Two novel delta‐endotoxin gene families cry26 and cry28 from Bacillus thuringiensis ssp. finitimus

Wojciechowska, Jana A; Lewitin, E.; Revina, L. P.; Zalunin, I. A.; Chestukhina, G. G.

doi:10.1016/s0014-5793(99)00650-x

Cited by 17 publications

(14 citation statements)

References 6 publications

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“…In addition to the complete toxin CDSs, pBtoxis also contains short sequences encoding fragments of toxins. pBt025 and pBt026 encode two segments with homologies to the center region of a Cry28Aa-like toxin, while pBt053 appears to encode a sequence with homology to the extreme C terminus of a Cry26Aa-like protein (49). In addition, the amino acid sequence encoded by pBt055 is similar at its C terminus to proteins encoded upstream of toxin genes (e.g., a hypothetical 29.1-kDa protein in the cry2Aa 5Ј region of Bacillus thuringiensis subsp.…”

Section: Resultsmentioning

confidence: 99%

Complete Sequence and Organization of pBtoxis, the Toxin-Coding Plasmid ofBacillus thuringiensissubsp.israelensis

Berry

O'Neil

Ben‐Dov

et al. 2002

Appl Environ Microbiol

276

236

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The entire 127,923-bp sequence of the toxin-encoding plasmid pBtoxis from Bacillus thuringiensis subsp. israelensis is presented and analyzed. In addition to the four known Cry and two known Cyt toxins, a third Cyt-type sequence was found with an additional C-terminal domain previously unseen in such proteins. Many plasmid-encoded genes could be involved in several functions other than toxin production. The most striking of these are several genes potentially affecting host sporulation and germination and a set of genes for the production and export of a peptide antibiotic.Isolates of Bacillus thuringiensis are the biological control agents most widely used to eradicate insect pests of crops or vectors of human disease. For the latter application, Bacillus thuringiensis subsp. israelensis is the bioinsecticide of choice in programs worldwide to control mosquitoes and blackfly vectors (29). The insect pathogenicity of this bacterium depends on the presence of the pBtoxis megaplasmid (13) that encodes all six of the previously described toxins in this isolate (Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, Cyt1Aa, and Cyt2Ba) (7,18). In addition, the plasmid carries several insertion sequences and encodes two further proteins (P19 and P20) with roles in promoting crystal formation and enhancing cell viability, probably by acting as chaperones (12,27,50). The pBtoxis plasmid has been partially mapped (6, 7), but the nucleotide sequence is limited to toxin genes and their flanking regions. Since the toxicity of the B. thuringiensis subsp. israelensis crystal is greater than that of any combination of the known toxins derived from it (9), it seems that other toxins or virulence factors may play a role in the activity of wild-type crystals. One possible source of such additional factors is the approximately 80% of the pBtoxis sequence that has not previously been analyzed. In order to understand fully this highly important virulence plasmid, we have therefore determined its entire nucleotide sequence as presented here. MATERIALS AND METHODSPlasmid preparation. The pBtoxis plasmid was prepared from B. thuringiensis subsp. israelensis strain 4Q2-72 (also known as 4Q5) and purified on a CsClethidium bromide density gradient as previously described (6).Sequencing and analysis. Plasmid DNA was sonicated and size fractionated on agarose gels. Two libraries were generated in pUC18 using insert sizes of 1.4 to 2 and 2 to 4 kb. Each clone was sequenced once from each end using ABI Big-Dye terminator chemistry on ABI3700 capillary sequencing machines. The final sequence was generated from 1,467 sequencing reads, giving 6.4-fold total coverage. All repeats were bridged by clone end read pairs or end-sequenced PCR products to confirm the assembly.The finished sequence was annotated using Artemis software (41). Potential coding sequences were identified by codon usage (34) and positional base preference methods and compared to the nonredundant protein databases using BLAST (3) and FASTA (38) software. The entire DNA sequence was also compared ...

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Section: Resultsmentioning

confidence: 99%

Complete Sequence and Organization of pBtoxis, the Toxin-Coding Plasmid ofBacillus thuringiensissubsp.israelensis

Berry

O'Neil

Ben‐Dov

et al. 2002

Appl Environ Microbiol

276

236

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“…In this regard, it is noteworthy that B. thuringiensis var. finitimus harbors both S-layer genes, sap and eag (Table 1) and its parasporal inclusion body is made of at least two Cry proteins, Cry26Aa1 and Cry28Aa1 (Wojciechowska et al 1999). We are planning to analyze the composition of the inclusion bodies in the 20 sap?, eag?…”

Section: Discussionmentioning

confidence: 99%

Mutually exclusive distribution of the sap and eag S-layer genes and the lytB/lytA cell wall hydrolase genes in Bacillus thuringiensis

Soufiane¹,

Suquet²,

Côté³

2011

Antonie van Leeuwenhoek

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Recently, two Bacillus thuringiensis strains were reported to synthesize parasporal inclusion bodies made not of the expected crystal (Cry) proteins but rather of the surface layer proteins (SLP) Sap (encoded by sap) and EA1 (encoded by eag), respectively. Whether the presence of the sap and eag genes is restricted to these two B. thuringiensis strains or ubiquitous in B. thuringiensis is unknown. We report here the distribution of the sap and eag genes in B. thuringiensis. Strains in the Bacillus cereus group were added for comparison purposes. We show that sap and eag are either present in tandem in 35% of the B. thuringiensis strains analysed and absent in 65% of the strains. When absent, a different tandem, the lytB/lytA cell wall hydrolase genes, is present. The distribution of the sap and eag S-layer and the lytB/lytA cell wall hydrolase genes is not species-specific in B. thuringiensis, B. cereus and Bacillus weihenstephanensis. Bacillus anthracis and Bacillus mycoides harbor sap and eag but not lytB/lytA. The sap, eag and lytB/lytA genes were absent in Bacillus pseudomycoides. Clearly, the distribution of the sap and eag S-layer and the lytB/lytA cell wall hydrolase genes in B. thuringiensis and in the Bacillus cereus group is mutually exclusive. We also showed that two genes involved in cell wall metabolism, csaA and csaB, are present not only upstream of the sap and eag S-layer genes, but also upstream of the lytB/lytA tandem in strains where sap and eag are absent. Bootstrapped neighbor-joining trees were inferred from the translated amino acid sequences of sap, eag and the tandem lytB/lytA, respectively.

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“…However, in a few strains, such as in B. thuringiensis subsp. finitimus strains [2], [3] and B. thuringiensis subsp. oyamensis strain LBIT-113 [4], the parasporal crystals are located between the exosporium and the spore coat and continue to adhere to the spore after mother cell lysis.…”

Section: Introductionmentioning

confidence: 99%

“…Two crystal protein encoding genes, cry26Aa1 and cry28Aa1 , were identified from B. thuringiensis subsp. finitimus B-1166 VKPM; encoding 131 and 125 kDa proteins respectively [3]. An unusual nontoxic strain of B. thuringiensis subsp.…”

Section: Introductionmentioning

confidence: 99%

Gene Clusters Located on Two Large Plasmids Determine Spore Crystal Association (SCA) in Bacillus thuringiensis Subsp. finitimus Strain YBT-020

Zhu

Shang

et al. 2011

PLoS ONE

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Crystals in Bacillus thuringiensis are usually formed in the mother cell compartment during sporulation and are separated from the spores after mother cell lysis. In a few strains, crystals are produced inside the exosporium and are associated with the spores after sporulation. This special phenotype, named ‘spore crystal association’ (SCA), typically occurs in B. thuringiensis subsp. finitimus. Our aim was to identify genes determining the SCA phenotype in B. thuringiensis subsp. finitimus strain YBT-020. Plasmid conjugation experiments indicated that the SCA phenotype in this strain was tightly linked with two large plasmids (pBMB26 and pBMB28). A shuttle bacterial artificial chromosome (BAC) library of strain YBT-020 was constructed. Six fragments from BAC clones were screened from this library and discovered to cover the full length of pBMB26; four others were found to cover pBMB28. Using fragment complementation testing, two fragments, each of approximately 35 kb and located on pBMB26 and pBMB28, were observed to recover the SCA phenotype in an acrystalliferous mutant, B. thuringiensis strain BMB171. Furthermore, deletion analysis indicated that the crystal protein gene cry26Aa from pBMB26, along with five genes from pBMB28, were indispensable to the SCA phenotype. Gene disruption and frame-shift mutation analyses revealed that two of the five genes from pBMB28, which showed low similarity to crystal proteins, determined the location of crystals inside the exosporium. Gene disruption revealed that the three remaining genes, similar to spore germination genes, contributed to the stability of the SCA phenotype in strain YBT-020. Our results thus identified the genes determining the SCA phenotype in B. thuringiensis subsp. finitimus.

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Two novel delta‐endotoxin gene families cry26 and cry28 from Bacillus thuringiensis ssp. finitimus

Cited by 17 publications

References 6 publications

Complete Sequence and Organization of pBtoxis, the Toxin-Coding Plasmid ofBacillus thuringiensissubsp.israelensis

Complete Sequence and Organization of pBtoxis, the Toxin-Coding Plasmid ofBacillus thuringiensissubsp.israelensis

Mutually exclusive distribution of the sap and eag S-layer genes and the lytB/lytA cell wall hydrolase genes in Bacillus thuringiensis

Gene Clusters Located on Two Large Plasmids Determine Spore Crystal Association (SCA) in Bacillus thuringiensis Subsp. finitimus Strain YBT-020

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