vectors and were expressed in Escherichia coli to yie ld active recombinant enzyme in each case. Complementation of a RubisC O-deletion strain of R. sphaeroides to photosynthetic growth by R. cap sulatus cbbLcbbS or cbbM was achieved using the broad host-range vecto r, pRK415, and R. sphaeroides expression vector pRPS-1.