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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wichroski, M. J.
Right arrow Articles by Ward, G. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wichroski, M. J.
Right arrow Articles by Ward, G. E.

 Previous Article

Eukaryotic Cell, October 2003, p. 1132-1136, Vol. 2, No. 5
1535-9778/03/$08.00+0     DOI: 10.1128/EC.2.5.1132-1136.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Biosynthesis of Glycosylphosphatidylinositol Is Essential to the Survival of the Protozoan Parasite Toxoplasma gondii

Michael J. Wichroski and Gary E. Ward*

Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405

Received 7 April 2003/ Accepted 7 August 2003


arrow
ABSTRACT
 
The PIGA gene from Toxoplasma gondii has been cloned and characterized. Like mammalian PIGA, the transmembrane and C-terminal domains are sufficient to direct localization to the parasite endoplasmic reticulum. A functional copy of PIGA is required for tachyzoite viability, demonstrating that glycosylphosphatidylinositol biosynthesis is an essential process in T. gondii.


arrow
TEXT
 
Glycosylphosphatidylinositol (GPI)-anchored proteins dominate the surface of the Toxoplasma gondii tachyzoite (3, 19) and have been implicated in both host cell attachment and modulation of the host immune response (7, 15, 19). To further our understanding of the synthesis, trafficking, and function(s) of GPIs and GPI-anchored proteins in T. gondii, we have begun to characterize the genes involved in the parasite's GPI biosynthetic pathway.

GPI biosynthesis is a conserved pathway among eukaryotes that occurs primarily in the endoplasmic reticulum (ER) and leads to the generation of both free GPIs and GPI-anchored proteins (reviewed in references 8, 9, 13, 17, and 21). In mammalian cells, the pathway is initiated on the cytosolic face of the ER with the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to phosphatidylinositol (PI) by the GPI-GlcNAc transferase complex (29-32). The GlcNAc transferase activity of the complex is thought to reside in the PI-glycan class A (PIGA) protein (18, 29). Loss-of-function mutations in PIGA result in a complete GPI deficiency, which consequently abolishes the surface expression of GPI-anchored proteins (reviewed in reference 2). The ability to tolerate a GPI deficiency is species specific and sometimes even life cycle stage specific (2, 10, 12, 14, 20, 22-25).

The complete PIGA cDNA of T. gondii strain RH(EP) (GenBank accession no. AY216495) was obtained and sequenced (using primers designed from an expressed sequence tag [EST1206547; http://ParaDB.cis.upenn.edu] that exhibited homology to the C-terminal region of multiple PIGA orthologues) from products of 5' and 3' rapid amplification of cDNA ends. Subsequent sequencing and characterization of the RH(EP) PIGA gene (GenBank accession no. AY216496) showed it to be a relatively large (~10 kb) single-copy gene harboring 11 introns. The predicted protein sequence of T. gondii PIGA (616 amino acids) exhibits significant similarity to sequences of other PIGA orthologues (Fig. 1), particularly between residues 22 and 383 (~50% identity and ~70% homology), a region which contains the putative GlcNAc transferase domain. As with other PIGA proteins, T. gondii PIGA harbors a potential transmembrane domain (residues 517 to 539) followed by a stretch of mostly hydrophilic residues (residues 541 to 616) extending to the C terminus (Fig. 1). T. gondii PIGA contains an insert of ~100 amino acids not found in other PIGA orthologues (residues 384 to 506; Fig. 1). This insert, which is also present in both genomic (http://toxodb.org/ToxoDB.shtml) and cDNA (data not shown) sequences of PIGA of T. gondii strain P(LK), exhibits no homology to any known proteins. Its functional significance, if any, is unknown.



View larger version (99K):
[in this window]
[in a new window]
  		FIG. 1. ClustalW sequence alignment of PIGA orthologues from T. gondii, Plasmodium falciparum (gi23495183), Arabidopsis thaliana (gi18407913), Homo sapiens (gi219994), Mus musculus (gi1402592), Paramecium tetraurelia (gi8571458), and Saccharomyces cerevisiae (gi9755344). Regions of homology are boxed, with identity denoted by dark shading and conserved amino acid changes denoted by light shading. The predicted T. gondii PIGA transmembrane domain (amino acids 517 to 539) is denoted by a thick horizontal line.

Stably expressed recombinant versions of T. gondii PIGA, containing either an N-terminal c-Myc epitope tag or a C-terminal green fluorescent protein (GFP) fusion, localized predominantly to ER-like structures encircling the nucleus of the tachyzoite (Fig. 2A and B). When transiently expressed, an engineered ER marker (Trypanosoma brucei GPI-phospholipase C [GPI-PLC] containing a secretory signal sequence and ER retention signal [secGPI-PLCHDEL]) localized to a perinuclear compartment (Fig. 2E) indistinguishable from that of PIGA.



View larger version (71K):
[in this window]
[in a new window]
  		FIG. 2. Subcellular localization of T. gondii PIGA. PIGA with a C-terminal fusion to GFP (A) or an N-terminal c-Myc epitope tag (B) localized to ER-like structures encircling the nucleus of the parasite. As a marker for ER localization, a recombinant version of T. brucei GPI-PLC, which normally localized to the periphery of the tachyzoite (C), was engineered to localize to the ER (secGPI-PLCHDEL) by the addition of an N-terminal secretory signal sequence and the C-terminal ER-retention motif, HDEL (E). When fused to the C terminus of GPI-PLC (GPI-PLCPIGA517-616), the putative transmembrane and ER-lumenal domains (amino acids 517 to 616) of T. gondii PIGA were sufficient to alter the localization of GPI-PLC to an ER-like localization (D) indistinguishable from that of PIGA. Bar, 5 µm. M, c=Myc; SS, signal sequence; H, HDEL; TM, transmembrane; DIC, differential interference contrast.

Previous studies have shown that the transmembrane domain of mammalian PIGA, together with the 23 residues immediately C terminal to this domain, is sufficient for ER targeting and/or retention (31). Like its mammalian counterpart, T. gondii PIGA lacks an obvious N-terminal signal sequence but appears to localize to the ER. To determine whether the transmembrane and C-terminal domains of T. gondii PIGA direct localization, residues 517 to 616 were fused to the C terminus of GPI-PLC (GPI-PLCPIGA517-616). In transient-expression studies, GPI-PLCPIGA517-616 exhibited a localization pattern similar to that of PIGA (Fig. 2D) and strikingly different from the peripheral localization of GPI-PLC lacking this domain (Fig. 2C), indicating that the signals required for ER targeting of T. gondii PIGA are also located within the transmembrane and C-terminal domains. Heterologously expressed GPI-PLC can induce a GPI deficiency in other parasites through the cleavage of GPI intermediates (10, 22). Stable expression of wild-type GPI-PLC exhibited little effect on tachyzoites (data not shown); however, it was not possible to generate clones stably expressing GPI-PLC targeted to the ER, suggesting that GPI-PLC expression at the site of GPI biosynthesis was lethal.

When chemical mutagenesis (27), insertional mutagenesis (6), and targeted gene disruption of PIGA, each coupled with selection (using Clostridium septicum alpha toxin) (11, 33) for a GPI-anchored protein deficiency, were used to attempt to generate GPI-deficient parasites, the results were uniformly unsuccessful, suggesting that GPI biosynthesis is required for tachyzoite viability. To show this conclusively, we attempted to disrupt PIGA in the presence of a second copy of the gene. A knockout vector (pHXGPRT/{Delta}PIGA5kb) was designed such that integration within the region located upstream of the internal deletion (type I) would result in a pseudodiploid (5, 26, 28) harboring two nonfunctional PIGA alleles (Fig. 3A).



View larger version (48K):
[in this window]
[in a new window]
  		FIG. 3. Targeted disruption of PIGA. (A) Schematic of the gene disruption strategy. The pHXGPRT/{Delta}PIGA5kb knockout vector was constructed such that single-crossover homologous recombination into the PIGA locus upstream of the deletion would generate a type I pseudodiploid with two nonfunctional PIGA alleles. To generate {Delta}PIGA5kb, the 5' end of PIGA was truncated within exon 1 (removing the 5' untranslated region and coding sequence corresponding to residues 1 to 47) and the 3' end was truncated into exon 11 (removing the coding sequence for the putative transmembrane and ER-lumenal domains [residues 519 to 616]). An internal sequence corresponding to a highly conserved region of PIGA (amino acids 303 to 368) was also deleted, and stop codons were engineered into all cloning junctions within the {Delta}PIGA5kb allele. Type I integration resulted in the harboring by the pairs upstream allele of the ~1.8-kb deletion (marked by {Delta}; large shaded box indicates the corresponding sequence in the wild-type allele), which could be detected by PCR (using primer set P) as a product of ~4.6 kb. The P primer set was found to be incapable of amplifying the predicted product (~6.5 kb) from wild-type genomic DNA. (B) Six independent stable transgenic populations (lane pairs 1 to 6) derived from either parental parasites (RH{Delta}HXGPRT) or parasites harboring a stable second copy of PIGA (RHPIGA-GFP) were screened for integration of the plasmid into PIGA (see text and elsewhere in this legend). {Delta}PIGA type I pseudodiploids were not detected in any of the six populations derived from parental parasites, while four out of six populations from RHPIGA-GFP parasites yielded a positive PCR product result (+). Lanes C, control primers; lanes P, pseudodiploid-specific primers; lanes M, molecular mass markers.

Parental [RH(EP){Delta}HXGPRT) (26, 28)] and RH(EP){Delta}HXGPRT/PIGA-GFP (referred to hereafter as RHPIGA-GFP) parasites were each transfected (16, 28) with undigested pHXGPRT/{Delta}PIGA5kb in six independent experiments. Stable expression of PIGA-GFP was confirmed by both immunofluorescence (Fig. 2A) and Western blot (data not shown) experiments. A forward primer directed against the 5' untranslated region of PIGA (which was not present in {Delta}PIGA5kb) and a reverse primer located directly downstream of the deleted region (primer set P) (Fig. 3A) were used for PCR to screen stable transgenic populations for the presence of type I {Delta}PIGA pseudodiploids. These primers yield a product of ~4.6 kb that is pseudodiploid specific, since amplification through the deleted region was not possible using genomic DNA as a template (Fig. 3B). As a control, the same upstream primer and a reverse primer located immediately upstream of the deleted region in the {Delta}PIGA5kb allele (primer set C) were used to amplify a product of ~4.6 kb from both the wild-type and pseudodiploid PIGA alleles (Fig. 3A). Type I {Delta}PIGA pseudodiploids were not detected in any of the six independent populations from the parental parasites. However, they were present in four of the six populations derived from RHPIGA-GFP parasites (Fig. 3B), indicating that a second copy of PIGA was sufficient to rescue the parasite upon disruption of the wild-type gene. Integration into the genomic locus was confirmed both by Southern blot analysis of independent clones and by the absence of a reverse transcription-PCR product corresponding to the PIGA transcript in these clones (data not shown).

These results demonstrate that GPI biosynthesis is essential for viability in T. gondii, and they identify the GPI biosynthetic pathway as a potential target for the development of new chemotherapeutics against this parasite. It is not clear whether the lethal consequences of PIGA disruption in T. gondii result from a deficiency in GPI-anchored proteins, free GPIs, or both. Future experiments aimed at disrupting components of the T. gondii GPI transamidase complex (1, 4, 12, 20, 34) should resolve this question and reveal the relative importance of GPI-anchored proteins in the T. gondii life cycle.


arrow
ACKNOWLEDGMENTS
 
We thank Kojo Mensa-Wilmot for helpful advice and for generously providing GPI-PLC constructs and antibodies. We also thank Dominique Soldati, Con Beckers, David Sibley, and Michael White for providing constructs, David Roos for helpful advice, and Mary Tierney, Doug Johnson, and members of the laboratory for critical reading of the manuscript.

This work was supported by PHS grants AI42355 (G.E.W.) and CA22435 (Vermont Cancer Center) and through the Vermont EPSCoR program under NSF grant EPS-9874685.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, 316 Stafford Hall, University of Vermont, Burlington, VT 05405. Phone: (802) 656-4868. Fax: (802) 656-8749. E-mail: gward{at}zoo.uvm.edu. Back


arrow
REFERENCES
 
    1
  1. Benghezal, M., A. Benachour, S. Rusconi, M. Aebi, and A. Conzelmann. 1996. Yeast Gpi8p is essential for GPI anchor attachment onto proteins. EMBO J. 15:6575-6583.[Medline]
    2. 2
  2. Bessler, M., A. Schaefer, and P. Keller. 2001. Paroxysmal nocturnal hemoglobinuria: insights from recent advances in molecular biology. Transfus. Med. Rev. 15:255-267.[Medline]
    3. 3
  3. Black, M. W., and J. C. Boothroyd. 2000. Lytic cycle of Toxoplasma gondii. Microbiol. Mol. Biol. Rev. 64:607-623.[Abstract/Free Full Text]
    4. 4
  4. Chen, R., S. Udenfriend, G. M. Prince, S. E. Maxwell, S. Ramalingam, L. D. Gerber, J. Knez, and M. E. Medof. 1996. A defect in glycosylphosphatidylinositol (GPI) transamidase activity in mutant K cells is responsible for their inability to display GPI surface proteins. Proc. Natl. Acad. Sci. USA 93:2280-2284.[Abstract/Free Full Text]
    5. 5
  5. Donald, R. G., and D. S. Roos. 1998. Gene knock-outs and allelic replacements in Toxoplasma gondii: HXGPRT as a selectable marker for hit-and-run mutagenesis. Mol. Biochem. Parasitol. 91:295-305.[CrossRef][Medline]
    6. 6
  6. Donald, R. G., and D. S. Roos. 1995. Insertional mutagenesis and marker rescue in a protozoan parasite: cloning of the uracil phosphoribosyltransferase locus from Toxoplasma gondii. Proc. Natl. Acad. Sci. USA 92:5749-5753.[Abstract/Free Full Text]
    7. 7
  7. Dzierszinski, F., M. Mortuaire, M. F. Cesbron-Delauw, and S. Tomavo. 2000. Targeted disruption of the glycosylphosphatidylinositol-anchored surface antigen SAG3 gene in Toxoplasma gondii decreases host cell adhesion and drastically reduces virulence in mice. Mol. Microbiol. 37:574-582.[CrossRef][Medline]
    8. 8
  8. Ferguson, M. A. 1999. The structure, biosynthesis and functions of glycosylphosphatidylinositol anchors, and the contributions of trypanosome research. J. Cell Sci. 112:2799-2809.[Abstract]
    9. 9
  9. Ferguson, M. A., J. S. Brimacombe, S. Cottaz, R. A. Field, L. S. Guther, S. W. Homans, M. J. McConville, A. Mehlert, K. G. Milne, J. E. Ralton, et al. 1994. Glycosyl-phosphatidylinositol molecules of the parasite and the host. Parasitology 108:S45-S54.
    10. 10
  10. Garg, N., M. Postan, K. Mensa-Wilmot, and R. L. Tarleton. 1997. Glycosylphosphatidylinositols are required for the development of Trypanosoma cruzi amastigotes. Infect. Immun. 65:4055-4060.[Abstract]
    11. 11
  11. Gordon, V. M., K. L. Nelson, J. T. Buckley, V. L. Stevens, R. K. Tweten, P. C. Elwood, and S. H. Leppla. 1999. Clostridium septicum alpha toxin uses glycosylphosphatidylinositol-anchored protein receptors. J. Biol. Chem. 274:27274-27280.[Abstract/Free Full Text]
    12. 12
  12. Hilley, J. D., J. L. Zawadzki, M. J. McConville, G. H. Coombs, and J. C. Mottram. 2000. Leishmania mexicana mutants lacking glycosylphosphatidylinositol (GPI):protein transamidase provide insights into the biosynthesis and functions of GPI-anchored proteins. Mol. Biol. Cell 11:1183-1195.[Abstract/Free Full Text]
    13. 13
  13. Ikezawa, H. 2002. Glycosylphosphatidylinositol (GPI)-anchored proteins. Biol. Pharm. Bull. 25:409-417.[CrossRef][Medline]
    14. 14
  14. Ilgoutz, S. C., J. L. Zawadzki, J. E. Ralton, and M. J. McConville. 1999. Evidence that free GPI glycolipids are essential for growth of Leishmania mexicana. EMBO J. 18:2746-2755.[CrossRef][Medline]
    15. 15
  15. Jacquet, A., L. Coulon, J. De Neve, V. Daminet, M. Haumont, L. Garcia, A. Bollen, M. Jurado, and R. Biemans. 2001. The surface antigen SAG3 mediates the attachment of Toxoplasma gondii to cell-surface proteoglycans. Mol. Biochem. Parasitol. 116:35-44.[CrossRef][Medline]
    16. 16
  16. Karsten, V., H. Qi, C. J. Beckers, and K. A. Joiner. 1997. Targeting the secretory pathway of Toxoplasma gondii. Methods 13:103-111.[CrossRef][Medline]
    17. 17
  17. Kinoshita, T., K. Ohishi, and J. Takeda. 1997. GPI-anchor synthesis in mammalian cells: genes, their products, and a deficiency. J. Biochem. (Tokyo) 122:251-257.[Abstract/Free Full Text]
    18. 18
  18. Kostova, Z., D. M. Rancour, A. K. Menon, and P. Orlean. 2000. Photoaffinity labelling with P3-(4-azidoanilido)uridine 5'-triphosphate identifies gpi3p as the UDP-GlcNAc-binding subunit of the enzyme that catalyses formation of GlcNAc-phosphatidylinositol, the first glycolipid intermediate in glycosylphosphatidylinositol synthesis. Biochem. J. 350:815-822.
    19. 19
  19. Lekutis, C., D. J. Ferguson, M. E. Grigg, M. Camps, and J. C. Boothroyd. 2001. Surface antigens of Toxoplasma gondii: variations on a theme. Int. J. Parasitol. 31:1285-1292.[CrossRef][Medline]
    20. 20
  20. Lillico, S., M. C. Field, P. Blundell, G. H. Coombs, and J. C. Mottram. 2003. Essential roles for GPI-anchored proteins in African trypanosomes revealed using mutants deficient in GPI8. Mol. Biol. Cell 14:1182-1194.[Abstract/Free Full Text]
    21. 21
  21. McConville, M. J., and A. K. Menon. 2000. Recent developments in the cell biology and biochemistry of glycosylphosphatidylinositol lipids (review). Mol. Membr. Biol. 17:1-16.[CrossRef][Medline]
    22. 22
  22. Mensa-Wilmot, K., J. H. LeBowitz, K. P. Chang, A. al-Qahtani, B. S. McGwire, S. Tucker, and J. C. Morris. 1994. A glycosylphosphatidylinositol (GPI)-negative phenotype produced in Leishmania major by GPI phospholipase C from Trypanosoma brucei: topography of two GPI pathways. J. Cell Biol. 124:935-947.[Abstract/Free Full Text]
    23. 23
  23. Nagamune, K., T. Nozaki, Y. Maeda, K. Ohishi, T. Fukuma, T. Hara, R. T. Schwarz, C. Sutterlin, R. Brun, H. Riezman, and T. Kinoshita. 2000. Critical roles of glycosylphosphatidylinositol for Trypanosoma brucei. Proc. Natl. Acad. Sci. USA 97:10336-10341.[Abstract/Free Full Text]
    24. 24
  24. Naik, R. S., E. A. Davidson, and D. C. Gowda. 2000. Developmental stage-specific biosynthesis of glycosylphosphatidylinositol anchors in intraerythrocytic Plasmodium falciparum and its inhibition in a novel manner by mannosamine. J. Biol. Chem. 275:24506-24511.[Abstract/Free Full Text]
    25. 25
  25. Naik, R. S., G. Krishnegowda, and D. C. Gowda. 2003. Glucosamine inhibits inositol acylation of the glycosylphosphatidylinositol anchors in intraerythrocytic Plasmodium falciparum. J. Biol. Chem. 278:2036-2042.[Abstract/Free Full Text]
    26. 26
  26. Pfefferkorn, E. R., and S. E. Borotz. 1994. Toxoplasma gondii: characterization of a mutant resistant to 6-thioxanthine. Exp. Parasitol. 79:374-382.[CrossRef][Medline]
    27. 27
  27. Pfefferkorn, E. R., and L. C. Pfefferkorn. 1979. Quantitative studies of the mutagenesis of Toxoplasma gondii. J. Parasitol. 65:364-370.[CrossRef][Medline]
    28. 28
  28. Roos, D. S., R. G. Donald, N. S. Morrissette, and A. L. Moulton. 1994. Molecular tools for genetic dissection of the protozoan parasite Toxoplasma gondii. Methods Cell Biol. 45:27-63.[Medline]
    29. 29
  29. Tiede, A., C. Nischan, J. Schubert, and R. E. Schmidt. 2000. Characterisation of the enzymatic complex for the first step in glycosylphosphatidylinositol biosynthesis. Int. J. Biochem. Cell Biol. 32:339-350.[CrossRef][Medline]
    30. 30
  30. Watanabe, R., N. Inoue, B. Westfall, C. H. Taron, P. Orlean, J. Takeda, and T. Kinoshita. 1998. The first step of glycosylphosphatidylinositol biosynthesis is mediated by a complex of PIG-A, PIG-H, PIG-C and GPI1. EMBO J. 17:877-885.[CrossRef][Medline]
    31. 31
  31. Watanabe, R., T. Kinoshita, R. Masaki, A. Yamamoto, J. Takeda, and N. Inoue. 1996. PIG-A and PIG-H, which participate in glycosylphosphatidylinositol anchor biosynthesis, form a protein complex in the endoplasmic reticulum. J. Biol. Chem. 271:26868-26875.[Abstract/Free Full Text]
    32. 32
  32. Watanabe, R., Y. Murakami, M. D. Marmor, N. Inoue, Y. Maeda, J. Hino, K. Kangawa, M. Julius, and T. Kinoshita. 2000. Initial enzyme for glycosylphosphatidylinositol biosynthesis requires PIG-P and is regulated by DPM2. EMBO J. 19:4402-4411.[CrossRef][Medline]
    33. 33
  33. Wichroski, M. J., J. A. Melton, C. G. Donahue, R. K. Tweten, and G. E. Ward. 2002. Clostridium septicum alpha-toxin is active against the parasitic protozoan Toxoplasma gondii and targets members of the SAG family of glycosylphosphatidylinositol-anchored surface proteins. Infect. Immun. 70:4353-4361.[Abstract/Free Full Text]
    34. 34
  34. Yu, J., S. Nagarajan, J. J. Knez, S. Udenfriend, R. Chen, and M. E. Medof. 1997. The affected gene underlying the class K glycosylphosphatidylinositol (GPI) surface protein defect codes for the GPI transamidase. Proc. Natl. Acad. Sci. USA 94:12580-12585.[Abstract/Free Full Text]

Eukaryotic Cell, October 2003, p. 1132-1136, Vol. 2, No. 5
1535-9778/03/$08.00+0     DOI: 10.1128/EC.2.5.1132-1136.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Leng, J., Butcher, B. A., Egan, C. E., Abi Abdallah, D. S., Denkers, E. Y. (2009). Toxoplasma gondii Prevents Chromatin Remodeling Initiated by TLR-Triggered Macrophage Activation. J. Immunol. 182: 489-497 [Abstract] [Full Text]  
  • Debierre-Grockiego, F., Campos, M. A., Azzouz, N., Schmidt, J., Bieker, U., Resende, M. G., Mansur, D. S., Weingart, R., Schmidt, R. R., Golenbock, D. T., Gazzinelli, R. T., Schwarz, R. T. (2007). Activation of TLR2 and TLR4 by Glycosylphosphatidylinositols Derived from Toxoplasma gondii. J. Immunol. 179: 1129-1137 [Abstract] [Full Text]  

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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wichroski, M. J.
Right arrow Articles by Ward, G. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wichroski, M. J.
Right arrow Articles by Ward, G. E.

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