1887

Abstract

The gene of KT2440 encoding the heat-shock sigma factor σ was cloned and sequenced, and the translated gene product was predicted to be a protein of 325 kDa. The unambiguous role of the gene as a sigma factor was confirmed because the cloned gene complemented the growth defect, at 37 and 42 °C, of an mutant strain. Primer extension analysis showed that in the gene is expressed from three promoters in cells growing at 30 °C. Two of them, P1 and P3, share homology with the σ-dependent promoters, while the third one, P2, shows a typical σ-consensus sequence. The pattern of transcription initiation of the gene did not change in response to different stresses, i.e. a sudden heat shock or the addition of aromatic compounds. However, the predicted secondary structure of the 5′ region of the mRNA derived from the three different promoters suggests regulation at the level of translation efficiency and/or mRNA half-life. An inverted repeat sequence located 20 bp downstream of the stop codon was shown to function as a terminator in growing at temperatures from 18 to 42 °C.

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2001-05-01
2020-01-27
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References

  1. Abril M. A., Timmis K. N., Ramos J. L, Michán C.. 1989; Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway. J Bacteriol171:6782–6790
    [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. 1990; Basic local alignment search tool. J Mol Biol215:403–410[CrossRef]
    [Google Scholar]
  3. Arsène F.. Tomoyasu T., Mogk A., Achirra C., Schulze-Specking A., Bukau B. 1999; Role of region C in regulation of heat shock gene-specific sigma factor of Escherichia coli , σ32. J Bacteriol181:3552–3561
    [Google Scholar]
  4. Ausubel F. M., Brent R. E., Kingston D. D., Moore J. G., Seidman J. A., Smith A., Struhl K. (editors) 1999; Current Protocols in Molecular Biology. New York: Wiley;
    [Google Scholar]
  5. Boyer H. W., Roulland-Dussoix D. 1969; A complementation analysis of the restriction and modification of DNA in Escherichia coli . J Mol Biol41:459–472[CrossRef]
    [Google Scholar]
  6. Bukau B. 1993; Regulation of the Escherichia coli heat-shock response. Mol Microbiol9:671–680[CrossRef]
    [Google Scholar]
  7. Calendar R., Erickson J. W., Halling C., Nolte A. 1988; Deletion and insertion mutations in the rpoH gene of Escherichia coli that produce functional sigma-32. J Bacteriol170:3479–3484
    [Google Scholar]
  8. Chomczynski P., Sacchi N. 1987; Single-step method of RNA isolation by acid guanidine thiocyanate-phenol-chloroform extraction. Anal Biochem162:156–159
    [Google Scholar]
  9. Cooper S., Ruettinger T. 1975; A temperature sensitive nonsense mutation affecting the synthesis of a major protein of Escherichia coli K12. Mol Gen Genet139:167–176
    [Google Scholar]
  10. van Dyk T. K.. Reed T. R., Vollmer A. C., LaRossa R. A. 1995; Synergistic induction of the heat shock response in Escherichia coli by simultaneous treatment with chemical inducers. J Bacteriol177:6001–6004
    [Google Scholar]
  11. Erickson J. W., Gross C. A. 1989; Identification of the sigma E subunit of Escherichia coli RNA polymerase: a second alternate sigma factor involved in high-temperature gene expression. Genes Dev3:1462–1471[CrossRef]
    [Google Scholar]
  12. Fickett J. W. 1982; Recognition of protein coding regions in DNA sequences. Nucleic Acids Res10:5303–5318[CrossRef]
    [Google Scholar]
  13. Franklin F. C. H., Bagdasarian M., Bagdasarian M. M., Timmis K. N. 1981; Molecular and functional analysis of the TOL plasmid pWW0 from Pseudomonas putida and cloning of genes for the entire regulated aromatic ring meta -cleavage pathway. Proc Natl Acad Sci USA78:7458–7462[CrossRef]
    [Google Scholar]
  14. Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. 1982; Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene18:289–296[CrossRef]
    [Google Scholar]
  15. Gross C. A. others 1996; Function and regulation of the heat shock proteins. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology , 2nd edn. pp1382–1399 Edited by Neidhardt F. C.. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  16. Köhler T.. Harayama S., Ramos J. L., Timmis K. N. 1989; Involvement of Pseudomonas putida RpoN σ factor in regulation of various metabolic functions. J Bacteriol171:4326–4333
    [Google Scholar]
  17. Kunz D. A., Chapman P. J. 1981; Catabolism of pseudocumene and 3-ethyltoluene by Pseudomonas putida ( arvilla ) mt-2: evidence for new functions of the TOL (pWW0) plasmid. J Bacteriol146:179–191
    [Google Scholar]
  18. Lonetto M., Gribskov M., Gross C. A. 1992; The σ70 family: sequence conservation and evolutionary relationships. J Bacteriol174:3843–3849
    [Google Scholar]
  19. McCarty J. S., Rudiger S., Schonfeld H. J., Schneider-Mergener J., Nakahigashi K., Yura T., Bukau B. 1996; Regulatory region C of the E. coli heat shock transcription factor, sigma32, constitutes a DnaK binding site and is conserved among eubacteria. J Mol Biol256:829–837[CrossRef]
    [Google Scholar]
  20. Macián F., Pérez-Roger I.. Armengod M. E. 1994; An improved vector system for constructing transcriptional lac Z fusions: analysis of regulation of the dnaA , dnaN , recF and gyrB genes of Escherichia coli . Gene145:17–24[CrossRef]
    [Google Scholar]
  21. Marqués S.. Ramos J. L., Timmis K. N. 1993; Analysis of the mRNA structure of the Pseudomonas putida TOL meta fission pathway operon around the transcription initiation point, the xylTE and the xylFJ regions. Biochim Biophys Acta1216:227–236[CrossRef]
    [Google Scholar]
  22. Marqués S.. Manzanera M., Gallegos M. T., Ramos J. L, González-Pérez M. M.. 1999; The XylS-dependent Pm promoter is transcribed in vivo by RNA polymerase with σ32 or σ38 depending on the growth phase. Mol Microbiol31:1105–1113[CrossRef]
    [Google Scholar]
  23. Miller J. H. 1972; Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  24. Morita M. T., Tanaka Y., Kodama T. S., Kyogoku Y., Yanagi H., Yura T. 1999; Translational induction of heat shock transcription factor sigma32: evidence for a built-in RNA thermosensor. Genes Dev13:655–665[CrossRef]
    [Google Scholar]
  25. Naczynski Z. M., Mueller C., Kropinski A. M. 1995; Cloning the gene for the heat shock response positive regulator (sigma 32 homolog) from Pseudomonas aeruginosa. Can J Microbiol41:75–87[CrossRef]
    [Google Scholar]
  26. Nagai H., Yano R., Erickson J. W., Yura T. 1990; Transcriptional regulation of the heat shock regulatory gene rpoH in Escherichia coli : involvement of a novel catabolite-sensitive promoter. J Bacteriol172:2710–2715
    [Google Scholar]
  27. Nagai H., Yuzawa H., Yura T. 1991; Interplay of two cis-acting mRNA regions in translational control of sigma 32 synthesis during the heat shock response of Escherichia coli . Proc Natl Acad Sci USA88:10515–10519[CrossRef]
    [Google Scholar]
  28. Nagai H., Yuzawa H., Kanemori M., Yura T. 1994; Distinct segment of σ32 polypeptide is involved in the DnaK-mediated negative control of heat shock response in Escherichia coli . Proc Natl Acad Sci USA91:10280–10284[CrossRef]
    [Google Scholar]
  29. Nakahigashi K., Yanagi H., Yura T. 1995; Isolation and sequence analysis of rpoH genes encoding sigma 32 homologues from Gram negative bacteria: conserved mRNA and protein segments for heat shock regulation. Nucleic Acids Res23:4383–4390
    [Google Scholar]
  30. Pallen M. 1999; RpoN-dependent transcription of rpoH ?. Mol Microbiol31:393[CrossRef]
    [Google Scholar]
  31. Pearson W., Lipman D. 1988; Improved tools for biological sequence comparison. Proc Natl Acad Sci USA85:2444–2448[CrossRef]
    [Google Scholar]
  32. Ramos J. L., Timmis K. N, Marqués S.. 1997; Transcriptional control of the Pseudomonas TOL plasmid catabolic operons is achieved through an interplay of host factors and plasmid encoded regulators. Annu Rev Microbiol51:341–373[CrossRef]
    [Google Scholar]
  33. Ramos-González M. I.. Molin S. 1998; Cloning, sequencing, and phenotypic characterization of the rpoS gene from Pseudomonas putida KT2440. J Bacteriol180:3421–3431
    [Google Scholar]
  34. Sahu G. K., Chowdhury R., Das J. 1997; The rpoH gene encoding σ32 homolog of Vibrio cholerae . Gene189:203–207[CrossRef]
    [Google Scholar]
  35. Sambrook J., Fritsch E. F., Maniatis T. 1989; Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  36. Spaink H. P., Okker R. J. H., Wijffelman C. A., Pees E., Lugtenberg B. J. J. 1987; Promoter in the nodulation region of the Rhizobium leguminosarum symbiosis plasmid pRL1J1. Plant Mol Biol9:27–39[CrossRef]
    [Google Scholar]
  37. Tomayasu T., Ogura T., Tatsuta T., Bukau B. 1998; Levels of DnaK and DnaJ provide tight control of heat shock gene expression and protein repair in Escherichia coli . Mol Microbiol30:567–581[CrossRef]
    [Google Scholar]
  38. Wang Q., Kaguni J. M. 1989; A novel sigma factor is involved in expression of the rpoH gene of Escherichia coli. J Bacteriol 171:4248–4283
    [Google Scholar]
  39. Worsey M. J., Williams P. A. 1975; Metabolism of toluene and the xylenes by Pseudomonas putida ( arvilla ) mt-2: evidence for a new function of the TOL plasmid. J Bacteriol124:7–13
    [Google Scholar]
  40. Wu J., Newton A. 1997; The Caulobacter heat shock sigma factor gene rpoH is positively autoregulated from a σ32-dependent promoter. J Bacteriol179:514–521
    [Google Scholar]
  41. Yanisch-Perron C., Vieira J., Messing J. 1985; Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene33:103–119[CrossRef]
    [Google Scholar]
  42. Yura T., Nagai H., Mori H. 1993; Regulation of the heat-shock response in bacteria. Annu Rev Microbiol47:321–350[CrossRef]
    [Google Scholar]
  43. Yuzawa H., Nagai H., Mori H., Yura T. 1993; Heat induction of sigma 32 synthesis mediated by mRNA secondary structure: a primary step of the heat shock response in Escherichia coli . Nucleic Acids Res21:5449–5455[CrossRef]
    [Google Scholar]
  44. Zuker M., Mathews D. H., Turner D. H. 1999; Algorithms and thermodynamics for RNA secondary structure prediction: a practical guide. In RNA Biochemistry and Biotechnology pp11–43NATO ASI Series Edited by Barciszewski J., Clark B. F. C.. Dordrecht: Kluwer;
    [Google Scholar]
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