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. 1997 Jan;179(2):487–495. doi: 10.1128/jb.179.2.487-495.1997

Cloning and characterization of two groESL operons of Rhodobacter sphaeroides: transcriptional regulation of the heat-induced groESL operon.

W T Lee 1, K C Terlesky 1, F R Tabita 1
PMCID: PMC178720  PMID: 8990302

Abstract

The nonsulfur purple bacterium Rhodobacter sphaeroides was found to contain two groESL operons. The groESL1 heat shock operon was cloned from a genomic library, and a 2.8-kb DNA fragment was sequenced and found to contain the groES and groEL genes. The deduced amino acid sequences of GroEL1 (cpn60) and GroES1 (cpn10) were in agreement with N-terminal sequences previously obtained for the isolated proteins (K. C. Terlesky and F. R. Tabita, Biochemistry 30:8181-8186, 1991). These sequences show a high degree of similarity to groESL genes isolated from other bacteria. Northern analysis indicated that the groESL1 genes were expressed as part of a 2.2-kb polycistronic transcript that is induced 13-fold after heat shock. Transcript size was not affected by heat shock; however, the amount of transcript was induced to its greatest extent 15 to 30 min after a 40 degrees C heat shock, from an initial temperature of 28 degrees C, and remained elevated up to 120 min. The R. sphaeroides groESL1 operon contains a putative hairpin loop at the start of the transcript that is present in other bacterial heat shock genes. Primer extension of the message showed that the transcription start site is at the start of this conserved hairpin loop. In this region were also found putative -35 and -10 sequences that are conserved upstream from other bacterial heat shock genes. Transcription of the groESL1 genes was unexpectedly low under photoautotrophic growth conditions. Thus far, it has not been possible to construct a groESL1 deletion strain, perhaps indicating that these genes are essential for growth. A second operon (groESL2) was also cloned from R. sphaeroides, using a groEL1 gene fragment as a probe; however, no transcript was observed for this operon under several different growth conditions. A groESL2 deletion strain was constructed, but there was no detectable change in the phenotype of this strain compared to the parental strain.

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Selected References

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  1. Babst M., Hennecke H., Fischer H. M. Two different mechanisms are involved in the heat-shock regulation of chaperonin gene expression in Bradyrhizobium japonicum. Mol Microbiol. 1996 Feb;19(4):827–839. doi: 10.1046/j.1365-2958.1996.438968.x. [DOI] [PubMed] [Google Scholar]
  2. Braig K., Otwinowski Z., Hegde R., Boisvert D. C., Joachimiak A., Horwich A. L., Sigler P. B. The crystal structure of the bacterial chaperonin GroEL at 2.8 A. Nature. 1994 Oct 13;371(6498):578–586. doi: 10.1038/371578a0. [DOI] [PubMed] [Google Scholar]
  3. Chandrasekhar G. N., Tilly K., Woolford C., Hendrix R., Georgopoulos C. Purification and properties of the groES morphogenetic protein of Escherichia coli. J Biol Chem. 1986 Sep 15;261(26):12414–12419. [PubMed] [Google Scholar]
  4. Dryden S. C., Kaplan S. Localization and structural analysis of the ribosomal RNA operons of Rhodobacter sphaeroides. Nucleic Acids Res. 1990 Dec 25;18(24):7267–7277. doi: 10.1093/nar/18.24.7267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ellis R. J. Chaperoning nascent proteins. Nature. 1994 Jul 14;370(6485):96–97. doi: 10.1038/370096a0. [DOI] [PubMed] [Google Scholar]
  6. Falcone D. L., Tabita F. R. Expression of endogenous and foreign ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) genes in a RubisCO deletion mutant of Rhodobacter sphaeroides. J Bacteriol. 1991 Mar;173(6):2099–2108. doi: 10.1128/jb.173.6.2099-2108.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fayet O., Ziegelhoffer T., Georgopoulos C. The groES and groEL heat shock gene products of Escherichia coli are essential for bacterial growth at all temperatures. J Bacteriol. 1989 Mar;171(3):1379–1385. doi: 10.1128/jb.171.3.1379-1385.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ferreyra R. G., Soncini F. C., Viale A. M. Cloning, characterization, and functional expression in Escherichia coli of chaperonin (groESL) genes from the phototrophic sulfur bacterium Chromatium vinosum. J Bacteriol. 1993 Mar;175(5):1514–1523. doi: 10.1128/jb.175.5.1514-1523.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fischer H. M., Babst M., Kaspar T., Acuña G., Arigoni F., Hennecke H. One member of a gro-ESL-like chaperonin multigene family in Bradyrhizobium japonicum is co-regulated with symbiotic nitrogen fixation genes. EMBO J. 1993 Jul;12(7):2901–2912. doi: 10.1002/j.1460-2075.1993.tb05952.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Georgopoulos C. P., Hendrix R. W., Casjens S. R., Kaiser A. D. Host participation in bacteriophage lambda head assembly. J Mol Biol. 1973 May 5;76(1):45–60. doi: 10.1016/0022-2836(73)90080-6. [DOI] [PubMed] [Google Scholar]
  11. Gibson J. L., Falcone D. L., Tabita F. R. Nucleotide sequence, transcriptional analysis, and expression of genes encoded within the form I CO2 fixation operon of Rhodobacter sphaeroides. J Biol Chem. 1991 Aug 5;266(22):14646–14653. [PubMed] [Google Scholar]
  12. Goloubinoff P., Christeller J. T., Gatenby A. A., Lorimer G. H. Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfoleded state depends on two chaperonin proteins and Mg-ATP. Nature. 1989 Dec 21;342(6252):884–889. doi: 10.1038/342884a0. [DOI] [PubMed] [Google Scholar]
  13. Goloubinoff P., Gatenby A. A., Lorimer G. H. GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli. Nature. 1989 Jan 5;337(6202):44–47. doi: 10.1038/337044a0. [DOI] [PubMed] [Google Scholar]
  14. Gor D., Mayfield J. E. Cloning and nucleotide sequence of the Brucella abortus groE operon. Biochim Biophys Acta. 1992 Feb 28;1130(1):120–122. doi: 10.1016/0167-4781(92)90476-g. [DOI] [PubMed] [Google Scholar]
  15. Gupta R. S. Evolution of the chaperonin families (Hsp60, Hsp10 and Tcp-1) of proteins and the origin of eukaryotic cells. Mol Microbiol. 1995 Jan;15(1):1–11. doi: 10.1111/j.1365-2958.1995.tb02216.x. [DOI] [PubMed] [Google Scholar]
  16. Hemmingsen S. M., Woolford C., van der Vies S. M., Tilly K., Dennis D. T., Georgopoulos C. P., Hendrix R. W., Ellis R. J. Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature. 1988 May 26;333(6171):330–334. doi: 10.1038/333330a0. [DOI] [PubMed] [Google Scholar]
  17. Hendrix R. W. Purification and properties of groE, a host protein involved in bacteriophage assembly. J Mol Biol. 1979 Apr 15;129(3):375–392. doi: 10.1016/0022-2836(79)90502-3. [DOI] [PubMed] [Google Scholar]
  18. Ishihama A., Ikeuchi T., Matsumoto A., Yamamoto S. A novel adenosine triphosphatase isolated from RNA polymerase preparations of Escherichia coli. II. Enzymatic properties and molecular structure. J Biochem. 1976 May;79(5):927–937. doi: 10.1093/oxfordjournals.jbchem.a131160. [DOI] [PubMed] [Google Scholar]
  19. Kanemori M., Mori H., Yura T. Effects of reduced levels of GroE chaperones on protein metabolism: enhanced synthesis of heat shock proteins during steady-state growth of Escherichia coli. J Bacteriol. 1994 Jul;176(14):4235–4242. doi: 10.1128/jb.176.14.4235-4242.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kim S. G., Batt C. A. Cloning and sequencing of the Lactococcus lactis subsp. lactis groESL operon. Gene. 1993 May 15;127(1):121–126. doi: 10.1016/0378-1119(93)90626-e. [DOI] [PubMed] [Google Scholar]
  21. Li M., Wong S. L. Cloning and characterization of the groESL operon from Bacillus subtilis. J Bacteriol. 1992 Jun;174(12):3981–3992. doi: 10.1128/jb.174.12.3981-3992.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Martin J., Mayhew M., Langer T., Hartl F. U. The reaction cycle of GroEL and GroES in chaperonin-assisted protein folding. Nature. 1993 Nov 18;366(6452):228–233. doi: 10.1038/366228a0. [DOI] [PubMed] [Google Scholar]
  23. Narberhaus F., Bahl H. Cloning, sequencing, and molecular analysis of the groESL operon of Clostridium acetobutylicum. J Bacteriol. 1992 May;174(10):3282–3289. doi: 10.1128/jb.174.10.3282-3289.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Penfold R. J., Pemberton J. M. An improved suicide vector for construction of chromosomal insertion mutations in bacteria. Gene. 1992 Sep 1;118(1):145–146. doi: 10.1016/0378-1119(92)90263-o. [DOI] [PubMed] [Google Scholar]
  25. Pridmore R. D. New and versatile cloning vectors with kanamycin-resistance marker. Gene. 1987;56(2-3):309–312. doi: 10.1016/0378-1119(87)90149-1. [DOI] [PubMed] [Google Scholar]
  26. Rusanganwa E., Gupta R. S. Cloning and characterization of multiple groEL chaperonin-encoding genes in Rhizobium meliloti. Gene. 1993 Apr 15;126(1):67–75. doi: 10.1016/0378-1119(93)90591-p. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Schmidt A., Schiesswohl M., Völker U., Hecker M., Schumann W. Cloning, sequencing, mapping, and transcriptional analysis of the groESL operon from Bacillus subtilis. J Bacteriol. 1992 Jun;174(12):3993–3999. doi: 10.1128/jb.174.12.3993-3999.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Segal G., Ron E. Z. Heat shock activation of the groESL operon of Agrobacterium tumefaciens and the regulatory roles of the inverted repeat. J Bacteriol. 1996 Jun;178(12):3634–3640. doi: 10.1128/jb.178.12.3634-3640.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Segal G., Ron E. Z. Heat shock transcription of the groESL operon of Agrobacterium tumefaciens may involve a hairpin-loop structure. J Bacteriol. 1993 May;175(10):3083–3088. doi: 10.1128/jb.175.10.3083-3088.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Terlesky K. C., Tabita F. R. Purification and characterization of the chaperonin 10 and chaperonin 60 proteins from Rhodobacter sphaeroides. Biochemistry. 1991 Aug 20;30(33):8181–8186. doi: 10.1021/bi00247a013. [DOI] [PubMed] [Google Scholar]
  32. Todd M. J., Viitanen P. V., Lorimer G. H. Dynamics of the chaperonin ATPase cycle: implications for facilitated protein folding. Science. 1994 Jul 29;265(5172):659–666. doi: 10.1126/science.7913555. [DOI] [PubMed] [Google Scholar]
  33. Wang X., Tabita F. R. Interaction of inactivated and active ribulose 1,5-bisphosphate carboxylase/oxygenase of Rhodobacter sphaeroides with nucleotides and the chaperonin 60 (GroEL) protein. J Bacteriol. 1992 Jun;174(11):3607–3611. doi: 10.1128/jb.174.11.3607-3611.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Watson G. M., Mann N. H., MacDonald G. A., Dunbar B. Identification and characterization of a GroEL homologue in Rhodobacter sphaeroides. FEMS Microbiol Lett. 1990 Nov;60(3):349–353. doi: 10.1016/0378-1097(90)90330-s. [DOI] [PubMed] [Google Scholar]
  35. Weaver K. E., Tabita F. R. Isolation and partial characterization of Rhodopseudomonas sphaeroides mutants defective in the regulation of ribulose bisphosphate carboxylase/oxygenase. J Bacteriol. 1983 Nov;156(2):507–515. doi: 10.1128/jb.156.2.507-515.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Webb R., Reddy K. J., Sherman L. A. Regulation and sequence of the Synechococcus sp. strain PCC 7942 groESL operon, encoding a cyanobacterial chaperonin. J Bacteriol. 1990 Sep;172(9):5079–5088. doi: 10.1128/jb.172.9.5079-5088.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  38. Yuan G., Wong S. L. Regulation of groE expression in Bacillus subtilis: the involvement of the sigma A-like promoter and the roles of the inverted repeat sequence (CIRCE). J Bacteriol. 1995 Oct;177(19):5427–5433. doi: 10.1128/jb.177.19.5427-5433.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yura T., Nagai H., Mori H. Regulation of the heat-shock response in bacteria. Annu Rev Microbiol. 1993;47:321–350. doi: 10.1146/annurev.mi.47.100193.001541. [DOI] [PubMed] [Google Scholar]
  40. Zeilstra-Ryalls J., Fayet O., Georgopoulos C. The universally conserved GroE (Hsp60) chaperonins. Annu Rev Microbiol. 1991;45:301–325. doi: 10.1146/annurev.mi.45.100191.001505. [DOI] [PubMed] [Google Scholar]
  41. Zuber U., Schumann W. CIRCE, a novel heat shock element involved in regulation of heat shock operon dnaK of Bacillus subtilis. J Bacteriol. 1994 Mar;176(5):1359–1363. doi: 10.1128/jb.176.5.1359-1363.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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