Fractionation of inducible alkane hydroxylase activity in Pseudomonas putida and characterization of hydroxylase-negative plasmid mutations.
The plasmid-determined inducible alkane hydroxylase of Pseudomonas putida resolved into particulate and soluble fractions. Spinach reductase and spinach ferredoxin could replace the soluble hydroxylase component. Two alkane hydroxylase mutants show in vitro complementation (S. Benson and J. Shapiro, J. Bacteriol., 123: 759-760, 1975): one, alk-7, lacks an active soluble component and the other, alk-181, lacks an active particulate component. Together with previous results on a particulate alcohol dehydrogenase enzyme (Benson and Shapiro, J. Bacteriol., 126: 794-798, 1976), these results allowed us to assay three plasmid-determined inducible activities: soluble alkane hydroxylase (alkA+), particulate alkane hydroxylase (alkB+), and particulate alcohol dehydrogenase (alkC+). Growth tests and in vitro complementation assays revealed three groups of plasmid mutations that block expression of alkane hydroxylase activity: alkA, which so far includes only the alk-7 mutation; alkB, which includes alk-181 and 11 other mutations; and a pleiotropic-negative class, which includes nine mutations that lead to loss of alkA+, alkB+, and alkC+ activities. Thus, the alk+ gene cluster found on IncP-2 plasmids contains at least four cistrons. We believe it is significant that two of these determined the presence of membrane proteins. The accompanying paper shows that these loci are part of a single regulon.
D000473:Alkanes / Q000378:metabolism; D002474:Cell-Free System; D005591:Chemical Fractionation; D005288:Ferredoxins / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010088:Oxidoreductases / Q000378:metabolism; D010944:Plants / Q000201:enzymology; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000378:metabolism; D014161:Transduction, Genetic
D000473:Alkanes; D005288:Ferredoxins; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases