Identification of Distinct Bacillus thuringiensis 4A4 Nematicidal Factors Using the Model Nematodes Pristionchus pacificus and Caenorhabditis elegans

Bacillus thuringiensis has been extensively used for the biological control of insect pests. Nematicidal B. thuringiensis strains have also been identified; however, virulence factors of such strains are poorly investigated. Here, we describe virulence factors of the nematicidal B. thuringiensis 4A4 strain, using the model nematodes Pristionchus pacificus and Caenorhabditis elegans. We show that B. thuringiensis 4A4 kills both nematodes via intestinal damage. Whole genome sequencing of B. thuringiensis 4A4 identified Cry21Ha, Cry1Ba, Vip1/Vip2 and β-exotoxin as potential nematicidal factors. Only Cry21Ha showed toxicity to C. elegans, while neither Cry nor Vip toxins were active against P. pacificus, when expressed in E. coli. Purified crystals also failed to intoxicate P. pacificus, while autoclaved spore-crystal mixture of B. thuringiensis 4A4 retained toxicity, suggesting that primary β-exotoxin is responsible for P. pacificus killing. In support of this, we found that a β-exotoxin-deficient variant of B. thuringiensis 4A4, generated by plasmid curing lost virulence to the nematodes. Thus, using two model nematodes we revealed virulence factors of the nematicidal strain B. thuringiensis 4A4 and showed the multifactorial nature of its virulence.

[1]  E. Ben-Dov,et al.  Bacillus thuringiensis subsp. israelensis and Its Dipteran-Specific Toxins , 2014, Toxins.

[2]  R. Sommer,et al.  Bacillus thuringiensis DB27 Produces Two Novel Protoxins, Cry21Fa1 and Cry21Ha1, Which Act Synergistically against Nematodes , 2014, Applied and Environmental Microbiology.

[3]  R. Sommer,et al.  Draft Genome Sequence of Highly Nematicidal Bacillus thuringiensis DB27 , 2014, Genome Announcements.

[4]  R. Sommer,et al.  New Role for DCR-1/Dicer in Caenorhabditis elegans Innate Immunity against the Highly Virulent Bacterium Bacillus thuringiensis DB27 , 2013, Infection and Immunity.

[5]  R. Sommer,et al.  The nematode Pristionchus pacificus as a model system for integrative studies in evolutionary biology , 2013, Molecular ecology.

[6]  M. Soberón,et al.  Cyt toxins produced by Bacillus thuringiensis: A protein fold conserved in several pathogenic microorganisms , 2013, Peptides.

[7]  Isabel Gómez,et al.  Evolution of Bacillus thuringiensis Cry toxins insecticidal activity , 2013, Microbial biotechnology.

[8]  M. Soberón,et al.  Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection. , 2013, FEMS microbiology reviews.

[9]  R. Sommer,et al.  Structure and glycolipid binding properties of the nematicidal protein Cry5B. , 2012, Biochemistry.

[10]  Ming Sun,et al.  Bacillus thuringiensis Metalloproteinase Bmp1 Functions as a Nematicidal Virulence Factor , 2012, Applied and Environmental Microbiology.

[11]  R. Sommer,et al.  System Wide Analysis of the Evolution of Innate Immunity in the Nematode Model Species Caenorhabditis elegans and Pristionchus pacificus , 2012, PloS one.

[12]  R. Sommer,et al.  Genome-Wide Analysis of Germline Signaling Genes Regulating Longevity and Innate Immunity in the Nematode Pristionchus pacificus , 2012, PLoS pathogens.

[13]  R. Sommer,et al.  The importance of being regular: Caenorhabditis elegans and Pristionchus pacificus defecation mutants are hypersusceptible to bacterial pathogens. , 2012, International journal for parasitology.

[14]  Ming Sun,et al.  Mining New Crystal Protein Genes from Bacillus thuringiensis on the Basis of Mixed Plasmid-Enriched Genome Sequencing and a Computational Pipeline , 2012, Applied and Environmental Microbiology.

[15]  S. Gill,et al.  Bacillus thuringiensis: A story of a successful bioinsecticide. , 2011, Insect biochemistry and molecular biology.

[16]  J. Schwartz,et al.  Global Functional Analyses of Cellular Responses to Pore-Forming Toxins , 2011, PLoS pathogens.

[17]  R. Sommer,et al.  A subset of naturally isolated Bacillus strains show extreme virulence to the free-living nematodes Caenorhabditis elegans and Pristionchus pacificus. , 2010, Environmental microbiology.

[18]  Ming Sun,et al.  Genome-wide Screening Reveals the Genetic Determinants of an Antibiotic Insecticide in Bacillus thuringiensis* , 2010, The Journal of Biological Chemistry.

[19]  B. Raymond,et al.  Bacillus thuringiensis: an impotent pathogen? , 2010, Trends in microbiology.

[20]  M. Soberón,et al.  Pore formation by Cry toxins. , 2010, Advances in experimental medicine and biology.

[21]  C. S. Hernández-Rodríguez,et al.  Screening and identification of vip genes in Bacillus thuringiensis strains , 2009, Journal of applied microbiology.

[22]  Ziniu Yu,et al.  New Strategy for Isolating Novel Nematicidal Crystal Protein Genes from Bacillus thuringiensis Strain YBT-1518 , 2008, Applied and Environmental Microbiology.

[23]  R. Sommer,et al.  Isolation of naturally associated bacteria of necromenic Pristionchus nematodes and fitness consequences , 2008, Journal of Experimental Biology.

[24]  H. Schulenburg,et al.  The genetics of pathogen avoidance in Caenorhabditis elegans , 2007, Molecular microbiology.

[25]  H. Schulenburg,et al.  The role of Caenorhabditis elegans insulin‐like signaling in the behavioral avoidance of pathogenic Bacillus thuringiensis , 2007, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[26]  S. Gill,et al.  Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. , 2007, Toxicon : official journal of the International Society on Toxinology.

[27]  Danielle L Huffman,et al.  Assays for toxicity studies in C. elegans with Bt crystal proteins. , 2006, Methods in molecular biology.

[28]  Cornelia I. Bargmann,et al.  Pathogenic bacteria induce aversive olfactory learning in Caenorhabditis elegans , 2005, Nature.

[29]  A. Dell,et al.  Glycolipids as Receptors for Bacillus thuringiensis Crystal Toxin , 2005, Science.

[30]  Cynthie Wong,et al.  Bacillus thuringiensis crystal proteins that target nematodes , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[31]  M. Porcar,et al.  Correlation between serovars of Bacillus thuringiensis and type I beta-exotoxin production. , 2003, Journal of invertebrate pathology.

[32]  V. Sanchis,et al.  Correspondence of High Levels of Beta-Exotoxin I and the Presence of cry1B in Bacillus thuringiensis , 2002, Applied and Environmental Microbiology.

[33]  A. Khan,et al.  Usefulness of staining parasporal bodies when screening for Bacillus thuringiensis. , 2002, Journal of invertebrate pathology.

[34]  R. D. de Maagd,et al.  How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. , 2001, Trends in genetics : TIG.

[35]  L. Marroquin,et al.  Bacillus thuringiensis (Bt) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans. , 2000, Genetics.

[36]  G. W. Warren.  Vegetative Insecticidal Proteins: Novel Proteins for Control of Corn Pests , 1997 .

[37]  M. Koziel,et al.  Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[38]  R. Sommer,et al.  Apoptosis and change of competence limit the size of the vulva equivalence group in Pristionchus pacificus: a genetic analysis , 1996, Current Biology.

[39]  A. Coomans,et al.  Effect of nematicidal Bacillus thuringiensis strains on free-living nematodes .1. Light microscopic observations, species and biological stage specificity and identification of resistant mutants of Caenorhabditis elegans , 1996 .