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. 1992 Aug;3(8):913–926. doi: 10.1091/mbc.3.8.913

A developmentally regulated Caulobacter flagellar promoter is activated by 3' enhancer and IHF binding elements.

J W Gober 1, L Shapiro 1
PMCID: PMC275648  PMID: 1392079

Abstract

The transcription of a group of flagellar genes is temporally and spatially regulated during the Caulobacter crescentus cell cycle. These genes all share the same 5' cis-regulatory elements: a sigma 54 promoter, a binding site for integration host factor (IHF), and an enhancer sequence, known as the ftr element. We have partially purified the ftr-binding proteins, and we show that they require the same enhancer sequences for binding as are required for transcriptional activation. We have also partially purified the Caulobacter homolog of IHF and demonstrate that it can facilitate in vitro integrase-mediated lambda recombination. Using site-directed mutagenesis, we provide the first demonstration that natural enhancer sequences and IHF binding elements that reside 3' to the sigma 54 promoter of a bacterial gene, flaNQ, are required for transcription of the operon, in vivo. The IHF protein and the ftr-binding protein is primarily restricted to the predivisional cell, the cell type in which these promoters are transcribed. flaNQ promoter expression is localized to the swarmer pole of the predivisional cell, as are other flagellar promoters that possess these regulatory sequences 5' to the start site. The requirement for an IHF binding site and an ftr-enhancer element in spatially transcribed flagellar promoters indicates that a common mechanism may be responsible for both temporal and polar transcription.

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Selected References

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

  1. Alley M. R., Maddock J. R., Shapiro L. Polar localization of a bacterial chemoreceptor. Genes Dev. 1992 May;6(5):825–836. doi: 10.1101/gad.6.5.825. [DOI] [PubMed] [Google Scholar]
  2. Champer R., Bryan R., Gomes S. L., Purucker M., Shapiro L. Temporal and spatial control of flagellar and chemotaxis gene expression during Caulobacter cell differentiation. Cold Spring Harb Symp Quant Biol. 1985;50:831–840. doi: 10.1101/sqb.1985.050.01.101. [DOI] [PubMed] [Google Scholar]
  3. Champer R., Dingwall A., Shapiro L. Cascade regulation of Caulobacter flagellar and chemotaxis genes. J Mol Biol. 1987 Mar 5;194(1):71–80. doi: 10.1016/0022-2836(87)90716-9. [DOI] [PubMed] [Google Scholar]
  4. Chen L. S., Mullin D., Newton A. Identification, nucleotide sequence, and control of developmentally regulated promoters in the hook operon region of Caulobacter crescentus. Proc Natl Acad Sci U S A. 1986 May;83(9):2860–2864. doi: 10.1073/pnas.83.9.2860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Claverie-Martin F., Magasanik B. Role of integration host factor in the regulation of the glnHp2 promoter of Escherichia coli. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1631–1635. doi: 10.1073/pnas.88.5.1631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Collado-Vides J., Magasanik B., Gralla J. D. Control site location and transcriptional regulation in Escherichia coli. Microbiol Rev. 1991 Sep;55(3):371–394. doi: 10.1128/mr.55.3.371-394.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Contreras I., Shapiro L., Henry S. Membrane phospholipid composition of Caulobacter crescentus. J Bacteriol. 1978 Sep;135(3):1130–1136. doi: 10.1128/jb.135.3.1130-1136.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dingwall A., Gober J. W., Shapiro L. Identification of a Caulobacter basal body structural gene and a cis-acting site required for activation of transcription. J Bacteriol. 1990 Oct;172(10):6066–6076. doi: 10.1128/jb.172.10.6066-6076.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dixon R. A. The genetic complexity of nitrogen fixation. The ninth Fleming lecture. J Gen Microbiol. 1984 Nov;130(11):2745–2755. doi: 10.1099/00221287-130-11-2745. [DOI] [PubMed] [Google Scholar]
  11. Ely B., Gerardot C. J., Fleming D. L., Gomes S. L., Frederikse P., Shapiro L. General nonchemotactic mutants of Caulobacter crescentus. Genetics. 1986 Nov;114(3):717–730. doi: 10.1093/genetics/114.3.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Evinger M., Agabian N. Caulobacter crescentus nucleoid: analysis of sedimentation behavior and protein composition during the cell cycle. Proc Natl Acad Sci U S A. 1979 Jan;76(1):175–178. doi: 10.1073/pnas.76.1.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Evinger M., Agabian N. Envelope-associated nucleoid from Caulobacter crescentus stalked and swarmer cells. J Bacteriol. 1977 Oct;132(1):294–301. doi: 10.1128/jb.132.1.294-301.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Galas D. J., Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157–3170. doi: 10.1093/nar/5.9.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gober J. W., Alley M. R., Shapiro L. Positional information during Caulobacter cell differentiation. Curr Opin Genet Dev. 1991 Oct;1(3):324–329. doi: 10.1016/s0959-437x(05)80295-3. [DOI] [PubMed] [Google Scholar]
  17. Gober J. W., Champer R., Reuter S., Shapiro L. Expression of positional information during cell differentiation of Caulobacter. Cell. 1991 Jan 25;64(2):381–391. doi: 10.1016/0092-8674(91)90646-g. [DOI] [PubMed] [Google Scholar]
  18. Gober J. W., Shapiro L. Integration host factor is required for the activation of developmentally regulated genes in Caulobacter. Genes Dev. 1990 Sep;4(9):1494–1504. doi: 10.1101/gad.4.9.1494. [DOI] [PubMed] [Google Scholar]
  19. Gober J. W., Xu H., Dingwall A. K., Shapiro L. Identification of cis and trans-elements involved in the timed control of a Caulobacter flagellar gene. J Mol Biol. 1991 Jan 20;217(2):247–257. doi: 10.1016/0022-2836(91)90539-i. [DOI] [PubMed] [Google Scholar]
  20. Gomes S. L., Shapiro L. Differential expression and positioning of chemotaxis methylation proteins in Caulobacter. J Mol Biol. 1984 Sep 25;178(3):551–568. doi: 10.1016/0022-2836(84)90238-9. [DOI] [PubMed] [Google Scholar]
  21. Gussin G. N., Ronson C. W., Ausubel F. M. Regulation of nitrogen fixation genes. Annu Rev Genet. 1986;20:567–591. doi: 10.1146/annurev.ge.20.120186.003031. [DOI] [PubMed] [Google Scholar]
  22. Hoover T. R., Santero E., Porter S., Kustu S. The integration host factor stimulates interaction of RNA polymerase with NIFA, the transcriptional activator for nitrogen fixation operons. Cell. 1990 Oct 5;63(1):11–22. doi: 10.1016/0092-8674(90)90284-l. [DOI] [PubMed] [Google Scholar]
  23. Kadonaga J. T., Tjian R. Affinity purification of sequence-specific DNA binding proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5889–5893. doi: 10.1073/pnas.83.16.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kosturko L. D., Daub E., Murialdo H. The interaction of E. coli integration host factor and lambda cos DNA: multiple complex formation and protein-induced bending. Nucleic Acids Res. 1989 Jan 11;17(1):317–334. doi: 10.1093/nar/17.1.317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  26. Kustu S., North A. K., Weiss D. S. Prokaryotic transcriptional enhancers and enhancer-binding proteins. Trends Biochem Sci. 1991 Nov;16(11):397–402. doi: 10.1016/0968-0004(91)90163-p. [DOI] [PubMed] [Google Scholar]
  27. Kustu S., Santero E., Keener J., Popham D., Weiss D. Expression of sigma 54 (ntrA)-dependent genes is probably united by a common mechanism. Microbiol Rev. 1989 Sep;53(3):367–376. doi: 10.1128/mr.53.3.367-376.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Landy A. Dynamic, structural, and regulatory aspects of lambda site-specific recombination. Annu Rev Biochem. 1989;58:913–949. doi: 10.1146/annurev.bi.58.070189.004405. [DOI] [PubMed] [Google Scholar]
  29. Loewy Z. G., Bryan R. A., Reuter S. H., Shapiro L. Control of synthesis and positioning of a Caulobacter crescentus flagellar protein. Genes Dev. 1987 Aug;1(6):626–635. doi: 10.1101/gad.1.6.626. [DOI] [PubMed] [Google Scholar]
  30. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  31. Mechulam Y., Blanquet S., Fayat G. Dual level control of the Escherichia coli pheST-himA operon expression. tRNA(Phe)-dependent attenuation and transcriptional operator-repressor control by himA and the SOS network. J Mol Biol. 1987 Oct 5;197(3):453–470. doi: 10.1016/0022-2836(87)90558-4. [DOI] [PubMed] [Google Scholar]
  32. Milhausen M., Agabian N. Caulobacter flagellin mRNA segregates asymmetrically at cell division. Nature. 1983 Apr 14;302(5909):630–632. doi: 10.1038/302630a0. [DOI] [PubMed] [Google Scholar]
  33. Miller H. I., Kirk M., Echols H. SOS induction and autoregulation of the himA gene for site-specific recombination in Escherichia coli. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6754–6758. doi: 10.1073/pnas.78.11.6754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Minnich S. A., Newton A. Promoter mapping and cell cycle regulation of flagellin gene transcription in Caulobacter crescentus. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1142–1146. doi: 10.1073/pnas.84.5.1142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Miskimins W. K., Roberts M. P., McClelland A., Ruddle F. H. Use of a protein-blotting procedure and a specific DNA probe to identify nuclear proteins that recognize the promoter region of the transferrin receptor gene. Proc Natl Acad Sci U S A. 1985 Oct;82(20):6741–6744. doi: 10.1073/pnas.82.20.6741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Moitoso de Vargas L., Kim S., Landy A. DNA looping generated by DNA bending protein IHF and the two domains of lambda integrase. Science. 1989 Jun 23;244(4911):1457–1461. doi: 10.1126/science.2544029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mullin D. A., Newton A. Ntr-like promoters and upstream regulatory sequence ftr are required for transcription of a developmentally regulated Caulobacter crescentus flagellar gene. J Bacteriol. 1989 Jun;171(6):3218–3227. doi: 10.1128/jb.171.6.3218-3227.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mullin D., Minnich S., Chen L. S., Newton A. A set of positively regulated flagellar gene promoters in Caulobacter crescentus with sequence homology to the nif gene promoters of Klebsiella pneumoniae. J Mol Biol. 1987 Jun 20;195(4):939–943. doi: 10.1016/0022-2836(87)90497-9. [DOI] [PubMed] [Google Scholar]
  39. Nash H. A., Robertson C. A. Purification and properties of the Escherichia coli protein factor required for lambda integrative recombination. J Biol Chem. 1981 Sep 10;256(17):9246–9253. [PubMed] [Google Scholar]
  40. Nathan P., Gomes S. L., Hahnenberger K., Newton A., Shapiro L. Differential localization of membrane receptor chemotaxis proteins in the Caulobacter predivisional cell. J Mol Biol. 1986 Oct 5;191(3):433–440. doi: 10.1016/0022-2836(86)90138-5. [DOI] [PubMed] [Google Scholar]
  41. Newton A., Ohta N., Ramakrishnan G., Mullin D., Raymond G. Genetic switching in the flagellar gene hierarchy of Caulobacter requires negative as well as positive regulation of transcription. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6651–6655. doi: 10.1073/pnas.86.17.6651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Newton A., Ohta N. Regulation of the cell division cycle and differentiation in bacteria. Annu Rev Microbiol. 1990;44:689–719. doi: 10.1146/annurev.mi.44.100190.003353. [DOI] [PubMed] [Google Scholar]
  43. Ninfa A. J., Mullin D. A., Ramakrishnan G., Newton A. Escherichia coli sigma 54 RNA polymerase recognizes Caulobacter crescentus flbG and flaN flagellar gene promoters in vitro. J Bacteriol. 1989 Jan;171(1):383–391. doi: 10.1128/jb.171.1.383-391.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ninfa A. J., Reitzer L. J., Magasanik B. Initiation of transcription at the bacterial glnAp2 promoter by purified E. coli components is facilitated by enhancers. Cell. 1987 Sep 25;50(7):1039–1046. doi: 10.1016/0092-8674(87)90170-x. [DOI] [PubMed] [Google Scholar]
  45. Ohta N., Chen L. S., Mullin D. A., Newton A. Timing of flagellar gene expression in the Caulobacter cell cycle is determined by a transcriptional cascade of positive regulatory genes. J Bacteriol. 1991 Feb;173(4):1514–1522. doi: 10.1128/jb.173.4.1514-1522.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Ohta N., Chen L. S., Swanson E., Newton A. Transcriptional regulation of a periodically controlled flagellar gene operon in Caulobacter crescentus. J Mol Biol. 1985 Nov 5;186(1):107–115. doi: 10.1016/0022-2836(85)90261-x. [DOI] [PubMed] [Google Scholar]
  47. POINDEXTER J. S. BIOLOGICAL PROPERTIES AND CLASSIFICATION OF THE CAULOBACTER GROUP. Bacteriol Rev. 1964 Sep;28:231–295. doi: 10.1128/br.28.3.231-295.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Popham D. L., Szeto D., Keener J., Kustu S. Function of a bacterial activator protein that binds to transcriptional enhancers. Science. 1989 Feb 3;243(4891):629–635. doi: 10.1126/science.2563595. [DOI] [PubMed] [Google Scholar]
  49. Ramakrishnan G., Newton A. FlbD of Caulobacter crescentus is a homologue of the NtrC (NRI) protein and activates sigma 54-dependent flagellar gene promoters. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2369–2373. doi: 10.1073/pnas.87.6.2369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Richet E., Abcarian P., Nash H. A. The interaction of recombination proteins with supercoiled DNA: defining the role of supercoiling in lambda integrative recombination. Cell. 1986 Sep 26;46(7):1011–1021. doi: 10.1016/0092-8674(86)90700-2. [DOI] [PubMed] [Google Scholar]
  51. Robertson C. A., Nash H. A. Bending of the bacteriophage lambda attachment site by Escherichia coli integration host factor. J Biol Chem. 1988 Mar 15;263(8):3554–3557. [PubMed] [Google Scholar]
  52. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Santero E., Hoover T., Keener J., Kustu S. In vitro activity of the nitrogen fixation regulatory protein NIFA. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7346–7350. doi: 10.1073/pnas.86.19.7346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Shapiro L., Mansour J., Shaw P., Henry S. Synthesis of specific membrane proteins is a function of DNA replication an phospholipid synthesis in Caulobacter crescentus. J Mol Biol. 1982 Aug 5;159(2):303–322. doi: 10.1016/0022-2836(82)90497-1. [DOI] [PubMed] [Google Scholar]
  55. Shore D., Stillman D. J., Brand A. H., Nasmyth K. A. Identification of silencer binding proteins from yeast: possible roles in SIR control and DNA replication. EMBO J. 1987 Feb;6(2):461–467. doi: 10.1002/j.1460-2075.1987.tb04776.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Stenzel T. T., Patel P., Bastia D. The integration host factor of Escherichia coli binds to bent DNA at the origin of replication of the plasmid pSC101. Cell. 1987 Jun 5;49(5):709–717. doi: 10.1016/0092-8674(87)90547-2. [DOI] [PubMed] [Google Scholar]
  57. Sundaresan V., Jones J. D., Ow D. W., Ausubel F. M. Klebsiella pneumoniae nifA product activates the Rhizobium meliloti nitrogenase promoter. Nature. 1983 Feb 24;301(5902):728–732. doi: 10.1038/301728a0. [DOI] [PubMed] [Google Scholar]
  58. Thompson J. F., Landy A. Empirical estimation of protein-induced DNA bending angles: applications to lambda site-specific recombination complexes. Nucleic Acids Res. 1988 Oct 25;16(20):9687–9705. doi: 10.1093/nar/16.20.9687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wedel A., Weiss D. S., Popham D., Dröge P., Kustu S. A bacterial enhancer functions to tether a transcriptional activator near a promoter. Science. 1990 Apr 27;248(4954):486–490. doi: 10.1126/science.1970441. [DOI] [PubMed] [Google Scholar]
  61. Xu H., Dingwall A., Shapiro L. Negative transcriptional regulation in the Caulobacter flagellar hierarchy. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6656–6660. doi: 10.1073/pnas.86.17.6656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. de Lorenzo V., Herrero M., Metzke M., Timmis K. N. An upstream XylR- and IHF-induced nucleoprotein complex regulates the sigma 54-dependent Pu promoter of TOL plasmid. EMBO J. 1991 May;10(5):1159–1167. doi: 10.1002/j.1460-2075.1991.tb08056.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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