Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Sep;171(9):4836–4843. doi: 10.1128/jb.171.9.4836-4843.1989

Induction of anaerobic gene expression in Rhodobacter capsulatus is not accompanied by a local change in chromosomal supercoiling as measured by a novel assay.

D N Cook 1, G A Armstrong 1, J E Hearst 1
PMCID: PMC210287  PMID: 2768190

Abstract

In the photosynthetic bacterium Rhodobacter capsulatus, the enzyme DNA gyrase has been implicated in the expression of genes for anaerobic metabolic processes such as nitrogen fixation and photosynthesis. To assess the involvement of supercoiling in anaerobic gene expression, we have developed an assay to detect in vivo changes in superhelicity of small regions of the bacterial chromosome. Our method is based on the preferential intercalaction of psoralen into supercoiled versus relaxed DNA, and we have demonstrated the sensitivity of the assay in vivo on chromosomal regions from 2 to 10 kilobases in size. In experiments with inhibitors of gyrase, the reactivity of individual chromosomal fragments to psoralen decreases by a factor of 1.8 compared with DNA from control cultures. We used our assay to determine whether there is a change in superhelicity near the genes coding for essential proteins for photosynthesis upon a shift from respiratory to anaerobic photosynthetic growth. For comparison, we also examined a restriction fragment containing the fbc operon, which codes for the subunits of cytochrome bc1, a membrane-bound electron transport complex utilized during both aerobic and anaerobic photosynthetic growth. During this shift in growth conditions, the puf and puh mRNAs, coding for structural polypeptides of the photosynthetic apparatus, underwent a six- to eightfold induction, while the amount of mRNA from the fbc locus remained constant. However, we detected no change in the superhelicity of either the genes for photosynthesis or those for the bc1 complex during this metabolic transition. Our data thus do not support a model in which stable changes in chromosomal superhelicity regulate anaerobic gene expression. We suggest instead that the requirement for DNA gyrase in the transcription of photosynthesis genes results from the requirement for a swivel near heavily transcribed regions of the chromosome.

Full text

PDF
4836

Images in this article

Selected References

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

  1. Bliska J. B., Cozzarelli N. R. Use of site-specific recombination as a probe of DNA structure and metabolism in vivo. J Mol Biol. 1987 Mar 20;194(2):205–218. doi: 10.1016/0022-2836(87)90369-x. [DOI] [PubMed] [Google Scholar]
  2. Brill S. J., Sternglanz R. Transcription-dependent DNA supercoiling in yeast DNA topoisomerase mutants. Cell. 1988 Jul 29;54(3):403–411. doi: 10.1016/0092-8674(88)90203-6. [DOI] [PubMed] [Google Scholar]
  3. Cimino G. D., Gamper H. B., Isaacs S. T., Hearst J. E. Psoralens as photoactive probes of nucleic acid structure and function: organic chemistry, photochemistry, and biochemistry. Annu Rev Biochem. 1985;54:1151–1193. doi: 10.1146/annurev.bi.54.070185.005443. [DOI] [PubMed] [Google Scholar]
  4. Daldal F., Davidson E., Cheng S. Isolation of the structural genes for the Rieske Fe-S protein, cytochrome b and cytochrome c1 all components of the ubiquinol: cytochrome c2 oxidoreductase complex of Rhodopseudomonas capsulata. J Mol Biol. 1987 May 5;195(1):1–12. doi: 10.1016/0022-2836(87)90322-6. [DOI] [PubMed] [Google Scholar]
  5. Dorman C. J., Barr G. C., Ni Bhriain N., Higgins C. F. DNA supercoiling and the anaerobic and growth phase regulation of tonB gene expression. J Bacteriol. 1988 Jun;170(6):2816–2826. doi: 10.1128/jb.170.6.2816-2826.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Drlica K. Biology of bacterial deoxyribonucleic acid topoisomerases. Microbiol Rev. 1984 Dec;48(4):273–289. doi: 10.1128/mr.48.4.273-289.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Drlica K., Snyder M. Superhelical Escherichia coli DNA: relaxation by coumermycin. J Mol Biol. 1978 Apr 5;120(2):145–154. doi: 10.1016/0022-2836(78)90061-x. [DOI] [PubMed] [Google Scholar]
  8. Gabellini N., Sebald W. Nucleotide sequence and transcription of the fbc operon from Rhodopseudomonas sphaeroides. Evaluation of the deduced amino acid sequences of the FeS protein, cytochrome b and cytochrome c1. Eur J Biochem. 1986 Feb 3;154(3):569–579. doi: 10.1111/j.1432-1033.1986.tb09437.x. [DOI] [PubMed] [Google Scholar]
  9. Gamper H. B., Hearst J. E. A topological model for transcription based on unwinding angle analysis of E. coli RNA polymerase binary, initiation and ternary complexes. Cell. 1982 May;29(1):81–90. doi: 10.1016/0092-8674(82)90092-7. [DOI] [PubMed] [Google Scholar]
  10. Gellert M., Mizuuchi K., O'Dea M. H., Nash H. A. DNA gyrase: an enzyme that introduces superhelical turns into DNA. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3872–3876. doi: 10.1073/pnas.73.11.3872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Giaever G. N., Wang J. C. Supercoiling of intracellular DNA can occur in eukaryotic cells. Cell. 1988 Dec 2;55(5):849–856. doi: 10.1016/0092-8674(88)90140-7. [DOI] [PubMed] [Google Scholar]
  12. Gray E. D. Studies on the adaptive formation of photosynthetic structures in Rhodopseudomonas spheroides. I. Synthesis of macromolecules. Biochim Biophys Acta. 1967 May 30;138(3):550–563. doi: 10.1016/0005-2787(67)90551-5. [DOI] [PubMed] [Google Scholar]
  13. Higgins C. F., Dorman C. J., Stirling D. A., Waddell L., Booth I. R., May G., Bremer E. A physiological role for DNA supercoiling in the osmotic regulation of gene expression in S. typhimurium and E. coli. Cell. 1988 Feb 26;52(4):569–584. doi: 10.1016/0092-8674(88)90470-9. [DOI] [PubMed] [Google Scholar]
  14. Hyde J. E., Hearst J. E. Binding of psoralen derivatives to DNA and chromatin: influence of the ionic environment on dark binding and photoreactivity. Biochemistry. 1978 Apr 4;17(7):1251–1257. doi: 10.1021/bi00600a019. [DOI] [PubMed] [Google Scholar]
  15. Johnston B. H., Johnson M. A., Moore C. B., Hearst J. E. Psoralen-DNA photoreaction: controlled production of mono- and diadducts with nanosecond ultraviolet laser pulses. Science. 1977 Aug 26;197(4306):906–908. doi: 10.1126/science.887929. [DOI] [PubMed] [Google Scholar]
  16. Kranz R. G., Haselkorn R. Anaerobic regulation of nitrogen-fixation genes in Rhodopseudomonas capsulata. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6805–6809. doi: 10.1073/pnas.83.18.6805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Liu L. F., Wang J. C. Supercoiling of the DNA template during transcription. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7024–7027. doi: 10.1073/pnas.84.20.7024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lockshon D., Morris D. R. Positively supercoiled plasmid DNA is produced by treatment of Escherichia coli with DNA gyrase inhibitors. Nucleic Acids Res. 1983 May 25;11(10):2999–3017. doi: 10.1093/nar/11.10.2999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Marrs B. Mobilization of the genes for photosynthesis from Rhodopseudomonas capsulata by a promiscuous plasmid. J Bacteriol. 1981 Jun;146(3):1003–1012. doi: 10.1128/jb.146.3.1003-1012.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Menzel R., Gellert M. Regulation of the genes for E. coli DNA gyrase: homeostatic control of DNA supercoiling. Cell. 1983 Aug;34(1):105–113. doi: 10.1016/0092-8674(83)90140-x. [DOI] [PubMed] [Google Scholar]
  21. Novak P. D., Maier R. J. Inhibition of hydrogenase synthesis by DNA gyrase inhibitors in Bradyrhizobium japonicum. J Bacteriol. 1987 Jun;169(6):2708–2712. doi: 10.1128/jb.169.6.2708-2712.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pettijohn D. E., Pfenninger O. Supercoils in prokaryotic DNA restrained in vivo. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1331–1335. doi: 10.1073/pnas.77.3.1331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pruss G. J. DNA topoisomerase I mutants. Increased heterogeneity in linking number and other replicon-dependent changes in DNA supercoiling. J Mol Biol. 1985 Sep 5;185(1):51–63. doi: 10.1016/0022-2836(85)90182-2. [DOI] [PubMed] [Google Scholar]
  24. Pruss G. J., Drlica K. DNA supercoiling and prokaryotic transcription. Cell. 1989 Feb 24;56(4):521–523. doi: 10.1016/0092-8674(89)90574-6. [DOI] [PubMed] [Google Scholar]
  25. Pruss G. J., Manes S. H., Drlica K. Escherichia coli DNA topoisomerase I mutants: increased supercoiling is corrected by mutations near gyrase genes. Cell. 1982 Nov;31(1):35–42. doi: 10.1016/0092-8674(82)90402-0. [DOI] [PubMed] [Google Scholar]
  26. Richardson S. M., Higgins C. F., Lilley D. M. DNA supercoiling and the leu-500 promoter mutation of Salmonella typhimurium. EMBO J. 1988 Jun;7(6):1863–1869. doi: 10.1002/j.1460-2075.1988.tb03019.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schloss J. A., Silflow C. D., Rosenbaum J. L. mRNA abundance changes during flagellar regeneration in Chlamydomonas reinhardtii. Mol Cell Biol. 1984 Mar;4(3):424–434. doi: 10.1128/mcb.4.3.424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sinden R. R., Carlson J. O., Pettijohn D. E. Torsional tension in the DNA double helix measured with trimethylpsoralen in living E. coli cells: analogous measurements in insect and human cells. Cell. 1980 Oct;21(3):773–783. doi: 10.1016/0092-8674(80)90440-7. [DOI] [PubMed] [Google Scholar]
  29. Sinden R. R., Pettijohn D. E. Chromosomes in living Escherichia coli cells are segregated into domains of supercoiling. Proc Natl Acad Sci U S A. 1981 Jan;78(1):224–228. doi: 10.1073/pnas.78.1.224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Snyder M., Drlica K. DNA gyrase on the bacterial chromosome: DNA cleavage induced by oxolinic acid. J Mol Biol. 1979 Jun 25;131(2):287–302. doi: 10.1016/0022-2836(79)90077-9. [DOI] [PubMed] [Google Scholar]
  31. Taylor D. P., Cohen S. N., Clark W. G., Marrs B. L. Alignment of genetic and restriction maps of the photosynthesis region of the Rhodopseudomonas capsulata chromosome by a conjugation-mediated marker rescue technique. J Bacteriol. 1983 May;154(2):580–590. doi: 10.1128/jb.154.2.580-590.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tsao Y. P., Wu H. Y., Liu L. F. Transcription-driven supercoiling of DNA: direct biochemical evidence from in vitro studies. Cell. 1989 Jan 13;56(1):111–118. doi: 10.1016/0092-8674(89)90989-6. [DOI] [PubMed] [Google Scholar]
  33. Tse-Dinh Y. C. Regulation of the Escherichia coli DNA topoisomerase I gene by DNA supercoiling. Nucleic Acids Res. 1985 Jul 11;13(13):4751–4763. doi: 10.1093/nar/13.13.4751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vos J. M., Hanawalt P. C. Processing of psoralen adducts in an active human gene: repair and replication of DNA containing monoadducts and interstrand cross-links. Cell. 1987 Aug 28;50(5):789–799. doi: 10.1016/0092-8674(87)90337-0. [DOI] [PubMed] [Google Scholar]
  35. Wang J. C. Interaction between DNA and an Escherichia coli protein omega. J Mol Biol. 1971 Feb 14;55(3):523–533. doi: 10.1016/0022-2836(71)90334-2. [DOI] [PubMed] [Google Scholar]
  36. Weaver P. F., Wall J. D., Gest H. Characterization of Rhodopseudomonas capsulata. Arch Microbiol. 1975 Nov 7;105(3):207–216. doi: 10.1007/BF00447139. [DOI] [PubMed] [Google Scholar]
  37. Worcel A., Burgi E. On the structure of the folded chromosome of Escherichia coli. J Mol Biol. 1972 Nov 14;71(2):127–147. doi: 10.1016/0022-2836(72)90342-7. [DOI] [PubMed] [Google Scholar]
  38. Wu H. Y., Shyy S. H., Wang J. C., Liu L. F. Transcription generates positively and negatively supercoiled domains in the template. Cell. 1988 May 6;53(3):433–440. doi: 10.1016/0092-8674(88)90163-8. [DOI] [PubMed] [Google Scholar]
  39. Yamamoto N., Droffner M. L. Mechanisms determining aerobic or anaerobic growth in the facultative anaerobe Salmonella typhimurium. Proc Natl Acad Sci U S A. 1985 Apr;82(7):2077–2081. doi: 10.1073/pnas.82.7.2077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhu Y. S., Hearst J. E. Transcription of oxygen-regulated photosynthetic genes requires DNA gyrase in Rhodobacter capsulatus. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4209–4213. doi: 10.1073/pnas.85.12.4209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zhu Y. S., Kaplan S. Effects of light, oxygen, and substrates on steady-state levels of mRNA coding for ribulose-1,5-bisphosphate carboxylase and light-harvesting and reaction center polypeptides in Rhodopseudomonas sphaeroides. J Bacteriol. 1985 Jun;162(3):925–932. doi: 10.1128/jb.162.3.925-932.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zsebo K. M., Hearst J. E. Genetic-physical mapping of a photosynthetic gene cluster from R. capsulata. Cell. 1984 Jul;37(3):937–947. doi: 10.1016/0092-8674(84)90428-8. [DOI] [PubMed] [Google Scholar]

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

RESOURCES