Transposition of a beta-lactamase locus from RP1 into Pseudomonas putida degradative plasmids

Benedik, Michael J.; Fennewald, Michael; Shapiro, James A.

doi:10.1128/jb.129.2.809-814.1977

Cited by 38 publications

(15 citation statements)

References 19 publications

(22 reference statements)

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“…Genetic crosses. Routine bacterial conjugations and transductions were carried out as previously described (2,11). To mobilize the alk-7 mutant of the Tra-OCT plasmid, we used a Tra+ IncP-2 drug resistance plasmid, R931 (12), in a triparental cross.…”

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confidence: 99%

Fractionation of inducible alkane hydroxylase activity in Pseudomonas putida and characterization of hydroxylase-negative plasmid mutations

Benson¹,

Fennewald²,

Shapiro³

et al. 1977

J Bacteriol

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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 pleiotropicnegative class, which includes nine mutations that lead to loss of alkA 4, alkB , and alkC+ activities. Thus, the alkgene 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.MATERIALS AND METHODS Bacterial strains. We used the Pseudomonas aeruginosa and Pseudomonas putida strains de-614 on July 16, 2020 by guest

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confidence: 99%

Fractionation of inducible alkane hydroxylase activity in Pseudomonas putida and characterization of hydroxylase-negative plasmid mutations

Benson¹,

Fennewald²,

Shapiro³

et al. 1977

J Bacteriol

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“…(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.…”

Section: Methodsmentioning

confidence: 99%

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

Section: Methodsmentioning

confidence: 99%

“…To determine trimethoprim resistance in P. putida, we use 1 mg in 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).…”

Section: Methodsmentioning

confidence: 99%

“…Transposable drug resistance elements are useful as mutagenic agents in the genetic analysis of physiological systems, and we have begun using them for the study of degradative plasmids of Pseudomonas (2,5). In this paper we report the isolation of mutation in the IncP-2 plasmid alk genes caused by insertion of the Tn7 trimethoprim and streptomycin resistance determinant (1).…”

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confidence: 99%

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

Fennewald

Shapiro

1979

J Bacteriol

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Conjugal crosses with Pseudomonas aeruginosa donors carrying the CAM-OCT and RP4::Tn 7 plasmids result in transfer of the Tn 7 trimethoprim resistance (Tp r ) determinant independently of RP4 markers. All Tp r exconjugants which lack RP4 markers have CAM-OCT genes and therefore must have received CAM-OCT::Tn 7 plasmids formed by transposition of Tn 7 from RP4::Tn 7 to CAM-OCT. Most crosses yield exconjugants carrying mutant CAM-OCT plasmids which no longer determine either camphor or alkane utilization and thus appear to carry Tn 7 inserts in the cam or alk loci, respectively. Transduction and reversion experiments indicated that at least 13 alkane-negative, camphor-positive, Tp r CAM-OCT::Tn 7 plasmids carry an alk ::Tn 7 mutation. Determination of linkage between the alk mutation and the Tp r determinant of Tn 7 on these plasmids is complicated by the presence of multiple copies of the Tn 7 element in the genome. Generalized transduction will remove Tn 7 from a CAM-OCT alk ::Tn 7 plasmid to yield alk + cells which carry no Tp r determinant on the CAM-OCT plasmid (as shown by transfer of the plasmid to a second strain). But the transduction to alk + does not remove all Tp r determinants from the genome of the recipient cell because the alkane-positive transductants remain trimethoprim resistant. Thus, it appears that copies of Tn 7 can accumulate in the genome of P. aeruginosa (CAM-OCT alk ::Tn 7 ) strains without leaving their original site. This result is consistent with transposition models that involve replication of the transposable element without excision from the original site.

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Perspectives for Genetic Engineering of Hydrocarbon Oxidizing Bacteria

Shapiro

Charbit

Benson

et al. 1981

Trends in the Biology of Fermentations for Fuels and Chemicals

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Transposition of a beta-lactamase locus from RP1 into Pseudomonas putida degradative plasmids

Cited by 38 publications

References 19 publications

Fractionation of inducible alkane hydroxylase activity in Pseudomonas putida and characterization of hydroxylase-negative plasmid mutations

Fractionation of inducible alkane hydroxylase activity in Pseudomonas putida and characterization of hydroxylase-negative plasmid mutations

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

Perspectives for Genetic Engineering of Hydrocarbon Oxidizing Bacteria

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