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 CO ₂ Requirement for Growth

Abstract

It has been previously established that Thiobacillus neapolitanus fixes CO2 by using a form I ribulose bisphosphate carboxylase/oxygenase (RuBisCO), 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 andcbbS were cloned and sequenced. Analysis of RNA showed thatcbbL and cbbS are cotranscribed 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 mutant is able to fix CO2, but requires a CO2supplement for growth. Separation of cellular proteins from both the wild type and the mutant on sucrose 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 antibodies raised against form I and form II RuBisCOs. The mutant does not possess carboxysomes. Smaller, empty inclusions are present, but biochemical analysis indicates that if they are carboxysome related, they are not functional, i.e., do not contain RuBisCO. Northern analysis showed that some of the shell components of the carboxysome are produced, which may explain the presence of these inclusions in the mutant.

Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component

“…Recombinant protein expression in yeast was accomplished using bait or prey expression vectors, pGBKT7 and pGADT7 respectively, provided as part of the Matchmaker GAL4 Two-Hybrid System 3 (Clontech, Palo Alto, CA). All carboxysome genes were individually amplified by PCR using pTnl template DNA [ 73 ]. Designed primers for the yeast two-hybrid system ( Table S6 ) were used to add endonuclease restriction sites Ndel and BamHI that flank the 5' and 3' ends of the PCR products, which allowed for the subcloning into either bait or prey vector DNA.…”

Microbial CO2 fixation potential in a tar-oil-contaminated porous aquifer

“…Two species of Thiobacillus, i.e. T. denitrificans (Baker et al, 1998;Beller et al, 2006) and T. thiophilus D24TN T (Kellermann & Griebler, 2009), were shown to be able to fix CO 2 via the Calvin cycle under anoxic conditions, using nitrate as electron acceptor and sulphide and/or thiosulphate as electron donor. Transcription of the cbbM and green-like cbbL genes, as well as the synthesis of RubisCO via enzyme activity tests, could be shown for T. thiophilus D24TN T (Kellermann & Griebler, 2009).…”

Diversity and function in microbial mats from the Lucky Strike hydrothermal vent field

“…kazani endosymbiont (Duperron et al, 2007a, b) and Solemya velum gill sulfur-oxidizing symbionts (Schwedock et al, 2004). In the cbbL gene library, sequences distantly affiliated with Chromatiales Alkalilimnicola ehrlichii MLHE-1 and Halothiobacillus neapolitanus (Baker et al, 1998) were also identified. We retrieved 38 form II RuBisCO (cbbM) genes and 12 transcript sequences (Fig.…”