Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Aug;171(8):4385–4394. doi: 10.1128/jb.171.8.4385-4394.1989

Construction of TnphoA gene fusions in Rhodobacter sphaeroides: isolation and characterization of a respiratory mutant unable to utilize dimethyl sulfoxide as a terminal electron acceptor during anaerobic growth in the dark on glucose.

M D Moore 1, S Kaplan 1
PMCID: PMC210216  PMID: 2546920

Abstract

We have constructed a suicide vector, pU1800, containing the transposable element TnphoA (Tn5 IS50L::phoA), for the purpose of producing protein fusions in vivo between the Escherichia coli alkaline phosphatase (APase) and proteins of the facultative photoheterotroph, Rhodobacter sphaeroides. We introduced TnphoA into the genome of R. sphaeroides at a coupled conjugation-transposition frequency of approximately 1 x 10(-6). Fusions giving rise to APase expression, as judged by blue-colony pigmentation when exconjugants were plated on growth medium containing the chromogenic indicator 5-bromo-4-chloro-3-indolyl phosphate, were observed in about 1% of the exconjugants. Numerous, distinguishable mutant phenotypes have been generated by this method, including those which lack the ability to use dimethyl sulfoxide as a terminal electron acceptor during anaerobic respiration, as well as those which are photosynthetically incompetent or altered in pigment synthesis, and others that express resistance to chlorate. The growth and spectral characteristics of several of these mutants, as well as the localization and quantitation of subcellular APase activity under different physiological conditions, have been examined. The presence of TnphoA in the host genome has been confirmed for each mutant analyzed, and specifically tagged DNA fragments containing TnphoA have been identified and localized; cosmids containing R. sphaeroides genomic DNA capable of complementing individual mutants have also been isolated. The usefulness of this approach in studying gene activity in R. sphaeroides is discussed.

Full text

PDF
4385

Images in this article

4389Image
on p.4389
4390Image
on p.4390

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abeliovich A., Kaplan S. Bacteriophages of Rhodopseudomonas spheroides: isolation and characterization of a Rhodopseudomonas spheroides bacteriophage. J Virol. 1974 Jun;13(6):1392–1399. doi: 10.1128/jvi.13.6.1392-1399.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allen L. N., Hanson R. S. Construction of broad-host-range cosmid cloning vectors: identification of genes necessary for growth of Methylobacterium organophilum on methanol. J Bacteriol. 1985 Mar;161(3):955–962. doi: 10.1128/jb.161.3.955-962.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baldwin T. O., Berends T., Bunch T. A., Holzman T. F., Rausch S. K., Shamansky L., Treat M. L., Ziegler M. M. Cloning of the luciferase structural genes from Vibrio harveyi and expression of bioluminescence in Escherichia coli. Biochemistry. 1984 Jul 31;23(16):3663–3667. doi: 10.1021/bi00311a014. [DOI] [PubMed] [Google Scholar]
  4. Bassford P. J., Jr, Silhavy T. J., Beckwith J. R. Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm. J Bacteriol. 1979 Jul;139(1):19–31. doi: 10.1128/jb.139.1.19-31.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bilous P. T., Weiner J. H. Molecular cloning and expression of the Escherichia coli dimethyl sulfoxide reductase operon. J Bacteriol. 1988 Apr;170(4):1511–1518. doi: 10.1128/jb.170.4.1511-1518.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Davis J., Donohue T. J., Kaplan S. Construction, characterization, and complementation of a Puf- mutant of Rhodobacter sphaeroides. J Bacteriol. 1988 Jan;170(1):320–329. doi: 10.1128/jb.170.1.320-329.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Donohue T. J., Cain B. D., Kaplan S. Alterations in the phospholipid composition of Rhodopseudomonas sphaeroides and other bacteria induced by Tris. J Bacteriol. 1982 Nov;152(2):595–606. doi: 10.1128/jb.152.2.595-606.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Donohue T. J., Chory J., Goldsand T. E., Lynn S. P., Kaplan S. Structure and physical map of Rhodopseudomonas sphaeroides bacteriophage RS1 DNA. J Virol. 1985 Jul;55(1):147–157. doi: 10.1128/jvi.55.1.147-157.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Donohue T. J., McEwan A. G., Kaplan S. Cloning, DNA sequence, and expression of the Rhodobacter sphaeroides cytochrome c2 gene. J Bacteriol. 1986 Nov;168(2):962–972. doi: 10.1128/jb.168.2.962-972.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fornari C. S., Watkins M., Kaplan S. Plasmid distribution and analyses in Rhodopseudomonas sphaeroides. Plasmid. 1984 Jan;11(1):39–47. doi: 10.1016/0147-619x(84)90005-2. [DOI] [PubMed] [Google Scholar]
  13. Fraley R. T., Lueking D. R., Kaplan S. The relationship of intracytoplasmic membrane assembly to the cell division cycle in Rhodopseudomonas sphaeroides. J Biol Chem. 1979 Mar 25;254(6):1980–1986. [PubMed] [Google Scholar]
  14. Hoffman C. S., Wright A. Fusions of secreted proteins to alkaline phosphatase: an approach for studying protein secretion. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5107–5111. doi: 10.1073/pnas.82.15.5107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jorgensen R. A., Rothstein S. J., Reznikoff W. S. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet. 1979;177(1):65–72. doi: 10.1007/BF00267254. [DOI] [PubMed] [Google Scholar]
  16. Kiley P. J., Varga A., Kaplan S. Physiological and structural analysis of light-harvesting mutants of Rhodobacter sphaeroides. J Bacteriol. 1988 Mar;170(3):1103–1115. doi: 10.1128/jb.170.3.1103-1115.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kwan H. S., Barrett E. L. Purification and properties of trimethylamine oxide reductase from Salmonella typhimurium. J Bacteriol. 1983 Sep;155(3):1455–1458. doi: 10.1128/jb.155.3.1455-1458.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  19. Lueking D. R., Fraley R. T., Kaplan S. Intracytoplasmic membrane synthesis in synchronous cell populations of Rhodopseudomonas sphaeroides. Fate of "old" and "new" membrane. J Biol Chem. 1978 Jan 25;253(2):451–457. [PubMed] [Google Scholar]
  20. Madigan M. T., Gest H. Growth of a photosynthetic bacterium anaerobically in darkness, supported by "oxidant-dependent" sugar fermentation. Arch Microbiol. 1978 May 30;117(2):119–122. doi: 10.1007/BF00402298. [DOI] [PubMed] [Google Scholar]
  21. Manoil C., Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Maquat L. E., Reznikoff W. S. In vitro analysis of the Escherichia coli RNA polymerase interaction with wild-type and mutant lactose promoters. J Mol Biol. 1978 Nov 15;125(4):467–490. doi: 10.1016/0022-2836(78)90311-x. [DOI] [PubMed] [Google Scholar]
  23. Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978 Jun 15;87(1):206–210. doi: 10.1016/0003-2697(78)90586-9. [DOI] [PubMed] [Google Scholar]
  24. Nano F. E., Kaplan S. Plasmid rearrangements in the photosynthetic bacterium Rhodopseudomonas sphaeroides. J Bacteriol. 1984 Jun;158(3):1094–1103. doi: 10.1128/jb.158.3.1094-1103.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Satoh T., Kurihara F. N. Purification and properties of dimethylsulfoxide reductase containing a molybdenum cofactor from a photodenitrifier, Rhodopseudomonas sphaeroides f.s. denitrificans. J Biochem. 1987 Jul;102(1):191–197. doi: 10.1093/oxfordjournals.jbchem.a122032. [DOI] [PubMed] [Google Scholar]
  26. Saunders V. A., Saunders J. R., Bennett P. M. Extrachromosomal deoxyribonucleic acid in wild-type and photosynthetically incompetent strains of Rhodopseudomonas spheroides. J Bacteriol. 1976 Mar;125(3):1180–1187. doi: 10.1128/jb.125.3.1180-1187.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schwartz D. C., Cantor C. R. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell. 1984 May;37(1):67–75. doi: 10.1016/0092-8674(84)90301-5. [DOI] [PubMed] [Google Scholar]
  28. Silvestro A., Pommier J., Giordano G. The inducible trimethylamine-N-oxide reductase of Escherichia coli K12: biochemical and immunological studies. Biochim Biophys Acta. 1988 Apr 28;954(1):1–13. doi: 10.1016/0167-4838(88)90049-0. [DOI] [PubMed] [Google Scholar]
  29. Southern E. Gel electrophoresis of restriction fragments. Methods Enzymol. 1979;68:152–176. doi: 10.1016/0076-6879(79)68011-4. [DOI] [PubMed] [Google Scholar]
  30. Styrvold O. B., Strøm A. R. Dimethylsulphoxide and trimethylamine oxide respiration of Proteus vulgaris. Evidence for a common terminal reductase system. Arch Microbiol. 1984 Nov;140(1):74–78. doi: 10.1007/BF00409774. [DOI] [PubMed] [Google Scholar]
  31. Tai S. P., Kaplan S. Intracellular localization of phospholipid transfer activity in Rhodopseudomonas sphaeroides and a possible role in membrane biogenesis. J Bacteriol. 1985 Oct;164(1):181–186. doi: 10.1128/jb.164.1.181-186.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Waterbury P. G., Lane M. J. Generation of lambda phage concatemers for use as pulsed field electrophoresis size markers. Nucleic Acids Res. 1987 May 11;15(9):3930–3930. doi: 10.1093/nar/15.9.3930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Weiner J. H., MacIsaac D. P., Bishop R. E., Bilous P. T. Purification and properties of Escherichia coli dimethyl sulfoxide reductase, an iron-sulfur molybdoenzyme with broad substrate specificity. J Bacteriol. 1988 Apr;170(4):1505–1510. doi: 10.1128/jb.170.4.1505-1510.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yen H. C., Marrs B. Growth of Rhodopseudomonas capsulata under anaerobic dark conditions with dimethyl sulfoxide. Arch Biochem Biophys. 1977 Jun;181(2):411–418. doi: 10.1016/0003-9861(77)90246-6. [DOI] [PubMed] [Google Scholar]
  35. van Niel C. B. THE CULTURE, GENERAL PHYSIOLOGY, MORPHOLOGY, AND CLASSIFICATION OF THE NON-SULFUR PURPLE AND BROWN BACTERIA. Bacteriol Rev. 1944 Mar;8(1):1–118. doi: 10.1128/br.8.1.1-118.1944. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES