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Molecular cloning and characterization of sequences from the regulatory cluster of the Pseudomonas plasmid alk system

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Summary

Alkane oxidation functions encoded by the Pseudomonas plasmid CAM-OCT are positively regulated by one or more products of a locus designated alkR. To characterize this locus in greater detail, molecular cloning and restriction mapping of sequences covering the alkR region have been carried out in Escherichia coli, followed by mobilization to Pseudomonas recipients for analysis of genetic content. Inserts from Pseudomonas (CAM-OCT) strains were cloned into vectors pLAFR1, the pLAFR1::Tn7S derivative pXJS5403, and the transposon vector Tn3Δ596. This has made it possible to: (1) construct a detailed restriction map of cloned fragments and the alkR region of CAM-OCT; (2) map insertion sites of the transposon Tn7S into alkR cistrons; and (3) analyze the ability of cloned sequences to complement or effect marker rescue of alkR nitrosoguanidine- and Tn7S-induced mutations. In addition, transcription of an alkB'-lacZ transcription fusion in the presence of a cloned 18.5 kb EcolRI alkR fragment and an inducer of the alk system confirmed that our cloned sequences contain functional alkR cistrons. The complementation/marker rescue results indicate that alkR is a complex locus and that the products of at least three cistrons are required for the complete AlkR+ phenotype. One of these cistrons is identified by mutations which alter a component of the inducer recognition system.

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References

  • Barth P, Grinter N (1977) Map of plasmid RP4 derived by insertion of transposon C. J Mol Biol 113:455–474

    Google Scholar 

  • Benson S (1978) Alkane oxidation in Pseudomonas putida. Ph.D. Thesis, University of Chicago

  • Benson S, Shapiro JA (1975) Induction of alkane hydroxylase proteins by unoxidised alkanes in Pseudomonas putida. J Bacteriol 123:759–760

    Google Scholar 

  • Benson S, Shapiro JA (1976) Plasmid-determined alcohol dehydrogenase activity in alkane-utilizing strains of Pseudomonas putida. J Bacteriol 126:794–798

    Google Scholar 

  • Benson S, Fennewald M, Shapiro JA, Huettner C (1977) Fractionation of inducible alkane hydroxylase activity in Pseudomonas putida and characterization of hydroxylase negative plasmid mutations. J Bacteriol 132:614–621

    Google Scholar 

  • Benson S, Oppuci M, Shapiro JA, Fennewald M (1979) Regulation of membrane peptides by the Pseudomonas plasmid alk region. J Bacteriol 140:754–762

    Google Scholar 

  • Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7:1513–1521

    Google Scholar 

  • Bolivar F, Rodriguez RL, Betlach MV, Boyer H (1977) Construction and characterization of new cloning vehicles. 1. Ampicillinresistant deriviatives of the plasmid pMB9. Gene 2:95–113

    Google Scholar 

  • Davis RW, Botstein D, Roth JR (1980) Advance bacterial genetics: a manual for genetic engineering. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Ditta G, Stanfield S, Corbin D, Helinski DR (1980) Broad hostrange DNA cloning system for Gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77:7347–7371

    Google Scholar 

  • Fennewald M (1978) Genetics of alkane utilization in Pseudomonas. Ph.D. Thesis, University of Chicago

  • Fennewald M, Shapiro JA (1977) Regulatory mutations of the Pseudomonas plasmid alk regulon. J Bacteriol 132:622–627

    Google Scholar 

  • Fennewald M, Shapiro JA (1979) Transposition of Tn7 in Pseudomonas aeruginosa and isolation of alk::Tn7 mutations. J Bacteriol 139:264–269

    Google Scholar 

  • Fennewald M, Benson S, Oppici M, Shapiro JA (1979) Insertion element analysis and mapping of the Pseudomonas plasmid alk regulon. J Bacteriol 133:940–952

    Google Scholar 

  • Grund A, Shapiro JA, Fennewald M, Bacha P, Leary J, Markbeiter K, Neider M, Toefer M (1975) Regulation of alkane oxidation in Pseudomonas putida. J Bacteriol 122:546–556

    Google Scholar 

  • Hauer B, Shapiro JA (1984) Control of Tn7 transposition. Mol Gen Genet 194:149–158

    Google Scholar 

  • Heffron F, Bedinger P, Champoux J, Falkow S (1977) Deletions affecting the transposition of an antibiotic-resistance gene. Proc Natl Acad Sci USA 74:702–706

    Google Scholar 

  • Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193–195

    Google Scholar 

  • Long SR, Buikema WJ, Ausubel FM (1982) Cloning of Rhizobium meliloti nodulation genes by direct complmentation of Nod− mutants. Nature 298:485–488

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual, Cold Spring Harbor,Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Meyer R, Shapiro JA (1980) Genetic organisation of the broadhost range plasmid R751. J Bacteriol 143:1362–1373

    Google Scholar 

  • Miller J (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Moore RJ, Krishnapillai V (1982) Tn7 and Tn501 insertions into P. aeruginosa plasmid R91-5: Mapping of two transfer regions. J Bacteriol 149:276–283

    Google Scholar 

  • Neider M, Shapiro JA (1975) Physiological function of the Pseudomonas putida (Pseudomonas oleovorans) alkane hydroxylase: Monoterminal oxidation of alkanes and fatty acids. J Bacteriol 122:92–98

    Google Scholar 

  • Owen DJ, Eggink G, Kok M, McBeth D, Yan Y, Shapiro JA (1984) Physical structure, genetic content and expression of the alkBAC operon. Mol Gen Genet 197:373–393

    Google Scholar 

  • Owen DJ, Shapiro JA (1985) Analysis of the pseudomonas OCT plasmid alk regulatory region (Abstract) Annual meeting Am Soc. Microbiol 1985, H158 p 134

  • Putnan M, Koch J (1974) Complications in the simplest cellular enzyme assay: lysis of Escherichia coli for the assay of β-galactosidase. Anal Biochem 63:350–360

    Google Scholar 

  • Schwartz RD, McCoy CJ (1973) Pseudomonas oleovorans hydroxylation-epoxidation system: additional strain improvements. Appl Microbiol 26:217–218

    Google Scholar 

  • Thomas CM, Stalker D, Guiney D, Helinski DR (1979) Essential regions for the replication and conjugal transfer of the broadhost-range plasmid RK2. In: Timmis KN, Puhler A (eds) Plasmids of medical, environmental and commercial importance. Elseview North Holland, Amsterdam, pp 375–385

    Google Scholar 

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  1. David J. Owen

    Present address: Laboratory for Cell, Molecular and Developmental Biology (M/C 066), Department of Biological Sciences, College of Liberal Arts and Sciences, Box 4348, 60680, Chicago, Ill, USA

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Cummings Life Science Center, University of Chicago, 920 East 58th Street, 60637, Chicago, IL, USA

    David J. Owen

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  1. David J. Owen
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Communicated by N.D.F. Grindley

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Owen, D.J. Molecular cloning and characterization of sequences from the regulatory cluster of the Pseudomonas plasmid alk system. Mol Gen Genet 203, 64–72 (1986). https://doi.org/10.1007/BF00330385

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  • DOI: https://doi.org/10.1007/BF00330385

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