INSERTION MUTATION OF THE FORM-I CBBL GENE ENCODING RIBULOSE-BISPHOSPHATE CARBOXYLASE OXYGENASE (RUBISCO) IN THIOBACILLUS-NEAPOLITANUS RESULTS IN EXPRESSION OF FORM-II RUBISCO, LOSS OF CARBOXYSOMES, AND AN INCREASED CO2 REQUIREMENT FOR GROWTH/

It has been previously established that Thiobacillus neapolitanus fixe s CO2 by using a form I ribulose bisphosphate carboxylase/oxygenase (R uBisCO), that much of the enzyme is sequestered into carboxysomes, and that the genes for the enzyme, cbbL and cbbS, are part of a putative carboxysome operon. In the present study, cbbL and cbbS were cloned an d sequenced. Analysis of RNA showed that cbbL and cbbS are cotranscrib ed on a message approximately 2,000 nucleotides in size. The insertion of a kanamycin resistance cartridge into cbbL resulted in a premature termination of transcription; a polar mutant was generated. The mutan t is able to fix CO2, but requires a CO2 supplement for growth. Separa tion of cellular proteins from both the wild type and the mutant on su crose gradients and subsequent analysis of the RuBisCO activity in the collected fractions showed that the mutant assimilates CO2 by using a form II RuBisCO. This was confirmed by immunoblot analysis using anti bodies raised against form I and form II RuBisCOs. The mutant does not possess carboxysomes. Smaller, empty inclusions are present, but bioc hemical analysis indicates that if they are carboxysome related, they are not functional, i.e., do not contain RuBisCO. Northern analysis sh owed that some of the shell components of the carboxysome are produced , which may explain the presence of these inclusions in the mutant.