• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. May 1997; 179(9): 2802–2809.
PMCID: PMC179038

A divergently transcribed open reading frame is located upstream of the Pseudomonas aeruginosa vfr gene, a homolog of Escherichia coli crp.

Abstract

The Pseudomonas aeruginosa homolog of the Escherichia coli global transcriptional regulator CRP (or CAP) was recently identified and designated Vfr (S. E. H. West, A. K. Sample, and L. J. Runyen-Janecky, J. Bacteriol. 176:7532-7542, 1994). Nucleotide sequence analysis of the region 5' to vfr identified a 423-bp open reading frame (ORF), which was designated orfX. The deduced amino acid sequence of ORFX was 53% identical and 87% similar to a divergent ORF of unknown function located 5' to the E. coli crp gene. When orfX was expressed from a phage T7 promoter in E. coli, a protein with an apparent molecular mass of approximately 18 kDa was produced. We constructed a chromosomal deletion of the region containing the 5' end of orfX (orfX'), vfr, and the 3' end of trpC (trpC') in P. aeruginosa strains PAO1 and PA103. The cloned vfr gene restored Vfr-dependent production of exotoxin A and protease in the PA103 orfX'-vfr-trpC' deletion mutant, suggesting that ORFX is not required for Vfr production or activity. To determine whether transcription of orfX and vfr are controlled by the same mechanisms that control transcription of the region of the divergent ORF (dorf) and of crp, we compared the vfr-orfX and crp-dorf intergenic regions. Using S1 nuclease analysis, we determined that the distance between the orfX and vfr transcriptional start sites was 105 bp. Thus, the P. aeruginosa orfX and vfr promoters are arranged in a back-to-back orientation rather than the face-to-face orientation of the dorf and crp promoters. A CRP recognition site is associated with each promoter in the crp-dorf intergenic region; binding of the CRP-cyclic AMP complex to the stronger dorf CRP recognition site activates transcription from the dorf promoter and represses transcription from the crp promoter. The vfr-orfX intergenic region does not contain an obvious CRP recognition site. In addition, vfr was not required for transcription of orfX. Unlike the dorf and crp mRNAs, the 5' ends of the orfX and vfr mRNAs were not complementary. Thus, the orfX mRNA cannot hybridize to the 5' end of the vfr mRNA to inhibit vfr transcription, a mechanism that has been postulated to control crp transcription in E. coli.

Full Text

The Full Text of this article is available as a PDF (375K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Aiba H. Autoregulation of the Escherichia coli crp gene: CRP is a transcriptional repressor for its own gene. Cell. 1983 Jan;32(1):141–149. [PubMed]
  • Beck CF, Warren RA. Divergent promoters, a common form of gene organization. Microbiol Rev. 1988 Sep;52(3):318–326. [PMC free article] [PubMed]
  • Berk AJ, Sharp PA. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. [PubMed]
  • Bhasin R, Freundlich M. The nucleotide sequence of the Escherichia coli crp divergent RNA and an overlapping ORF. Biochim Biophys Acta. 1991 Dec 2;1129(1):109–111. [PubMed]
  • Botsford JL, Harman JG. Cyclic AMP in prokaryotes. Microbiol Rev. 1992 Mar;56(1):100–122. [PMC free article] [PubMed]
  • Chung DW, Collier RJ. Enzymatically active peptide from the adenosine diphosphate-ribosylating toxin of Pseudomonas aeruginosa. Infect Immun. 1977 Jun;16(3):832–841. [PMC free article] [PubMed]
  • Cossart P, Gicquel-Sanzey B. Regulation of expression of the crp gene of Escherichia coli K-12: in vivo study. J Bacteriol. 1985 Jan;161(1):454–457. [PMC free article] [PubMed]
  • Gralla JD. Promoter recognition and mRNA initiation by Escherichia coli E sigma 70. Methods Enzymol. 1990;185:37–54. [PubMed]
  • Hanamura A, Aiba H. Molecular mechanism of negative autoregulation of Escherichia coli crp gene. Nucleic Acids Res. 1991 Aug 25;19(16):4413–4419. [PMC free article] [PubMed]
  • Hanamura A, Aiba H. A new aspect of transcriptional control of the Escherichia coli crp gene: positive autoregulation. Mol Microbiol. 1992 Sep;6(17):2489–2497. [PubMed]
  • Holloway BW, Krishnapillai V, Morgan AF. Chromosomal genetics of Pseudomonas. Microbiol Rev. 1979 Mar;43(1):73–102. [PMC free article] [PubMed]
  • Kolb A, Busby S, Buc H, Garges S, Adhya S. Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem. 1993;62:749–795. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Liu PV. Exotoxins of Pseudomonas aeruginosa. I. Factors that influence the production of exotoxin A. J Infect Dis. 1973 Oct;128(4):506–513. [PubMed]
  • Maxam AM, Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. [PMC free article] [PubMed]
  • Ohman DE, Sadoff JC, Iglewski BH. Toxin A-deficient mutants of Pseudomonas aeruginosa PA103: isolation and characterization. Infect Immun. 1980 Jun;28(3):899–908. [PMC free article] [PubMed]
  • Okamoto K, Freundlich M. Mechanism for the autogenous control of the crp operon: transcriptional inhibition by a divergent RNA transcript. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5000–5004. [PMC free article] [PubMed]
  • Okamoto K, Hara S, Bhasin R, Freundlich M. Evidence in vivo for autogenous control of the cyclic AMP receptor protein gene (crp) in Escherichia coli by divergent RNA. J Bacteriol. 1988 Nov;170(11):5076–5079. [PMC free article] [PubMed]
  • Olsen RH, DeBusscher G, McCombie WR. Development of broad-host-range vectors and gene banks: self-cloning of the Pseudomonas aeruginosa PAO chromosome. J Bacteriol. 1982 Apr;150(1):60–69. [PMC free article] [PubMed]
  • Pearson WR, Lipman DJ. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. [PMC free article] [PubMed]
  • Pugsley AP. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. [PMC free article] [PubMed]
  • Ross W, Gosink KK, Salomon J, Igarashi K, Zou C, Ishihama A, Severinov K, Gourse RL. A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase. Science. 1993 Nov 26;262(5138):1407–1413. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Schweizer HP. Escherichia-Pseudomonas shuttle vectors derived from pUC18/19. Gene. 1991 Jan 2;97(1):109–121. [PubMed]
  • Schweizer HP. Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol Microbiol. 1992 May;6(9):1195–1204. [PubMed]
  • Schweizer HD. Small broad-host-range gentamycin resistance gene cassettes for site-specific insertion and deletion mutagenesis. Biotechniques. 1993 Nov;15(5):831–834. [PubMed]
  • Simons RW, Houman F, Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53(1):85–96. [PubMed]
  • Smith AW, Iglewski BH. Transformation of Pseudomonas aeruginosa by electroporation. Nucleic Acids Res. 1989 Dec 25;17(24):10509–10509. [PMC free article] [PubMed]
  • Tabor S, Richardson CC. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. [PMC free article] [PubMed]
  • West SE, Iglewski BH. Codon usage in Pseudomonas aeruginosa. Nucleic Acids Res. 1988 Oct 11;16(19):9323–9335. [PMC free article] [PubMed]
  • West SE, Kaye SA, Hamood AN, Iglewski BH. Characterization of Pseudomonas aeruginosa mutants that are deficient in exotoxin A synthesis and are altered in expression of regA, a positive regulator of exotoxin A. Infect Immun. 1994 Mar;62(3):897–903. [PMC free article] [PubMed]
  • West SE, Sample AK, Runyen-Janecky LJ. The vfr gene product, required for Pseudomonas aeruginosa exotoxin A and protease production, belongs to the cyclic AMP receptor protein family. J Bacteriol. 1994 Dec;176(24):7532–7542. [PMC free article] [PubMed]
  • West SE, Schweizer HP, Dall C, Sample AK, Runyen-Janecky LJ. Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene. 1994 Oct 11;148(1):81–86. [PubMed]
  • Wretlind B, Pavlovskis OR. Genetic mapping and characterization of Pseudomonas aeruginosa mutants defective in the formation of extracellular proteins. J Bacteriol. 1984 Jun;158(3):801–808. [PMC free article] [PubMed]

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

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...