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. 1997 Aug;65(8):3091–3099. doi: 10.1128/iai.65.8.3091-3099.1997

The anthrax toxin activator gene atxA is associated with CO2-enhanced non-toxin gene expression in Bacillus anthracis.

A R Hoffmaster 1, T M Koehler 1
PMCID: PMC175436  PMID: 9234759

Abstract

The Bacillus anthracis toxin genes, cya, lef, and pag, can be viewed as a regulon, in which transcription of all three genes is activated in trans by the same regulatory gene, atxA, in response to the same signal, CO2. In atxA+ strains, toxin gene expression is increased 5- to 20-fold in cells grown in 5% CO2 relative to cells grown in air. CO2-enhanced toxin gene transcription is not observed in atx4-null mutants. Here, we used two independent techniques to obtain evidence for additional CO2-induced atxA-regulated genes. First, total protein preparations from atxA4+ and atxA isolates grown in 5% CO2 and in air were examined by two-dimensional electrophoresis. Comparison of the resulting protein patterns indicated that synthesis of non-toxin proteins is influenced by growth in elevated CO2 and the toxin gene regulator, atxA. Second, we generated random transcriptional lacZ fusions in B. anthracis with transposon Tn917-LTV3. Transposon-insertion libraries were screened for mutants expressing CO2-enhanced atxA-dependent beta-galactosidase activity. DNA sequence analysis of transposon insertion sites in 17 mutants carrying CO2- and atxA-regulated fusions revealed 10 mutants carrying independent insertions on the 185-kb toxin plasmid pXO1 which did not map to the toxin genes. The tcr-lacZ fusion mutants (tcr for toxin coregulated) were Tox+, indicating that these genes may not be involved in anthrax toxin gene activation. Our data indicate a clear association of atxA with CO2-enhanced gene expression in B. anthracis and provide evidence that atxA regulates genes other than the structural genes for the anthrax toxin proteins.

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Selected References

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  1. Bartkus J. M., Leppla S. H. Transcriptional regulation of the protective antigen gene of Bacillus anthracis. Infect Immun. 1989 Aug;57(8):2295–2300. doi: 10.1128/iai.57.8.2295-2300.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Battisti L., Green B. D., Thorne C. B. Mating system for transfer of plasmids among Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis. J Bacteriol. 1985 May;162(2):543–550. doi: 10.1128/jb.162.2.543-550.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Camilli A., Portnoy A., Youngman P. Insertional mutagenesis of Listeria monocytogenes with a novel Tn917 derivative that allows direct cloning of DNA flanking transposon insertions. J Bacteriol. 1990 Jul;172(7):3738–3744. doi: 10.1128/jb.172.7.3738-3744.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chakraborty T., Leimeister-Wächter M., Domann E., Hartl M., Goebel W., Nichterlein T., Notermans S. Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene. J Bacteriol. 1992 Jan;174(2):568–574. doi: 10.1128/jb.174.2.568-574.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dai Z., Koehler T. M. Regulation of anthrax toxin activator gene (atxA) expression in Bacillus anthracis: temperature, not CO2/bicarbonate, affects AtxA synthesis. Infect Immun. 1997 Jul;65(7):2576–2582. doi: 10.1128/iai.65.7.2576-2582.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dai Z., Sirard J. C., Mock M., Koehler T. M. The atxA gene product activates transcription of the anthrax toxin genes and is essential for virulence. Mol Microbiol. 1995 Jun;16(6):1171–1181. doi: 10.1111/j.1365-2958.1995.tb02340.x. [DOI] [PubMed] [Google Scholar]
  7. Domann E., Zechel S., Lingnau A., Hain T., Darji A., Nichterlein T., Wehland J., Chakraborty T. Identification and characterization of a novel PrfA-regulated gene in Listeria monocytogenes whose product, IrpA, is highly homologous to internalin proteins, which contain leucine-rich repeats. Infect Immun. 1997 Jan;65(1):101–109. doi: 10.1128/iai.65.1.101-109.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fouet A., Mock M. Differential influence of the two Bacillus anthracis plasmids on regulation of virulence gene expression. Infect Immun. 1996 Dec;64(12):4928–4932. doi: 10.1128/iai.64.12.4928-4932.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Green B. D., Battisti L., Koehler T. M., Thorne C. B., Ivins B. E. Demonstration of a capsule plasmid in Bacillus anthracis. Infect Immun. 1985 Aug;49(2):291–297. doi: 10.1128/iai.49.2.291-297.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Koehler T. M., Dai Z., Kaufman-Yarbray M. Regulation of the Bacillus anthracis protective antigen gene: CO2 and a trans-acting element activate transcription from one of two promoters. J Bacteriol. 1994 Feb;176(3):586–595. doi: 10.1128/jb.176.3.586-595.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Leimeister-Wächter M., Domann E., Chakraborty T. The expression of virulence genes in Listeria monocytogenes is thermoregulated. J Bacteriol. 1992 Feb;174(3):947–952. doi: 10.1128/jb.174.3.947-952.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Leppla S. H. Production and purification of anthrax toxin. Methods Enzymol. 1988;165:103–116. doi: 10.1016/s0076-6879(88)65019-1. [DOI] [PubMed] [Google Scholar]
  13. McIver K. S., Heath A. S., Scott J. R. Regulation of virulence by environmental signals in group A streptococci: influence of osmolarity, temperature, gas exchange, and iron limitation on emm transcription. Infect Immun. 1995 Nov;63(11):4540–4542. doi: 10.1128/iai.63.11.4540-4542.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  15. Perez-Casal J., Caparon M. G., Scott J. R. Mry, a trans-acting positive regulator of the M protein gene of Streptococcus pyogenes with similarity to the receptor proteins of two-component regulatory systems. J Bacteriol. 1991 Apr;173(8):2617–2624. doi: 10.1128/jb.173.8.2617-2624.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pezard C., Weber M., Sirard J. C., Berche P., Mock M. Protective immunity induced by Bacillus anthracis toxin-deficient strains. Infect Immun. 1995 Apr;63(4):1369–1372. doi: 10.1128/iai.63.4.1369-1372.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Portnoy D. A., Chakraborty T., Goebel W., Cossart P. Molecular determinants of Listeria monocytogenes pathogenesis. Infect Immun. 1992 Apr;60(4):1263–1267. doi: 10.1128/iai.60.4.1263-1267.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ristroph J. D., Ivins B. E. Elaboration of Bacillus anthracis antigens in a new, defined culture medium. Infect Immun. 1983 Jan;39(1):483–486. doi: 10.1128/iai.39.1.483-486.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sirard J. C., Mock M., Fouet A. Molecular tools for the study of transcriptional regulation in Bacillus anthracis. Res Microbiol. 1995 Nov-Dec;146(9):729–737. doi: 10.1016/0923-2508(96)81069-2. [DOI] [PubMed] [Google Scholar]
  20. Sirard J. C., Mock M., Fouet A. The three Bacillus anthracis toxin genes are coordinately regulated by bicarbonate and temperature. J Bacteriol. 1994 Aug;176(16):5188–5192. doi: 10.1128/jb.176.16.5188-5192.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. THORNE C. B., BELTON F. C. An agar-diffusion method for titrating Bacillus anthracis immunizing antigen and its application to a study of antigen production. J Gen Microbiol. 1957 Oct;17(2):505–516. doi: 10.1099/00221287-17-2-505. [DOI] [PubMed] [Google Scholar]
  22. Thorne C. B. Transducing bacteriophage for Bacillus cereus. J Virol. 1968 Jul;2(7):657–662. doi: 10.1128/jvi.2.7.657-662.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Uchida I., Hornung J. M., Thorne C. B., Klimpel K. R., Leppla S. H. Cloning and characterization of a gene whose product is a trans-activator of anthrax toxin synthesis. J Bacteriol. 1993 Sep;175(17):5329–5338. doi: 10.1128/jb.175.17.5329-5338.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Uchida I., Makino S., Sasakawa C., Yoshikawa M., Sugimoto C., Terakado N. Identification of a novel gene, dep, associated with depolymerization of the capsular polymer in Bacillus anthracis. Mol Microbiol. 1993 Aug;9(3):487–496. doi: 10.1111/j.1365-2958.1993.tb01710.x. [DOI] [PubMed] [Google Scholar]
  25. Vietri N. J., Marrero R., Hoover T. A., Welkos S. L. Identification and characterization of a trans-activator involved in the regulation of encapsulation by Bacillus anthracis. Gene. 1995 Jan 11;152(1):1–9. doi: 10.1016/0378-1119(94)00662-c. [DOI] [PubMed] [Google Scholar]
  26. Welkos S. L., Lowe J. R., Eden-McCutchan F., Vodkin M., Leppla S. H., Schmidt J. J. Sequence and analysis of the DNA encoding protective antigen of Bacillus anthracis. Gene. 1988 Sep 30;69(2):287–300. doi: 10.1016/0378-1119(88)90439-8. [DOI] [PubMed] [Google Scholar]
  27. 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]

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