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Complementation of a Rhodopseudomonas sphaeroides ribulose bisphosphate carboxylase-oxygenase regulatory mutant from a genomic library.

ABSTRACT

A genomic library containing HindIII partial digests of Rhodopseudomonas sphaeroides HR DNA was constructed in the broad-host-range cosmid cloning vector pVK102. With a portion of this library as donor in complementation studies with the ribulose 1,5-bisphosphate carboxylase-oxygenase regulatory mutant R. sphaeroides KW25/11, a fragment of DNA which is capable of partially complementing this mutant was isolated. In four independent matings, Aut+ transconjugants which contained a hybrid plasmid carrying the same 28-kilobase-pair insert were isolated. While complemented strains were capable of growing at rates equal to that of the wild type under photoautotrophic conditions, they were not able to match wild-type levels of ribulose 1,5-biphosphate carboxylase-oxygenase activity or of form I ribulose 1,5-bisphosphate carboxylase-oxygenase protein. In addition, there is some indication that recombination may be necessary for optimal complementation to occur. The size of the complementing fragment was further reduced to 2.7 kilobase pairs by using vectors constructed for subcloning.