Physiological and genetic aspects of the utilisation of methylated amines in M. methylotrophus.
thesisposted on 19.11.2015, 09:10 by Judeline Winifred. Horton
M. methulotrophus is a Gram negative obligate methylotroph depending on the presence of reduced carbon compounds containing one or more carbon atoms, but containing no carbon-carbon bonds. This organism can synthesize all its cellular constituents from methanol, trimethylamine, dimethylamine, or methylamine. Conversion of methanol and the methylated amines to cell carbon involves the ultimate oxidation to formaldehyde and ammonia. While the methanol dehydrogenase is produced constitutively, the enzymes involved in the assimilation of the methylated amines are inducible. All three enzymes of the trimethylamine pathway are always induced regardless of the methylated amine substrate. Transposon mutagenesis was used to generate mutations in M.methylotrophus and an antibiotic selection procedure used to isolate mutants defective specifically in the trimethylamine pathway. Two mutants were characterised and subjected to further study. The mutant tmd 3 was unable to utilise trimethylamine, dimethylamine or methylamine as substrates and was shown to lack trimethylamine dehydrogenase by polyacrylamide gel electrophoresis. Enzyme studies confirmed the lack of the dehydrogenase within the mutant cells. The mutant mad 1, unable to use methylamine as a substrate, was shown via enzyme studies to contain trimethylamine dehydrogenase, and dimethylamine dehydrogenase, but to lack methylamine dehydrogenase activity. Molecular cloning of wild-type M. methylotrophus DNA in a broad host-range plasmid vector was used to isolate DNA fragments that could replace the mad 1 defect. An 11kb fragment was isolated that fully restored methylamine dehydrogenase activity to mad 1 cells. A 2.5kb fragment was subcloned and shown, by Southern blotting with 32P-labelled In5 DNA as a probe, to contain the site of integration of the In5 insertion, located to within a few hundred base pairs of the end. DNA sequencing, now in progress, has generated 600 base pairs of sequence from either end of the subcloned fragment, within which several regions of interest were noted.