Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Oct;179(20):6448–6452. doi: 10.1128/jb.179.20.6448-6452.1997

Transcriptional characterization of the Rickettsia prowazekii major macromolecular synthesis operon.

E I Shaw 1, G L Marks 1, H H Winkler 1, D O Wood 1
PMCID: PMC179562  PMID: 9335295

Abstract

Recent studies have demonstrated that Rickettsia prowazekii can regulate transcription of selected genes at the level of initiation. However, little information concerning the existence of operons and coordinate gene regulation in this obligate intracellular parasitic bacterium is available. To address these issues, we have focused on the rpoD gene linkage group (greA-open reading frame 23 [ORF23]-dnaG-rpoD), which includes the rickettsial analog (ORF23-dnaG-rpoD) of the major macromolecular synthesis operon (MMSO). The rickettsial MMSO consists of an ORF coding for a protein of unknown function the structural genes for DNA primase (dnaG) and the major sigma factor of RNA polymerase (rpoD). RNase protection assays (RPA) were used to determine if these genes are organized into an operon controlled by multiple promoters and the quantities of transcripts produced by these genes relative to each other. RPA with a probe spanning the 270-base greA-ORF23 intervening region identified a putative transcriptional promoter within the intervening sequence. Multiple RPA probes spanning the next 4,041 bases of the linkage group demonstrated the presence of a continuous transcript and thus the existence of an operon. A probe spanning the dnaG-rpoD region revealed that two additional mRNA fragments were also protected, which enabled us to identify additional putative promoters for rpoD within dnaG. Primer extension determined that the 5' ends of the three transcripts consist separately of adenine (located 227 bases upstream of ORF23) and uracil and adenine (located 336 and 250 bases upstream of rpoD, respectively). Quantitation of transcripts produced by the three ORFs determined the relative amounts of transcripts (ORF23 to dnaG to rpoD) to be 1:2.7:5.1.

Full Text

The Full Text of this article is available as a PDF (449.1 KB).

Selected References

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

  1. Andersson S. G., Eriksson A. S., Näslund A. K., Andersen M. S., Kurland C. G. The Rickettsia prowazekii genome: a random sequence analysis. Microb Comp Genomics. 1996;1(4):293–315. [PubMed] [Google Scholar]
  2. Aniskovitch L. P., Winkler H. H. Instability of Rickettsia prowazekii RNA polymerase-promoter complexes. J Bacteriol. 1995 Nov;177(21):6301–6303. doi: 10.1128/jb.177.21.6301-6303.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aniskovitch L. P., Winkler H. H. Rickettsia prowazekii sigma factor sigma 73 can be overexpressed in Escherichia coli and promotes RNA polymerase binding and transcription. Microbiology. 1996 Apr;142(Pt 4):901–906. doi: 10.1099/00221287-142-4-901. [DOI] [PubMed] [Google Scholar]
  4. Burton Z. F., Gross C. A., Watanabe K. K., Burgess R. R. The operon that encodes the sigma subunit of RNA polymerase also encodes ribosomal protein S21 and DNA primase in E. coli K12. Cell. 1983 Feb;32(2):335–349. doi: 10.1016/0092-8674(83)90453-1. [DOI] [PubMed] [Google Scholar]
  5. Cai J., Pang H., Wood D. O., Winkler H. H. The citrate synthase-encoding gene of Rickettsia prowazekii is controlled by two promoters. Gene. 1995 Sep 22;163(1):115–119. doi: 10.1016/0378-1119(95)00365-d. [DOI] [PubMed] [Google Scholar]
  6. Cai J., Winkler H. H. Identification of tlc and gltA mRNAs and determination of in situ RNA half-life in Rickettsia prowazekii. J Bacteriol. 1993 Sep;175(17):5725–5727. doi: 10.1128/jb.175.17.5725-5727.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cai J., Winkler H. H. Transcriptional regulation in the obligate intracytoplasmic bacterium Rickettsia prowazekii. J Bacteriol. 1996 Sep;178(18):5543–5545. doi: 10.1128/jb.178.18.5543-5545.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dasch G. A., Weiss E. Characterization of the Madrid E strain of Rickettsia prowazekii purified by renografin density gradient centrifugation. Infect Immun. 1977 Jan;15(1):280–286. doi: 10.1128/iai.15.1.280-286.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ding H. F., Winkler H. H. Characterization of the DNA-melting function of the Rickettsia prowazekii RNA polymerase. J Biol Chem. 1993 Feb 25;268(6):3897–3902. [PubMed] [Google Scholar]
  10. Ding H. F., Winkler H. H. Purification and partial characterization of the DNA-dependent RNA polymerase from Rickettsia prowazekii. J Bacteriol. 1990 Oct;172(10):5624–5630. doi: 10.1128/jb.172.10.5624-5630.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ding H. F., Winkler H. H. The molar ratio of sigma 73 to core polymerase in the obligate intracellular bacterium, Rickettsia prowazekii. Mol Microbiol. 1994 Mar;11(5):869–873. doi: 10.1111/j.1365-2958.1994.tb00365.x. [DOI] [PubMed] [Google Scholar]
  12. Emory S. A., Belasco J. G. The ompA 5' untranslated RNA segment functions in Escherichia coli as a growth-rate-regulated mRNA stabilizer whose activity is unrelated to translational efficiency. J Bacteriol. 1990 Aug;172(8):4472–4481. doi: 10.1128/jb.172.8.4472-4481.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Eremeeva M. E., Roux V., Raoult D. Determination of genome size and restriction pattern polymorphism of Rickettsia prowazekii and Rickettsia typhi by pulsed field gel electrophoresis. FEMS Microbiol Lett. 1993 Aug 15;112(1):105–112. doi: 10.1111/j.1574-6968.1993.tb06431.x. [DOI] [PubMed] [Google Scholar]
  14. Erickson B. D., Burton Z. F., Watanabe K. K., Burgess R. R. Nucleotide sequence of the rpsU-dnaG-rpoD operon from Salmonella typhimurium and a comparison of this sequence with the homologous operon of Escherichia coli. Gene. 1985;40(1):67–78. doi: 10.1016/0378-1119(85)90025-3. [DOI] [PubMed] [Google Scholar]
  15. Kajitani M., Fukuda R., Ishihama A. Autogenous and post-transcriptional regulation of Escherichia coli RNA polymerase synthesis in vitro. Mol Gen Genet. 1980;179(3):489–496. doi: 10.1007/BF00271738. [DOI] [PubMed] [Google Scholar]
  16. Lupski J. R., Godson G. N. DNA----DNA, and DNA----RNA----protein: orchestration by a single complex operon. Bioessays. 1989 May;10(5):152–157. doi: 10.1002/bies.950100504. [DOI] [PubMed] [Google Scholar]
  17. Lupski J. R., Godson G. N. The rpsU-dnaG-rpoD macromolecular synthesis operon of E. coli. Cell. 1984 Dec;39(2 Pt 1):251–252. doi: 10.1016/0092-8674(84)90001-1. [DOI] [PubMed] [Google Scholar]
  18. Lupski J. R., Ruiz A. A., Godson G. N. Promotion, termination, and anti-termination in the rpsU-dnaG-rpoD macromolecular synthesis operon of E. coli K-12. Mol Gen Genet. 1984;195(3):391–401. doi: 10.1007/BF00341439. [DOI] [PubMed] [Google Scholar]
  19. Marks G. L., Winkler H. H., Wood D. O. Isolation and characterization of the gene coding for the major sigma factor of Rickettsia prowazekii DNA-dependent RNA polymerase. Gene. 1992 Nov 2;121(1):155–160. doi: 10.1016/0378-1119(92)90175-o. [DOI] [PubMed] [Google Scholar]
  20. Marks G. L., Wood D. O. Characterization of the gene coding for the Rickettsia prowazekii DNA primase analogue. Gene. 1993 Jan 15;123(1):121–125. doi: 10.1016/0378-1119(93)90550-m. [DOI] [PubMed] [Google Scholar]
  21. Metzger R., Brown D. P., Grealish P., Staver M. J., Versalovic J., Lupski J. R., Katz L. Characterization of the macromolecular synthesis (MMS) operon from Listeria monocytogenes. Gene. 1994 Dec 30;151(1-2):161–166. doi: 10.1016/0378-1119(94)90649-1. [DOI] [PubMed] [Google Scholar]
  22. Mironov V. N., Van Montagu M., Inzé D. High throughput RNase protection assay. Nucleic Acids Res. 1995 Aug 25;23(16):3359–3360. doi: 10.1093/nar/23.16.3359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pang H., Winkler H. H. The concentrations of stable RNA and ribosomes in Rickettsia prowazekii. Mol Microbiol. 1994 Apr;12(1):115–120. doi: 10.1111/j.1365-2958.1994.tb01000.x. [DOI] [PubMed] [Google Scholar]
  24. Rowen L., Kornberg A. Primase, the dnaG protein of Escherichia coli. An enzyme which starts DNA chains. J Biol Chem. 1978 Feb 10;253(3):758–764. [PubMed] [Google Scholar]
  25. 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]
  26. Smiley B. L., Lupski J. R., Svec P. S., McMacken R., Godson G. N. Sequences of the Escherichia coli dnaG primase gene and regulation of its expression. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4550–4554. doi: 10.1073/pnas.79.15.4550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Taylor W. E., Straus D. B., Grossman A. D., Burton Z. F., Gross C. A., Burgess R. R. Transcription from a heat-inducible promoter causes heat shock regulation of the sigma subunit of E. coli RNA polymerase. Cell. 1984 Sep;38(2):371–381. doi: 10.1016/0092-8674(84)90492-6. [DOI] [PubMed] [Google Scholar]
  28. Versalovic J., Koeuth T., Britton R., Geszvain K., Lupski J. R. Conservation and evolution of the rpsU-dnaG-rpoD macromolecular synthesis operon in bacteria. Mol Microbiol. 1993 Apr;8(2):343–355. doi: 10.1111/j.1365-2958.1993.tb01578.x. [DOI] [PubMed] [Google Scholar]
  29. Wang L. F., Doi R. H. Nucleotide sequence and organization of Bacillus subtilis RNA polymerase major sigma (sigma 43) operon. Nucleic Acids Res. 1986 May 27;14(10):4293–4307. doi: 10.1093/nar/14.10.4293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wang L. F., Price C. W., Doi R. H. Bacillus subtilis dnaE encodes a protein homologous to DNA primase of Escherichia coli. J Biol Chem. 1985 Mar 25;260(6):3368–3372. [PubMed] [Google Scholar]
  31. Weiss E., Coolbaugh J. C., Williams J. C. Separation of viable Rickettsia typhi from yolk sac and L cell host components by renografin density gradient centrifugation. Appl Microbiol. 1975 Sep;30(3):456–463. doi: 10.1128/am.30.3.456-463.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Winkler H. H., Miller E. T. Phospholipid composition of Rickettsia prowazeki grown in chicken embryo yolk sacs. J Bacteriol. 1978 Oct;136(1):175–178. doi: 10.1128/jb.136.1.175-178.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Winkler H. H. Rickettsia species (as organisms). Annu Rev Microbiol. 1990;44:131–153. doi: 10.1146/annurev.mi.44.100190.001023. [DOI] [PubMed] [Google Scholar]
  34. Wood D. O., Atkinson W. H., Sikorski R. S., Winkler H. H. Expression of the Rickettsia prowazekii citrate synthase gene in Escherichia coli. J Bacteriol. 1983 Jul;155(1):412–416. doi: 10.1128/jb.155.1.412-416.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yajnik V., Godson G. N. Selective decay of Escherichia coli dnaG messenger RNA is initiated by RNase E. J Biol Chem. 1993 Jun 25;268(18):13253–13260. [PubMed] [Google Scholar]

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

RESOURCES