Cloning of cbb gene promoters from the sulfur-oxidizing bacteria Thiobacillus thioparus and Halothiobacillus neapolitanus, and assessment of their promoter efficacies

To determine the transcriptional potential of cbb genes involved in CO₂ fixation, the promoters of two sulfur-oxidizing bacteria, Thiobacillus thioparus DSM 505 and Halothiobacillus neapolitanus DSM 15147 were cloned. The unknown upstream sequence of the DSM 505 cbbL gene was successfully amplified through three rounds of thermal asymmetric interlaced PCR, yielding the fragments UP_{505-cbbL-Short} and UP_{505-cbbL-Long}. Sequence analysis confirmed that these two fragments contained the upstream sequence of the DSM 505 cbbL gene and that UP_{505-cbbL-Long} contained a promoter sequence. A series of fragments containing potential DSM 505 cbbL promoters (P_{505-cbbL-Y+I-1}, P_505-cbbL-Y+I, P_{505-cbbL-Y+I+1}, P_{505-cbbL-J+I-1}, P_505-cbbL-J+I and P_{505-cbbL-J+I+1}) and fragments containing the known promoters of DSM 15147 cbb genes were inserted into the promoter-reporter plasmid pUC18-GFP, which contained the green fluorescent protein (GFP) gene. Sequencing results confirmed that all the promoter fragments were successfully cloned and transformed into Escherichia coli Top10. The promoter efficacies of the promoter fragments were then assessed by measuring the fluorescence intensities of the recombinant strains. For DSM 15147, the promoter efficacy of the cbbL promoter was much higher than that of the cbbM promoter, which corresponded with the transcription efficiencies of the cbb genes. For DSM 505, the promoter fragments P_{505-cbbL-Y+I+1} and P_{505-cbbL-J+I+1} were able to initiate GFP expression. The promoter efficacy of the DSM 505 cbbL promoter was higher than that of the DSM 15147 cbbL promoter, indicating that the cbbL gene transcription and CO₂ assimilation potentials of DSM 505 may be higher than those of DSM 15147. Copyright © 2019 Friends Science Publishers.