The genetics and biochemistry of a propane-utilizing "Rhodococcus rhodochrous"
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 assessed. Finally, a model for the pathway of propane oxidation in R. rhodochrous PNKbl is also presented showing oxidation via terminal and subterminal carbon atoms.