The regulation of gene expression in sulphate reducing bacteria

Cadby, Ian Thomas (2014). The regulation of gene expression in sulphate reducing bacteria. University of Birmingham. Ph.D.

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Abstract

\(Desulfovibrio desulfuricans\) ATCC27774 can use both nitrate and sulphate as terminal electron acceptors for growth. Contrary to expectations, the less favourable terminal electron acceptor, sulphate, is utilised preferentially. It has been proposed that the nap gene cluster of \(D. desulfuricans\), which encodes nitrate reduction, is regulated at the transcription level by both sulphate repression and nitrate induction. Transcription factors of the CRP/FNR family have been implicated in this regulation.
At the start of this study a bioinformatic analysis identified five CRP-homologues in \(D. desulfuricans\), two of which, HcpR and HcpR2, were predicted to bind discrete regulatory motifs and influence the transcription of \(nap\), \(hcpR\) and the gene for the hybrid cluster protein, \(hcp\). Physiological and qRT-PCR experiments revealed that expression of \(hcpR\) is induced in the presence of nitrite whereas expression of \(hcpR2\) is constitutive. Furthermore, tolerance to nitrosative stress can be pre-induced by exposure to nitrite.
In electromobility shift and footprinting assays, HcpR bound to CRP-like DNA targets upstream of the \(nap\) and \(hcpR\) operons: HcpR2 bound to a different target upstream of \(hcp\). HcpR was shown to heterolgously affect transcription at CRP-regulated promoters in an \(Escherichia\) \(coli\) host. The pET expression system was used to overproduce HcpR, the recombinant protein was purified and western analysis with a rabbit anti-HcpR antiserum revealed that nitric oxide is the inducer of HcpR synthesis. Once induced, significant levels of HcpR persisted in the absence of effector for many generations.
The results suggest that the primary function of HcpR is auto-regulation of the \(hcpR\) operon. This transcription unit includes genes similar to \(nimA\) and \(wrbA\) and their expression is believed to be essential for protecting \(D. desulfuricans\) against nitrosative stress. Contrary to the predictions of the bioinformatic analysis, expression of \(nap\) did not correlate with \(hcpR\) levels. An alternative hypothesis is proposed in which \(nap\) and \(nrf\) are primarily regulated by a sigma-54-dependent mechanism.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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