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Cloning and expression of Bacillus subtilis ribosomal RNA gene promoters in Escherichia coli

University of British Columbia

In coli, ribosomal RNA (rRNA) is synthesized in proportion to the cellular growth rate squared, a phenomenon known as growth rate dependent regulation. The promoters of rRNA operons, which consist of two tandemly arranged -35, -10 regions about 120 bp apart, mediate this characteristic form of regulation. To provide insight into how this regulation is achieved and to extend previous observations from studies with E. coli to the rRNA operons of other species, the promoter region from the B. subtilis rrnB operon was cloned onto a transcription fusion plasmid such that the synthesis of chloramphenicol acetytransferase (CAT) was directed by the subtilis promoters. Expression in E. coli showed that CAT specific activity increased in a growth rate dependent manner. The synthesis of CAT mRNA, the CAT mRNA half-life, and plasmid copy number were all measured directly to establish the validity of using CAT specific activity as a measure of promoter function. Only the downstream P2 promoter of the subtilis P1-P2 tandem pair was shown to be growth rate regulated; in contrast, the P1 promoter of the native E. coli rrnB operon was responsive to growth rate. Deletion of an A-T rich sequence upstream of the B. subtilis P1 promoter had no effect on the overall level of P1 or P2 transcription. These results indicated that the general mechanisms conferring growth rate regulation were conserved between coli and B. subtilis, although differences were noted at the level of the individual P1 and P2 promoters. In an attempt to develop a similar operon fusion system for use in B. subtilis a number of bi-functional shuttle vectors were constructed by fusing subtills plasmids to an coli replicon carrying a promoterless catechol 2,3 dioxygenase gene. However, it was found that overexpression of this protein was lethal to E. coli cell3 and rRNA promoters could not be maintained unless their transcriptional activity was curtailed. Furthermore, such bi-f unctional vectors were highly unstable in E. coli or after transfer to subtills. Nevertheless, data obtained using one of these vectors indicated that B. subtilis rRNA promoters did not possess the antitermination function associated with the analogous coli promoters, illustrating an additional functional difference between E. coli and subtilis rRNA promoters.

Text

2010-08-01

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http://hdl.handle.net/2429/27066

Microbiology and Immunology

University of British Columbia

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