The genetics and biochemistry of a propane-utilizing "Rhodococcus rhodochrous"
Ashraf, William (1990) The genetics and biochemistry of a propane-utilizing "Rhodococcus rhodochrous". PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b1409455~S15
The pathways of terminal and subterminal propane oxidation have been
investigated in a propane-utilizing R. rhodochrous PNKbl. NTG-generated
pleiotrophic mutants, characterized by their inability to utilize
propane have been isolated. Several classes of mutants have been
obtained which are unable to metabolize potential propane oxidation
intermediates, e. g. propanol (alcA- or alcB-), propanal (ald-), acetone
(ket-), propanoate (oate-) and acetate (ace-). Only ket- mutants
retained the ability to metabolize propane. Mutants defective in the
first step of propane metabolism (aik-), were also unable to metabolize
acetol (a potential subterminal intermediate). Mutant analysis suggests
that propane is oxidized via terminal and subterminal pathways in
R. rhodochrous PNKbl. However, acetone (a potential subterminal
intermediate) does not appear to have a role in propane metabolism.
A propane-specific 86 kDa NAD+-dependent secondary alcohol dehydrogenase
has been purified to homogeneity. This enzyme oxidizes a range of
primary and secondary aliphatic alochols (C2 to C8). It is also
responsible for both propan-l-ol and propan-2-ol dehydrogenase
activities measured in cell-free extracts of propane-grown cells.
Western-blot analysis has shown that it is induced during growth on
propane, propan-2-ol, acetol and acetate (subterminal intermediates);
but not propan-l-ol, propanal propanoate (terminal intermediates) or
acetone. This technique has also demonstrated that a conserved NAD+-dependent alcohol dehydrogenase was induced in Rhodococcus - Nocardia
bacteria after growth on propane.
SDS-PAGE revealed proteins specific to cells grown on propane and
acetol, which may be components of a novel propane/acetol oxygenase
system. Oxygenase activity, as demonstrated by the epoxidation of
propene, was induced after growth on propane and acetol. NADPHdependent
acetol oxygenase activity was also detected. These results
suggest a relationship between the metabolism of propane and acetol.
Mutants unable to utilize propan-l-ol or propan-2-ol (aicA- and aicB- respectively)
were examined by assaying for NAD+-dependent propan-l-ol
and propan-2-ol dehydrogenase activities, by using SDS-PAGE analysis of
cell-free extracts and comparing the pattern and distribution of
pol peptides with the wild-type, and by Western-blot analysis of the
NAD -dependent secondary alcohol dehydrogenase synthesized by aicmutants.
Results demonstrated the aic- mutants had generally lower
NAD+-dependent alcohol dehydrogenase activities altered polypeptide
patterns and that alcB mutants synthesized NAD-dependent secondary
alcohol dehydrogenase which had altered electrophoretic mobility after
non-denaturing PAGE. The latter result may explain the inability of
these mutants to utilize propan-2-ol as a growth substrate.
The development of a plasmid transformation and gene transfer system for
R. rhodochrous PNKbl based on previously published methods has also been
Finally, a model for the pathway of propane oxidation in R. rhodochrous
PNKbl is also presented showing oxidation via terminal and subterminal
|Item Type:||Thesis or Dissertation (PhD)|
|Subjects:||Q Science > QR Microbiology|
|Library of Congress Subject Headings (LCSH):||Rhodococcus, Propane -- Oxidation, Propane -- Metabolism|
|Official Date:||March 1990|
|Institution:||University of Warwick|
|Theses Department:||Department of Biological Sciences|
|Supervisor(s)/Advisor:||Murrell, J. C. (J. Colin)|
|Sponsors:||Science and Engineering Research Council (Great Britain) (SERC)|
|Extent:||xvi, 223 p.|
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