Movahedzadeh, Farahnaz; (1996) Regulation of mycobacterium tuberculosis RECA expression. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The SOS response involves the coordinated expression of more than 20 genes in response to DNA damage and makes an important contribution to the survival of bacteria in hostile environments. The key regulatory components of the SOS response are RecA and Lex A, the activator and repressor respectively. The recA gene of M. tuberculosis has previously been cloned and sequenced (Davis et al., 1991). However, nothing was known about the regulation of this system in mycobacteria. In this study the expression of the recA gene was shown to be inducible in response to various DNA damaging agents by using a transcriptional fusion to the reporter gene chloramphenicol-acetyl transferase (CAT). Furthermore, by producing constructs containing various lengths of upstream sequence I have been able to identify the key regulatory elements involved in the induction. A segment of DNA around 300 bp upstream of the coding region was shown to be required for expression. This stretch of DNA contains a putative LexA-binding site, based on homology to the "Cheo box" binding site of Bacillus subtilis (but unlike the "SOS box" of Escherichia coli). I have also cloned and sequenced the lexA gene of Mycobacterium tuberculosis and this too has a "Cheo box" like sequence within its upstream region. Primer extension analysis has been used to identify promoter sequences involved in recA and lexA expression. The LexA protein was overexpressed, purified and demonstrated to bind to the Cheo boxes.

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