Microbial respiration can be highly diverse and adaptable, with many bacteria able to respond to changes in their environment promptly and efficiently. The regulation of respiratory enzymes by highly responsive and precise transcriptional regulators confers distinct advantage for survival in sometimes harsh and extreme conditions. The organohalide-respiring bacterium Desulfitobacterium hafniense DCB-2 is able to utilise a wide range of electron acceptors and respiratory processes through tight regulation of respiratory machinery. An example of this tight regulation of respiratory machinery can been seen by biochemical analysis of the CRP-FNR-type transcriptional regulator family CprK, of which five are present in the strain. CprK1 is able to sense the presence of the physiological ligand, 3-chloro-4-hydroxyphenylacetic acid (CHPA), of reductive dehalogenase CprA1 with nM affinity. In this work we demonstrate that CprK1 is able to distinguish between the chlorinated CprA1 substrate CHPA and the non-chlorinated product 4-hydroxyphenylacetic acid (HPA) by ‘pKa interrogation’ of the 4-hydroxy moiety and by the atomic radius of the ortho-moiety. Through the use of in vitro biophysical and in vivo transcriptional response assays, we show that CprK1 is able to sense a number of halogenated phenols, including phenylacetic acids and nitrophenols. We also demonstrate that a 4-hydroxyl group is essential for CprK1 activation. In Chapter 4, an attempt to modify the effector sensitivity of CprK1 is performed by site-specific and random mutagenesis, and mutant selection assays are developed. We show that CprK1 is highly resistant to effector specificity modifications, with seemingly minor or conservative amino acid changes removing CprK1’s ability to initiate transcription. In Chapter 5, the CprK1 paralogue, CprK4 from D. hafniense DCB-2 is characterised by in vitro biophysical and in vivo transcriptional response assays in order to assess its potential as a biosensor. We show that CprK4 is able to bind cis-regulatory DNA elements dehaloboxes 7 and 10 in the absence of effector by Surface Plasmon Resonance (SPR) protein array; however, we were unable to identify its effectors reliably. Due to the unknown nature of CprK4’s effector, it is still unclear whether CprK4 could be a valuable biosensor.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:603195 |
| Date | January 2014 |
| Creators | Kemp, Laura |
| Contributors | Munro, Andrew; Leys, David |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/functional-studies-of-cprk-a-transcriptional-regulator-of-organohalide-respiration(d1f3ecd2-78b2-4a91-b4a0-98761c487a11).html |
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