This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Steidl, R.
Right arrow Articles by Jayaswal, R. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Steidl, R.
Right arrow Articles by Jayaswal, R. K.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, August 2008, p. 2923-2925, Vol. 52, No. 8
0066-4804/08/$08.00+0     doi:10.1128/AAC.00273-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Staphylococcus aureus Cell Wall Stress Stimulon Gene-lacZ Fusion Strains: Potential for Use in Screening for Cell Wall-Active Antimicrobials{triangledown}

Rebecca Steidl, Stacy Pearson, Robert E. Stephenson, Nagender Ledala, Sutthirat Sitthisak, Brian J. Wilkinson, and Radheshyam K. Jayaswal*

Department of Biological Sciences, Illinois State University, Normal, Illinois 61790-4120

Received 27 February 2008/ Returned for modification 9 April 2008/ Accepted 30 May 2008


arrow
ABSTRACT
 
lacZ fusion strains were constructed using the promoters of five cell wall stress stimulon genes: pbp2, tcaA, vraSR, sgtB, and lytR. All fusion strains were induced only in the presence of cell wall-active antibiotics, suggesting the potential of these strains for use in high-throughput screening for new cell wall-active agents.


arrow
TEXT
 
Staphylococcus aureus is a medically important bacterium responsible for a number of diseases and is the leading cause of both nosocomial and community-acquired infections (1, 13, 14). Antibiotic resistance has developed rapidly in S. aureus, and now methicillin-resistant strains of S. aureus are encountered worldwide. Various strategies have been employed to search for new antibacterial drugs (5, 6, 7, 8, 11, 12, 17, 18, 23, 25). We propose a gene expression assay as a strategy for the screening and identification of potential cell wall-active antimicrobial agents. This approach is based on a fusion between a target gene promoter and a reporter gene (2, 3, 7, 9, 21, 24, 28, 31). In the past decade, microarray techniques have been used to pinpoint bacterial genes as potential novel targets for antibiotic discovery. Recently, several laboratories have used DNA microarray analyses to show that various genes involved in cell wall synthesis are upregulated by cell wall-active antibiotics (15, 30, 33). Here, we fused the promoters of five genes (pbp2, tcaA, vraSR, lytR, and sgtB) which were significantly upregulated by cell wall-active antibiotics (30) to promoterless lacZ genes (4) in S. aureus SH1000 (10). To demonstrate the potential utility of these strains for drug discovery, we determined the specificity of gene induction by measuring β-galactosidase activity after treatment with various chemicals and incubation under different environmental conditions.

S. aureus cells were grown in tryptic soy broth/agar at 37°C with the appropriate antibiotics. The promoter and lacZ gene transcriptional fusions of pbp2 (Ppbp2::lacZ), vraSR (PvraSR::lacZ), tcaA(PtcaA::lacZ), lytR(PlytR::lacZ), and sgtB(PsgtB::lacZ) were constructed as described earlier (22, 27). The promoter fragments were subcloned upstream of a promoterless lacZ gene in the shuttle vector pAZ106 (4) and transferred into S. aureus RN4220 by electroporation (20). The lacZ fusion constructs were transferred by phage 80{alpha}-mediated transduction from S. aureus RN4220 into S. aureus SH1000 (22). Overnight cultures of the fusion strains were diluted 100-fold in tryptic soy broth and grown to an optical density at 600 nm (OD600) of about 0.3 at 37°C. Potential inducing agents were added to the cultures, and the cultures were incubated for an additional 2 h, after which β-galactosidase activity was determined. β-Galactosidase activity was measured colorimetrically using o-nitrophenyl-β-D-galactoside as the substrate (19). For potential high-throughput screening, we used a modified assay for 96-well plates in a total reaction volume of 50 µl, using methylumbelliferyl-β-glucuronide as the substrate (29). Other molecular techniques were performed as described by Novick (20) and Sambrook and Russell (26).

To demonstrate that the expression of the lacZ gene was dose dependent, all promoter-lacZ fusion strains were incubated with various concentrations of oxacillin (Fig. 1A). Overnight cultures of the Ppbp2::lacZ clone were diluted 1:100 in tryptic soy broth and grown to an OD600 of approximately 0.3. Various concentrations of oxacillin, ranging from 0.075 µg/ml to 8.0 µg/ml, were then added, and the cultures were incubated with shaking at 37°C. Cultures were collected after 0.5, 1, and 2 h, and β-galactosidase assays were performed. The induction of β-galactosidase could be seen after 0.5 h, with an oxacillin concentration as low as 0.3 µg/ml, and the highest substantial pbp2 induction was seen after 2 h, with an oxacillin concentration of 1.2 µg/ml, which is also the MIC of oxacillin for SH1000. Similarly, dose-dependent β-galactosidase assays were performed for all of the promoter-lacZ fusion strains, with oxacillin as the inducing agent (Fig. 1B). For all strains, induction was shown with oxacillin concentrations as low as 0.3 µg/ml. Most strains exhibited an ~4-fold induction, with the exception of the PsgtB::lacZ strain, which exhibited a much lower basal β-galactosidase activity and a 13-fold induction (Fig. 1B). All strains also exhibited an increase in β-galactosidase expression as the oxacillin concentration was raised from 1.2 µg/ml to 8 µg/ml. Induction was seen in all fusion strains by all cell wall-active antibiotics tested, i.e., D-cycloserine, bacitracin, and vancomycin (Table 1). The largest overall induction was found using D-cycloserine as the inducing agent with the PsgtB::lacZ clone. Induction with bacitracin was modest but present in all strains, resulting in ≥2-fold induction.


Figure 1
View larger version (37K):
[in this window]
[in a new window]

  		FIG. 1. (A) Effect of oxacillin on β-galactosidase expression in the Ppbp2::lacZ clone when incubated for 0.5, 1, and 2 h. An analysis of the β-galactosidase expression from SH1000 Ppbp2::lacZ fusion strains in response to oxacillin is shown. Overnight cultures of Ppbp2::lacZ were grown to an OD600 of 0.3, various concentrations of oxacillin were added, and the cultures were incubated with shaking for 0.5 ({blacksquare}), 1 (Figure 1), and 2 h ({square}). Cells were harvested, and β-galactosidase activities were determined. Error bars represent the standard deviations for triplicate experiments. (B) Effect of oxacillin on β-galactosidase expression in all lacZ fusion clones. An analysis of the β-galactosidase expression from SH1000 promoter-lacZ fusion strains in response to oxacillin is shown. Overnight cultures of Ppbp2::lacZ ({square}), PvraSR::lacZ (Figure 1), PtcaA::lacZ (Figure 1), PlytR::lacZ (Figure 1), and PsgtB::lacZ (Figure 1) fusion reporter strains were incubated with various concentrations of oxacillin for 2 h. After antibiotic treatment, cells were harvested and β-galactosidase activities were determined as described in the text. Error bars represent the standard deviations for triplicate experiments.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Effect of various inducing agents on the expression of β-galactosidase in lacZ fusion constructs

To test whether the induction of promoters was specific to cell wall-active antibiotics, various classes of antibiotics, such as the translational inhibitors erythromycin, chloramphenicol, streptomycin, and tetracycline, the transcriptional inhibitor rifampin, the cell membrane permeability-altering antibiotic nisin, and the folic acid biosynthesis inhibitor trimethoprim, were added to growing cultures of the promoter-lacZ fusion strains at various MICs. As shown in Table 1, no significant induction was observed. Northern blot analysis further confirmed that protein synthesis inhibitors did not result in the transcription of cell wall stress stimulon genes (data not shown). These results clearly suggest that the induction of β-galactosidase in fusion strains is highly specific to cell wall-active antibiotics.

To show that the induction was not a general stress response, the effects of various environmental conditions were tested. As shown in Table 1, no induction was seen by the cell wall-lytic enzymes lysozyme (5 µg/ml) and lysostaphin (0.25 µg/ml). Unlike cell wall-active antibiotics, which inhibit the synthesis of the cell wall, both of these agents cause enzymatic degradation of peptidoglycan. Also, no induction was seen with Triton X-100 (1%), a mild detergent (cell membrane disrupter). Other environmental stress conditions, such as pH (5 to 9), temperature (25 to 42°C), osmotic stress (1.5 M NaCl), and oxidative conditions (20 mM hydrogen peroxide or 50 µM paraquat), did not lead to induction (data not shown). Thus, the induction of all lacZ fusion strains was found to be unaffected by general stress conditions.

Among the clones we studied, PsgtB::lacZ showed a 5- to 15-fold induction by cell wall-active antibiotics, with small standard deviations and a consistent basal level of β-galactosidase activity. sgtB is a member gene of the cell wall stress stimulon, under the control of the VraSR system in S. aureus (15, 16), and catalyzes peptidoglycan transglycosylase activity (32). The sgtB promoter strain may offer the best potential whole-cell screen for cell wall-active agents, with the advantages of screening directly in S. aureus and avoiding compounds active against cell-free targets that are inactive against whole cells (33).


arrow
ACKNOWLEDGMENTS
 
We thank Anthony Otsuka for critical reading of the manuscript.

This work has been partially supported by a grant from the NIH to R.K.J.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biological Sciences, Illinois State University, Normal, IL 61790-4120. Phone: (309) 438-5128. Fax: (309) 438-3722. E-mail: drjay{at}ilstu.edu Back

{triangledown} Published ahead of print on 9 June 2008. Back


arrow
REFERENCES
 
    1
  1. Archer, G. L. 1998. Staphylococcus aureus: a well-armed pathogen. Clin. Infect. Dis. 26:1179-1181.[Medline]
    2. 2
  2. Bechor, O., D. R. Smulski, T. K. Van Dyk, R. A. LaRossa, and S. Belkin. 2002. Recombinant microorganisms as environmental biosensors: pollutants detection by Escherichia coli bearing fabA'::lux fusions. J. Biotechnol. 94:125-132.[CrossRef][Medline]
    3. 3
  3. Cao, M., T. Wang, R. Ye, and J. D. Helmann. 2002. Antibiotics that inhibit cell wall biosynthesis induce expression of the Bacillus subtilis {sigma}W and {sigma}M regulons. Mol. Microbiol. 45:1267-1276.[CrossRef][Medline]
    4. 4
  4. Chan, P. F., S. J. Foster, E. Ingham, and M. O. Clements. 1998. The Staphylococcus aureus alternative sigma factor {sigma}B controls the environmental stress response but not starvation survival or pathogenicity in a mouse abscess model. J. Bacteriol. 180:6082-6089.[Abstract/Free Full Text]
    5. 5
  5. DeVito, J. A., J. A. Mills, V. G. Liu, A. Agarwal, C. F. Sizemore, Z. Yao, D. M. Stoughton, M. G. Cappiello, M. D. Barbosa, L. A. Foster, and D. L. Pompliano. 2002. An array of target-specific screening strains for antibacterial discovery. Nat. Biotechnol. 20:478-483.[CrossRef][Medline]
    6. 6
  6. Donadio, S., L. Carrano, L. Brandi, S. Serina, A. Soffientini, E. Raimondi, N. Montanini, M. Sosio, and C. O. Gualerzi. 2002. Targets and assays for discovering novel antibacterial agents. J. Biotechnol. 99:175-185.[CrossRef][Medline]
    7. 7
  7. Falk, S. P., A. T. Ulijasz, and B. Weisblum. 2007. Differential assay for high-throughput screening of antibacterial compounds. J. Biomol. Screen. 12:1102-1108.[Abstract/Free Full Text]
    8. 8
  8. Fernandes, P. 2006. Antibacterial discovery and development—the failure of success? Nat. Biotechnol. 24:1497-1503.[CrossRef][Medline]
    9. 9
  9. Grissom-Arnold, J., W. E. Alborn, Jr., T. I. Nicas, and S. R. Jaskunas. 1997. Induction of VanA vancomycin resistance genes in Enterococcus faecalis: use of a promoter fusion to evaluate glycopeptide and nonglycopeptide induction signals. Microb. Drug Resist. 3:53-64.[Medline]
    10. 10
  10. Horsburgh, M. J., J. L. Aish, I. J. White, L. Shaw, J. K. Lithgow, and S. J. Foster. 2002. {sigma}B modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from Staphylococcus aureus 8325-4. J. Bacteriol. 184:5457-5467.[Abstract/Free Full Text]
    11. 11
  11. Hughes, D. 2003. Exploiting genomics, genetics and chemistry to combat antibiotic resistance. Nat. Rev. Genet. 4:432-441.[CrossRef][Medline]
    12. 12
  12. Ji, Y., B. Zhang, S. F. Van Horn, P. Warren, G. Woodnutt, M. K. Burnham, and M. Rosenberg. 2001. Identification of critical staphylococcal genes using conditional phenotypes generated by antisense RNA. Science 293:2266-2269.[Abstract/Free Full Text]
    13. 13
  13. Kennedy, A. D., M. Otto, K. R. Braughton, A. R. Whitney, L. Chen, B. Mathema, J. R. Mediavilla, K. A. Byrne, L. D. Parkins, F. C. Tenover, B. N. Kreiswirth, J. M. Musser, and F. R. DeLeo. 2008. Epidemic community-associated methicillin-resistant Staphylococcus aureus: recent clonal expansion and diversification. Proc. Natl. Acad. Sci. USA 105:1327-1332.[Abstract/Free Full Text]
    14. 14
  14. Klevens, R. M., M. A. Morrison, S. K. Fridkin, A. Reingold, S. Petit, K. Gershman, S. Ray, L. H. Harrison, R. Lynfield, G. Dumyati, J. M. Townes, A. S. Craig, G. Fosheim, L. K. McDougal, and F. C. Tenover. 2006. Community-associated methicillin-resistant Staphylococcus aureus and healthcare risk factors. Emerg. Infect. Dis. 12:1991-1993.[Medline]
    15. 15
  15. Kuroda, M., H. Kuroda, T. Oshima, F. Takeuchi, H. Mori, and K. Hiramatsu. 2003. Two-component system VraSR positively modulates the regulation of cell-wall biosynthesis pathway in Staphylococcus aureus. Mol. Microbiol. 49:807-821.[CrossRef][Medline]
    16. 16
  16. McAleese, F., S. W. Wu, K. Sieradzki, P. Dunman, E. Murphy, S. Projan, and A. Tomasz. 2006. Overexpression of genes of the cell wall stimulon in clinical isolates of Staphylococcus aureus exhibiting vancomycin-intermediate-S. aureus-type resistance to vancomycin. J. Bacteriol. 188:1120-1133.[Abstract/Free Full Text]
    17. 17
  17. McDevitt, D., and M. Rosenberg. 2001. Exploiting genomics to discover new antibiotics. Trends Microbiol. 9:611-617.[CrossRef][Medline]
    18. 18
  18. Miesel, L., J. Greene, and T. A. Black. 2003. Genetic strategies for antibacterial drug discovery. Nat. Rev. Genet. 4:442-456.[CrossRef][Medline]
    19. 19
  19. Miller, J. M. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
    20. 20
  20. Novick, R. P. 1991. Genetic systems in staphylococci. Methods Enzymol. 204:587-636.[Medline]
    21. 21
  21. O'Neill, A. J., K. Miller, B. Oliva, and I. Chopra. 2004. Comparison of assays for detection of agents causing membrane damage in Staphylococcus aureus. J. Antimicrob. Chemother. 54:1127-1129.[Abstract/Free Full Text]
    22. 22
  22. Pechous, R., N. Ledala, B. J. Wilkinson, and R. K. Jayaswal. 2004. Regulation of the expression of cell wall stress stimulon member gene msrA1 in methicillin-susceptible or -resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 48:3057-3063.[Abstract/Free Full Text]
    23. 23
  23. Projan, S. J. 2003. Why is big Pharma getting out of antibacterial drug discovery? Curr. Opin. Microbiol. 6:427-430.[CrossRef][Medline]
    24. 24
  24. Quesada, A. R., A. Canedo, M. A. Moreno, and J. L. Fernandez-Puentes. 1996. A microtitre plate-based assay for the screening of beta-lactams. Lett. Appl. Microbiol. 22:303-306.[Medline]
    25. 25
  25. Rosamond, J., and A. Allsop. 2000. Harnessing the power of the genome in the search for new antibiotics. Science 287:1973-1976.[Abstract/Free Full Text]
    26. 26
  26. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
    27. 27
  27. Singh, V. K., R. K. Jayaswal, and B. J. Wilkinson. 2001. Cell wall-active antibiotic induced proteins of Staphylococcus aureus identified using a proteomic approach. FEMS Microbiol. Lett. 199:79-84.[Medline]
    28. 28
  28. Sun, D., S. Cohen, N. Mani, C. Murphy, and D. M. Rothstein. 2002. A pathway-specific cell based screening system to detect bacterial cell wall inhibitors. J. Antibiot. (Tokyo) 55:279-287.[Medline]
    29. 29
  29. Ulijasz, A. T., A. Grenader, and B. Weisblum. 1996. A vancomycin-inducible LacZ reporter system in Bacillus subtilis: induction by antibiotics that inhibit cell wall synthesis and by lysozyme. J. Bacteriol. 178:6305-6309.[Abstract/Free Full Text]
    30. 30
  30. Utaida, S., P. M. Dunman, D. Macapagal, E. Murphy, S. J. Projan, V. K. Singh, R. K. Jayaswal, and B. J. Wilkinson. 2003. Genome-wide transcriptional profiling of the response of Staphylococcus aureus to cell-wall-active antibiotics reveals a cell-wall-stress stimulon. Microbiology 149:2719-2732.[Abstract/Free Full Text]
    31. 31
  31. Van Dyk, T. K., Y. Wei, M. K. Hanafey, M. Dolan, M. J. Reeve, J. A. Rafalski, L. B. Rothman-Denes, and R. A. LaRossa. 2001. A genomic approach to gene fusion technology. Proc. Natl. Acad. Sci. USA 98:2555-2560.[Abstract/Free Full Text]
    32. 32
  32. Wang, Q. M., R. B. Peery, R. B. Johnson, W. E. Alborn, W.-K. Yeh, and P. L. Skatrud. 2001. Identification and characterization of a monofunctional glycosyltransferase from Staphylococcus aureus. J. Bacteriol. 183:4779-4785.[Abstract/Free Full Text]
    33. 33
  33. Wilkinson, B. J., A. Muthaiyan, and R. K. Jayaswal. 2005. The cell wall stress stimulon of Staphylococcus aureus and other gram-positive bacteria. Curr. Med. Chem. Anti-Infect. Agents 4:259-276.[CrossRef]

Antimicrobial Agents and Chemotherapy, August 2008, p. 2923-2925, Vol. 52, No. 8
0066-4804/08/$08.00+0     doi:10.1128/AAC.00273-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Steidl, R.
Right arrow Articles by Jayaswal, R. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Steidl, R.
Right arrow Articles by Jayaswal, R. K.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»