information indicate that CYP450s are prevalent in members of the bacterial phylum
Deinococcus-Thermus as well as the archaeal family Halobacteriaceae that belong to the
phyulm Euryarchaeota. A property shared by these phylogenetically distant extremophiles is
the production of carotenoid pigments. It became the purpose of this study to use genome
sequence information to clone and study new CYP450s from the genera Thermus and
Halobacterium and to explore the role of these CYP450s in pigment production.
The non-pigmented thermophilic bacterium Thermus scotoductus SA-01 was screened by
PCR for the presence of a cytochrome P450 monooxygenase (CYP450). No CYP450 could
be found and subsequent genome sequencing confirmed this finding. However, a CYP450
gene (CYP175A) was isolated from the closely related yellow pigmented strain Thermus sp.
NMX2.A1 using oligonucleotides based on the DNA sequence of the β-carotene gene
cluster from three Thermus strains.
The genome sequence of T. scotoductus SA-01, revealed a ferredoxin (Fdx) and ferredoxin
reductase (FNR) that were almost identical to those of Thermus thermophilus HB27. In T.
thermophilus HB27 the Fdx and FNR are the native redox partners for CYP175A1, a β-
carotene hydroxylase. After heterologous expression in Escherichia coli, we attempted to
hydroxylate β-carotene with the CYP450 from Thermus sp. NMX2.A1 and the redox partners
of T. scotoductus SA-01 using cell free extracts, but no products were detected.
Thirty two CYP450s have been identified in the sequenced genomes of thirteen extremely
halophilic archaea. Initial attempts to clone and heterologously express a CYP174A2-
homologue from a Haloarcula LK-1 strain in E. coli and Pseudomonas fluorescens were
unsuccessful. In order to study the physiological role of CYP450s in halophilic archaea and
to create a strain that can be used for heterologous expression of CYP450s from halophiles
CYP174A1 was deleted from H. salinarum R1. CYP174A1 is the only CYP450 in H.
salinarum R1 and H. salinarum R1 is a genetically tractable strain.
Upon culturing the wildtype and deletion strains, a difference in red pigmentation of
stationary phase cultures was observed; implying that CYP174A1 might play a role in carotenoid synthesis. Microarray analyses revealed that the bop gene, which codes for
bacterioopsin (BO) was severely repressed in stationary phase cultures of the deletion strain
and sucrose gradient experiments showed a consequent loss of purple membrane (PM) in
the deletion strain. The classical causes of bop repression e.g. insertion elements in the bop
open reading frame as well as in the brz gene was ruled out by PCR screening. In addition
to bop repression, the neighboring vng1459 and vng1468 genes (both part of the bopregulon)
were also down regulated, but the genes normally involved in regulation of the bop
gene were not affected. Currently the functions of vng1459 and vng1468 are unknown.
Retinal, together with BO, is a key component of bacteriorhodopsin (BR) and essential for
PM synthesis. Retinal is formed by the central cleavage of β-carotene which can be
achieved by monooxygenases or dioxygenases.The Blh and Brp proteins in H. bacterium
salinarum are very closely related to a confirmed bacterial 15,15â²-β-carotene dioxygenase
and studies have shown that deletion of both brp and blh results in complete abolishment of
retinal and BR. It is therefore unlikely that CYP174A1 plays a role in retinal biosynthesis.
Another possible function for CYP174A1 might be the hydroxylation of β-carotene, since it is
known that H. salinarum strains produce hydroxylated carotenoids such as transastaxanthin,
but no genes encoding typical β-carotene hydroxylases or ketolases have been
identified in the genomes of H. salinarum strains. This will imply that hydroxylated
carotenoids play a role in the regulation of bop.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-08152012-145358 |
| Date | 15 August 2012 |
| Creators | Müller, Walter Joseph |
| Contributors | Dr LA Piater, Prof J Albertyn, Prof E van Heerden, Prof MS Smit |
| Publisher | University of the Free State |
| Source Sets | South African National ETD Portal |
| Language | en-uk |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.uovs.ac.za//theses/available/etd-08152012-145358/restricted/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University Free State or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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