Simultaneous identification of two cyclohexanone oxidation genes from an environmental Brevibacterium isolate using mRNA differential display.
The technique of mRNA differential display was used to identify simultaneously two metabolic genes involved in the degradation of cyclohexanone in a new halotolerant Brevibacterium environmental isolate. In a strategy based only on the knowledge that cyclohexanone oxidation was inducible in this strain, the mRNA population of cells exposed to cyclohexanone was compared to that of control cells using reverse transcription-PCR reactions primed with a collection of 81 arbitrary oligonucleotides. Three DNA fragments encoding segments of flavin monooxygenases were isolated with this technique, leading to the identification of the genes of two distinct cyclohexanone monooxygenases, the enzymes responsible for the oxidation of cyclohexanone. Each monooxygenase was expressed in Escherichia coli and characterized. This work validates the application of mRNA differential display for the discovery of new microbial metabolic genes.
Brzostowicz
P C
PC
E. I. DuPont de Nemours Co., Central Research and Development, Wilmington, Delaware 19800-0328, USA.
Gibson
K L
KL
Thomas
S M
SM
Blasko
M S
MS
Rouvière
P E
PE
D000595:Amino Acid Sequence; D001951:Brevibacterium / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003512:Cyclohexanones / Q000378:metabolism*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D019105:Random Amplified Polymorphic DNA Technique; D020133:Reverse Transcriptase Polymerase Chain Reaction
D003512:Cyclohexanones; D012333:RNA, Messenger; C036468:cyclohexanone; D010105:Oxygenases; C019961:cyclohexanone oxygenase