A Bacillus thuringiensis S-Layer Protein Involved in Toxicity against Epilachna varivestis (Coleoptera: Coccinellidae)

Abstract

The use of Bacillus thuringiensis as a biopesticide is a viable alternative for insect control since the insecticidal Cry proteins produced by these bacteria are highly specific; harmless to humans, vertebrates, and plants; and completely biodegradable. In addition to Cry proteins, B. thuringiensis produces a number of extracellular compounds, including S-layer proteins (SLP), that contribute to virulence. The S layer is an ordered structure representing a proteinaceous paracrystalline array which completely covers the surfaces of many pathogenic bacteria. In this work, we report the identification of an S-layer protein by the screening of B. thuringiensis strains for activity against the coleopteran pest Epilachna varivestis (Mexican bean beetle; Coleoptera: Coccinellidae). We screened two B. thuringiensis strain collections containing unidentified Cry proteins and also strains isolated from dead insects. Some of the B. thuringiensis strains assayed against E. varivestis showed moderate toxicity. However, a B. thuringiensis strain (GP1) that was isolated from a dead insect showed a remarkably high insecticidal activity. The parasporal crystal produced by the GP1 strain was purified and shown to have insecticidal activity against E. varivestis but not against the lepidopteran Manduca sexta or Spodoptera frugiperda or against the dipteran Aedes aegypti. The gene encoding this protein was cloned and sequenced. It corresponded to an S-layer protein highly similar to previously described SLP in Bacillus anthracis (EA1) and Bacillus licheniformis (OlpA). The phylogenetic relationships among SLP from different bacteria showed that these proteins from Bacillus cereus, Bacillus sphaericus, B. anthracis, B. licheniformis, and B. thuringiensis are arranged in the same main group, suggesting similar origins. This is the first report that demonstrates that an S-layer protein is directly involved in toxicity to a coleopteran pest.

ABacillus anthracisS-Layer Homology Protein That Binds Heme and Mediates Heme Delivery to IsdC

“…The finding that BslK also contains a NEAT domain prompted an investigation into whether this potential surface protein partakes in heme binding and transfer. Treatment of B. anthracis cells expressing a His-tagged form of BslK with guanidine led to the release of BslK H6 into the supernatant, a finding consistent with the extraction of noncovalently associated S-layer proteins from the bacterial surface as reported by others (32,42,53,63,70). Immunofluorescence microscopy using Texas Red-conjugated anti-hexahistidyl antibodies also indicated that BslK H6 localized to the bacterial surface (Y. Tarlovsky and A. W. Maresso, unpublished observations).…”

“…To determine if cell-associated BslK H6 is a surface protein, we treated BslK H6 -expressing cells with guanidine-HCl (GN), a chaotropic agent known to induce the release of noncovalently attached cell envelope proteins such as S-layer polypeptides from the surface of bacteria (42,63,70). B. anthracis cells were grown in the presence of IPTG, and bacterial sediments were treated with 4 M guanidine for 5 min.…”

Proteomic Analysis of Bacillus thuringiensis at Different Growth Phases by Using an Automated Online Two-Dimensional Liquid Chromatography-Tandem Mass Spectrometry Strategy

“…Cell adhesion is the major role of SLPs. An S-layer protein coming from B. thuringiensis GP1 strains has also been reported to have high insecticidal activity against the coleopteran pest Epilachna varivestis (38). In the present article, except for the SLPs that may play roles in cell integrity and shape maintenance or cell adhesion, we identified an S-layer protein, EA1 (Q63FB8), which belongs to B. cereus.…”

Phylogenetic Analysis and Heterologous Expression of Surface Layer Protein SlpC ofBacillus sphaericusC3-41

“…3,4) S-layers play key roles in the interaction between bacterial cells and environment as protective coats, structures involved in cell adhesion and surface recognition, molecular sieves, attachment structures for high-molecular-mass extracellular proteins such as amylases, and in the sorption of toxic heavy metal ions. [5][6][7] Furthermore, it has been indicated that the S-layer might act as a virulence factor in several pathogenic bacteria, such as the S-layer protein produced by Bacillus thuringiensis GP1, which shows insecticidal activity against the coleopteran Epilachna varivestis, 8) and the S-layer of Lactobacillus acidophilus which mediates the adherence of the strains to avian intestinal epithelial cells. 9) B. sphaericus is a naturally occurring gram-positive soil bacterium that represents a strictly aerobic group of mesophilic endospore-forming bacteria.…”