Regulatory mutations of the Pseudomonas plasmid alk regulon

Abstract

Pseudomonas putida strains with plasmids carrying pleiotropic alk mutations gave rise to alkane-positive "revertants," which differ from wild type. Some had restricted ability to utilize alkane and primary alcohol growth substrates, and others could grow on undecane and dodecanol, which are not utilized by alk strains. These revertants showed altered responses to normal inducers of alkA+, alkB-, and alkC+ activities. Some revertants were constitutive for these activities. Constitutive mutants could also be isolated directly from wild type, but they appeared spontaneously at a frequency of less than 2 x 10'. Regulatory mutations of all three types, pleiotropic negative, altered inducer specificity. and constitutive, were tightly linked in transduction crosses with a polar alkB mutation. These results demonstrate that the IncP-2 plasmid alk gene cluster constitutes a regulon. They also permit the identification of at least one cistron whose gene product participates in inducer recognition and suggest that the alkABC regulon is not under simple repressor control.

The AlkB Monooxygenase of Pseudomonas oleovorans

“…Plasmid pGEc47, as a derivative of broad-host-range plasmid RK2 has a copy number of 3-5 in E. coli (Figurski et al, 1979), whereas the OCT plasmid, an IncP2 plasmid, has a copy number of 1-2 in l? oleovorans (Fennewald and Shapiro, 1977;Hansen and Olsen, 1978). Possible additional causes for the difference in alkB mRNA levels between the E. coli alk' recombinants remain to be elucidated.…”

Transposition of Tn 7 in Pseudomonas aeruginosa and Isolation of alk ::Tn 7 Mutations

“…(Presumably this reflects lysis of sensitive recipients by spontaneously liberated F116L.) Phage F116L has been described previously (2,8,11); F11C is a clear-plaque mutant which gives better plate lysates and is not impaired in transduction of lysogenic recipients.…”

“…Transduction and conjugation methods. Transduction and conjugation methods have been described previously (2,4,8).…”

“…Media and growth tests. In general, media and growth tests have been described previously (4,8,16). To determine and select trimethoprim resistance in P. aeruginosa, we use 1 mg of trimethoprim in either complete agar or minimal PA agar per ml.…”

“…P. putida is intrinsically resist-Tn7 TRANSPOSITION BEHAVIOR aThe P. putida strains are derived from PpGl of Gunsalus and have been described in our previous publications (2,16). b The P. aeruginosa strains all derive from the PAC line of P. H. Clarke and their use as hosts for CAM-OCT plasnids has been described previously (8). The detailed genetic analysis and alkane utilization characteristics of strains PpS1117 through -1132, -1170, -1345 and PAS485, -486, -487 will be described separately (Fennewald et al., manuscript in preparation).…”