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. 1996 Sep;178(18):5508–5512. doi: 10.1128/jb.178.18.5508-5512.1996

Physiological changes and alk gene instability in Pseudomonas oleovorans during induction and expression of alk genes.

Q Chen 1, D B Janssen 1, B Witholt 1
PMCID: PMC178375  PMID: 8808943

Abstract

The alk genes of Pseudomonas oleovorans, which is able to metabolize alkanes and alkenes, are organized in alkST and alkBFGHJKL clusters, in which the expression of alkBFGHJKL is positively regulated by AlkS. Growth of the wild-type strain GPo1 and P. oleovorans GPo12 alk recombinants on octane resulted in changes of cellular physiology and morphology. These changes, which included lower growth rates and a reduction of the number of CFU due to filamentation, were also seen when the cells were grown on aqueous medium, and the alk genes were induced with dicyclopropylketone, a gratuitous inducer of the alk genes. These effects were seen only for recombinants carrying both alkST and alkBFGHJKL operons. Deletion of parts of either alkB or alkJ, which encode two major Alk proteins located in the cytoplasmic membrane, modified but did not eliminate the effects described above, suggesting that they were due to induction and expression of several alk genes. Continuous growth of the cells in the presence of dicyclopropylketone for about 10 generations led to inactivation, but not elimination, of the alk genes. This resulted in a return of the recombinants to normal physiology and growth.

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Selected References

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  1. BAPTIST J. N., GHOLSON R. K., COON M. J. Hydrocarbon oxidation by a bacterial enzyme system. I. Products of octane oxidation. Biochim Biophys Acta. 1963 Jan 1;69:40–47. doi: 10.1016/0006-3002(63)91223-x. [DOI] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brosius J. Toxicity of an overproduced foreign gene product in Escherichia coli and its use in plasmid vectors for the selection of transcription terminators. Gene. 1984 Feb;27(2):161–172. doi: 10.1016/0378-1119(84)90137-9. [DOI] [PubMed] [Google Scholar]
  4. Chen Q., Janssen D. B., Witholt B. Growth on octane alters the membrane lipid fatty acids of Pseudomonas oleovorans due to the induction of alkB and synthesis of octanol. J Bacteriol. 1995 Dec;177(23):6894–6901. doi: 10.1128/jb.177.23.6894-6901.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Duetz W. A., Winson M. K., van Andel J. G., Williams P. A. Mathematical analysis of catabolic function loss in a population of Pseudomonas putida mt-2 during non-limited growth on benzoate. J Gen Microbiol. 1991 Jun;137(6):1363–1368. doi: 10.1099/00221287-137-6-1363. [DOI] [PubMed] [Google Scholar]
  6. Eggink G., Engel H., Meijer W. G., Otten J., Kingma J., Witholt B. Alkane utilization in Pseudomonas oleovorans. Structure and function of the regulatory locus alkR. J Biol Chem. 1988 Sep 15;263(26):13400–13405. [PubMed] [Google Scholar]
  7. Eggink G., Engel H., Vriend G., Terpstra P., Witholt B. Rubredoxin reductase of Pseudomonas oleovorans. Structural relationship to other flavoprotein oxidoreductases based on one NAD and two FAD fingerprints. J Mol Biol. 1990 Mar 5;212(1):135–142. doi: 10.1016/0022-2836(90)90310-I. [DOI] [PubMed] [Google Scholar]
  8. Eggink G., Lageveen R. G., Altenburg B., Witholt B. Controlled and functional expression of the Pseudomonas oleovorans alkane utilizing system in Pseudomonas putida and Escherichia coli. J Biol Chem. 1987 Dec 25;262(36):17712–17718. [PubMed] [Google Scholar]
  9. Enzymatic -oxidation. VI. Isolation of homogeneous reduced diphosphopyridine nucleotide-rubredoxin reductase. J Biol Chem. 1972 Apr 10;247(7):2109–2116. [PubMed] [Google Scholar]
  10. Favre-Bulle O., Schouten T., Kingma J., Witholt B. Bioconversion of n-octane to octanoic acid by a recombinant Escherichia coli cultured in a two-liquid phase bioreactor. Biotechnology (N Y) 1991 Apr;9(4):367–371. doi: 10.1038/nbt0491-367. [DOI] [PubMed] [Google Scholar]
  11. Kok M., Oldenhuis R., van der Linden M. P., Meulenberg C. H., Kingma J., Witholt B. The Pseudomonas oleovorans alkBAC operon encodes two structurally related rubredoxins and an aldehyde dehydrogenase. J Biol Chem. 1989 Apr 5;264(10):5442–5451. [PubMed] [Google Scholar]
  12. Kok M., Oldenhuis R., van der Linden M. P., Raatjes P., Kingma J., van Lelyveld P. H., Witholt B. The Pseudomonas oleovorans alkane hydroxylase gene. Sequence and expression. J Biol Chem. 1989 Apr 5;264(10):5435–5441. [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Lageveen R. G., Huisman G. W., Preusting H., Ketelaar P., Eggink G., Witholt B. Formation of Polyesters by Pseudomonas oleovorans: Effect of Substrates on Formation and Composition of Poly-(R)-3-Hydroxyalkanoates and Poly-(R)-3-Hydroxyalkenoates. Appl Environ Microbiol. 1988 Dec;54(12):2924–2932. doi: 10.1128/aem.54.12.2924-2932.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nieboer M., Kingma J., Witholt B. The alkane oxidation system of Pseudomonas oleovorans: induction of the alk genes in Escherichia coli W3110 (pGEc47) affects membrane biogenesis and results in overexpression of alkane hydroxylase in a distinct cytoplasmic membrane subfraction. Mol Microbiol. 1993 Jun;8(6):1039–1051. doi: 10.1111/j.1365-2958.1993.tb01649.x. [DOI] [PubMed] [Google Scholar]
  16. Peterson J. A., Basu D., Coon M. J. Enzymatic omega-oxidation. I. Electon carriers in fatty acid and hydrocarbon hydroxylation. J Biol Chem. 1966 Nov 10;241(21):5162–5164. [PubMed] [Google Scholar]
  17. Summers D. K. The kinetics of plasmid loss. Trends Biotechnol. 1991 Aug;9(8):273–278. doi: 10.1016/0167-7799(91)90089-z. [DOI] [PubMed] [Google Scholar]
  18. Witholt B. Method for isolating mutants overproducing nicotinamide adenine dinucleotide and its precursors. J Bacteriol. 1972 Jan;109(1):350–364. doi: 10.1128/jb.109.1.350-364.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. van Beilen J. B., Eggink G., Enequist H., Bos R., Witholt B. DNA sequence determination and functional characterization of the OCT-plasmid-encoded alkJKL genes of Pseudomonas oleovorans. Mol Microbiol. 1992 Nov;6(21):3121–3136. doi: 10.1111/j.1365-2958.1992.tb01769.x. [DOI] [PubMed] [Google Scholar]

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