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. 1997 Jul;65(7):2576–2582. doi: 10.1128/iai.65.7.2576-2582.1997

Regulation of anthrax toxin activator gene (atxA) expression in Bacillus anthracis: temperature, not CO2/bicarbonate, affects AtxA synthesis.

Z Dai 1, T M Koehler 1
PMCID: PMC175364  PMID: 9199422

Abstract

Anthrax toxin gene expression in Bacillus anthracis is dependent on the presence of atxA, a trans-acting regulatory gene located on the resident 185-kb plasmid pXO1. In atxA+ strains, expression of the toxin genes (pag, lef, and cya) is enhanced by two physiologically significant signals: elevated CO2/bicarbonate and temperature. To determine whether increased toxin gene expression in response to these signals is associated with increased atxA expression, we monitored steady-state levels of atxA mRNA and AtxA protein in cells cultured in different conditions. We purified histidine-tagged AtxA [AtxA(His)] from Escherichia coli and used anti-AtxA(His) serum to detect AtxA in protein preparations from B. anthracis cells. AtxA was identified as a protein with an apparent size of 56 kDa in cytoplasmic fractions of B. anthracis cells. Our data indicate that atxA expression is not influenced by CO2/bicarbonate levels. However, the steady-state level of atxA mRNA in cells grown in elevated CO2/bicarbonate at 37 degrees C is five- to sixfold higher than that observed in cells grown in the same conditions at 28 degrees C. A corresponding difference in AtxA protein was also seen at the different growth temperatures. When atxA was cloned on a multicopy plasmid in B. anthracis, AtxA levels corresponding to the atxA gene copy number were observed. However, this strain produced significantly less pag mRNA and protective antigen protein than the parental strain harboring atxA in single copy on pXO1. These results indicate that increased AtxA expression does not lead to a corresponding increase in pag expression. Our data strongly suggest that an additional factor(s) is involved in regulation of pag and that the relative amounts of such a factor(s) and AtxA are important for optimal toxin gene expression.

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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. Cataldi A., Fouet A., Mock M. Regulation of pag gene expression in Bacillus anthracis: use of a pag-lacZ transcriptional fusion. FEMS Microbiol Lett. 1992 Nov 1;77(1-3):89–93. doi: 10.1016/0378-1097(92)90137-d. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Dorman C. J. 1995 Flemming Lecture. DNA topology and the global control of bacterial gene expression: implications for the regulation of virulence gene expression. Microbiology. 1995 Jun;141(Pt 6):1271–1280. doi: 10.1099/13500872-141-6-1271. [DOI] [PubMed] [Google Scholar]
  5. Granger D. L., Perfect J. R., Durack D. T. Virulence of Cryptococcus neoformans. Regulation of capsule synthesis by carbon dioxide. J Clin Invest. 1985 Aug;76(2):508–516. doi: 10.1172/JCI112000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Grodberg J., Dunn J. J. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J Bacteriol. 1988 Mar;170(3):1245–1253. doi: 10.1128/jb.170.3.1245-1253.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Iwanaga M., Yamamoto K. New medium for the production of cholera toxin by Vibrio cholerae O1 biotype El Tor. J Clin Microbiol. 1985 Sep;22(3):405–408. doi: 10.1128/jcm.22.3.405-408.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kass E. H., Kendrick M. I., Tsai Y. C., Parsonnet J. Interaction of magnesium ion, oxygen tension, and temperature in the production of toxic-shock-syndrome toxin-1 by Staphylococcus aureus. J Infect Dis. 1987 Apr;155(4):812–815. doi: 10.1093/infdis/155.4.812. [DOI] [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. 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]
  12. Rohde J. R., Fox J. M., Minnich S. A. Thermoregulation in Yersinia enterocolitica is coincident with changes in DNA supercoiling. Mol Microbiol. 1994 Apr;12(2):187–199. doi: 10.1111/j.1365-2958.1994.tb01008.x. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. 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]
  17. 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]
  18. 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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