A Novel Bacillus thuringiensis Cry-Like Protein from a Rare Filamentous Strain Promotes Crystal Localization within the Exosporium

Abstract

dMutation of a novel cry-like gene (cry256) from Bacillus thuringiensis resulted in a protein crystal, normally located within the spore's exosporium, being found predominately outside the exosporium. The cry256 gene codes for a 3-domain Cry-like protein that does not correspond to any of the known Cry protein holotypes. During sporulation, the bacterium Bacillus thuringiensis produces a protein crystal comprised of one or more members of the Cry family of protein toxins. The crystal is normally located outside the exosporium, which surrounds the spore (1); however, relatively few strains of B. thuringiensis have been shown to localize their crystals within the exosporium (2, 3). With the aim of understanding how some crystals are localized within the exosporium, Debro et al. showed that in B. thuringiensis subsp. finitimus, the genes necessary for localizing crystals within the exosporium where located on two plasmids (4). Zhu et al. later identified these genes (5) and showed that not all B. thuringiensis organisms used these genes to localize crystals within their exosporium. Building on this previous work, we undertook a study to identify genes responsible for crystal localization in a novel filamentous strain of B. thuringiensis called Bt2-56 ( Fig. 1A and A=) (6,14).Inactivation of gene cry256 causes loss of crystals from the exosporium. Genes in Bt2-56 were mutated by random transposon insertion using plasmid pMarA (7). Mutants with crystals located outside the exosporium were identified by viewing stained preparations of sporulated mutants (8). Approximately 84% of the crystals from a mutant called M1-11 were observed outside the exosporium (Fig. 1B and B=).Inverse PCR using primers left-tn1 and oIPCR2a (Table 1) was used to amplify M1-11 genomic DNA flanking the transposable element, indicating that transposons had integrated into both a histidine kinase gene and a cry-like gene that we call cry256. The DNA sequence of both strands of the entire cry256 gene and its immediate flanking regions was then determined by "walking" the gene, using primers designed from previously sequenced DNA regions (GenBank accession number JQ670887). The DNA sequence of cry256 from Bt2-56 (the unmutated parent strain of M1-11) was also determined, using an Illumina GAIIx sequencer at the University of Houston's IMD Sequencing Center. The 36-bp sequencing reads were assembled using Velvet (9) and visualized with the software program Tablet (10). DNA translations were performed using a Web-based DNA-to-protein tool (11; the Web application utilized is located at http://insilico.ehu.es /translate/). The transposon was found to have inserted into a region that separated the last 49 amino acids of the protein encoded by cry256 from the rest of protein (see file S1 in the supplemental material [the insertion site highlighted in red at position 1528]). Aside from the transposon, the DNA sequence of cry256 in mutant M1-11 was identical to that obtained for the wild-type cry256 gene in Bt2-56.Since the cause-and-effect relationship ...

Bacillus thuringiensis DB27 Produces Two Novel Protoxins, Cry21Fa1 and Cry21Ha1, Which Act Synergistically against Nematodes

“…SEM images confirmed this phenotype (Fig. 2E) and showed striking similarity to the SCA of a rare filamentous B. thuringiensis strain (26). In contrast to wild-type B. thuringiensis DB27, the plasmid-cured variant did not produce any Cry protein crystals, as shown in light microscopy ( Fig.…”

“…This phenotype has been previously described as spore-crystal association (SCA) (25). While many studies have concentrated on identification of genes responsible for this phenotype (25,26,44), the functional significance of SCA is not yet clear. Considering that Cry proteins are not stable in the environment, the exosporium may be used as a protective membrane.…”

Surviving Between Hosts: Sporulation and Transmission

“…B. thuringiensis produces an insecticidal crystal toxin during sporulation and under the control of the sporulation transcription factors that direct mother cell gene expression (σ E and/or σ K ) (116). The crystal is typically in close association with the spore, and can be underneath or outside of the exosporium (117)(118)(119). Unlike what usually results from the disease caused by the C. perfringens enterotoxin, B. thuringiensis kills its insect host and then replicates in the cadaver.…”

The stationary phase regulator CpcR activates cry gene expression in non‐sporulating cells of Bacillus thuringiensis

“…The crystal inclusion is generally formed in the mother cell compartment of the bacteria during sporulation or stationary phase (Deng, Peng, Song, & Lereclus, ). However, different situations have been observed in a few strains: (i) a crystal formed between the exosporium and spore coat (Ammons et al, ; Debro, Fitz‐James, & Aronson, ; Zhu et al, ) and (ii) the production of spores and crystals in two distinct subpopulations of bacterial cells (Deng et al, ; Srinivas, Vennison, Sudha, Balasubramanian, & Sekar, ). This latter phenotype was observed in two particular B. thuringiensis strains: yunnanensis (Srinivas et al, ) and LM1212 (Deng et al, ).…”