Increased toxicity of modified mosquitocidal binary toxins of Bacillus sphaericus expressed in Escherichia coli

ABSTRACT Alanine residues were substituted by site-directed mutagenesis at selected sites of the N- and C-terminal regions of the binary toxin (51- and 42-kDa peptides) of B. sphaericus 1593M, and the mutant toxins were cloned and expressed in Escherichia coli. Bioassays with mosquito larvae, using binary toxins derived from individual mutants, showed that the substitution of alanine at some sites in both the 51-kDa and the 42-kDa peptides resulted in a total loss of activity. Surprisingly, after mixing two nontoxic derivatives of the same peptide, i.e., one mutated at the N-terminal end and the other mutated at the C-terminal end of either the 51-kDa or the 42-kDa peptide, the toxicity was restored. This result indicates that the altered binary toxins can functionally complement each other by forming oligomers.

Download Full-text

UV protectants for the biopesticide based on Bacillus sphaericus Neide and their role in protecting the binary toxins from UV radiation

Journal of Invertebrate Pathology ◽

10.1016/j.jip.2008.12.003 ◽

2009 ◽

Vol 100 (3) ◽

pp. 147-152 ◽

Cited By ~ 14

Author(s):

A.B. Hadapad ◽

R.S. Hire ◽

N. Vijayalakshmi ◽

T.K. Dongre

Keyword(s):

Uv Radiation ◽

Bacillus Sphaericus ◽

Binary Toxins

Download Full-text

Medium Optimization for Improved Production of Dihydrolipohyl Dehydrogenase from Bacillus sphaericus PAD-91 in Escherichia coli

Molecular Biotechnology ◽

10.1007/s12033-017-0013-z ◽

2017 ◽

Vol 59 (7) ◽

pp. 260-270 ◽

Cited By ~ 2

Author(s):

Hamid Shahbazmohammadi ◽

Eskandar Omidinia

Keyword(s):

Escherichia Coli ◽

Medium Optimization ◽

Bacillus Sphaericus

Download Full-text

A new tubRZ operon involved in the maintenance of the Bacillus sphaericus mosquitocidal plasmid pBsph

Microbiology ◽

10.1099/mic.0.075465-0 ◽

2014 ◽

Vol 160 (6) ◽

pp. 1112-1124 ◽

Cited By ~ 16

Author(s):

Yong Ge ◽

Xiaomin Hu ◽

Ni Zhao ◽

Tingyu Shi ◽

Quanxin Cai ◽

...

Keyword(s):

Point Mutations ◽

Bacillus Sphaericus ◽

Plasmid Replication ◽

Electron And Fluorescence Microscopy ◽

The Stability ◽

Mutant Forms ◽

Dna Complex ◽

Binary Toxins

pBsph is a mosquitocidal plasmid first identified from Bacillus sphaericus, encoding binary toxins (Bin toxins) that are highly toxic to mosquito larvae. This plasmid plays an important role in the maintenance and evolution of the bin genes in B. sphaericus. However, little is known about its replication and partitioning. Here, we identified a 2.4 kb minimal replicon of pBsph plasmid that contained an operon encoding TubR-Bs and TubZ-Bs, each of which was shown to be required for plasmid replication. TubR-Bs was shown to be a transcriptional repressor of tubRZ-Bs genes and could bind cooperatively to the replication origin of eleven 12 bp degenerate repeats in three blocks, and this binding was essential for plasmid replication. TubZ-Bs exhibited GTPase activities and interacted with TubR-Bs : DNA complex to form a ternary nucleoprotein apparatus. Electron and fluorescence microscopy revealed that TubZ-Bs assembled filaments both in vitro and in vivo, and two point mutations in TubZ-Bs (T114A and Y260A) that severely impaired the GTPase and polymerization activities were found to be defective for plasmid maintenance. Further investigation demonstrated that overproduction of TubZ-Bs-GFP or its mutant forms significantly reduced the stability of pBsph. Taken together, these results suggested that TubR-Bs and TubZ-Bs are involved in the replication and probably in the partitioning of pBsph plasmid, increasing our understanding of the genetic particularity of TubZ systems.

Download Full-text

Interaction of the Bacillus sphaericus mosquito larvicidal proteins

Canadian Journal of Microbiology ◽

10.1139/m90-151 ◽

1990 ◽

Vol 36 (12) ◽

pp. 870-878 ◽

Cited By ~ 38

Author(s):

Elizabeth W. Davidson ◽

Coreen Oei ◽

Marian Meyer ◽

Allan L. Bieber ◽

John Hindley ◽

...

Keyword(s):

Escherichia Coli ◽

Sequence Analysis ◽

Key Words ◽

Culex Quinquefasciatus ◽

Bacillus Sphaericus ◽

Insecticidal Proteins ◽

Posterior Midgut ◽

Sds Page ◽

Reduced Activity

Genes for 51.4- and 41.9-kDa insecticidal proteins of Bacillus sphaericus were separately cloned and expressed in Escherichia coli. Both proteins were required for toxicity. Approximately equal numbers of cells containing the 51.4- and 41.9-kDa proteins produced the greatest toxicity; excess 41.9-kDa protein did not affect toxicity, whereas excess 51.4-kDa protein reduced activity. Larvae were killed when 41.9-kDa protein was fed up to 24 h after the 51.4-kDa protein, but not when the order of feeding was reversed. Radiolabelled toxins bound in approximately equal amounts to the gastric caecum and posterior midgut of Culex quinquefasciatus larvae. Radiolabelled 51.4-kDa protein was rapidly degraded by ca. 12–13 kDa in the larval gut, while 41.9-kDa protein was degraded by 1–2 kDa. Nonreduced toxin extracted from B. sphaericus produced a band on SDS–PAGE of ca. 68–74 kDa that contained both 51.4- and 41.9-kDa proteins based on sequence analysis, and a band of ca. 51 kDa that contained primarily 41.9-kDa protein. Escherichia coli containing 51.4-kDa protein enhanced toxicity of the latter eluted SDS-PAGE band. These proteins may associate very strongly, and trace amounts of 51.4-kDa protein in preparations of 41.9-kDa protein from B. sphaericus may be responsible for the previously reported toxicity of the latter. Key words: Bacillus sphaericus, toxin, mosquito, cloning.

Download Full-text

Cloning the modification methylase gene of Bacillus sphaericus R in Escherichia coli

Gene ◽

10.1016/0378-1119(80)90051-7 ◽

1980 ◽

Vol 10 (3) ◽

pp. 219-225 ◽

Cited By ~ 63

Author(s):

Éva Szomolányi ◽

Antal Kiss ◽

Pál Venetianer

Keyword(s):

Escherichia Coli ◽

Bacillus Sphaericus ◽

Methylase Gene

Download Full-text

Cloning and expression in Escherichia coli of two DNA fragments from Bacillus sphaericus encoding mosquito-larvicidal activity

Journal of Biotechnology ◽

10.1016/0168-1656(88)90036-3 ◽

1988 ◽

Vol 7 (1) ◽

pp. 71-82 ◽

Cited By ~ 4

Author(s):

Augustine E. Souza ◽

Vidya Rajan ◽

Kunthala Jayaraman

Keyword(s):

Escherichia Coli ◽

Larvicidal Activity ◽

Bacillus Sphaericus ◽

Cloning And Expression ◽

Dna Fragments ◽

Expression In Escherichia Coli ◽

Mosquito Larvicidal Activity

Download Full-text

The 8-amino-7-oxopelargonate synthase from Bacillus sphaericus. Purification and preliminary characterization of the cloned enzyme overproduced in Escherichia coli

Biochemical Journal ◽

10.1042/bj2830327 ◽

1992 ◽

Vol 283 (2) ◽

pp. 327-331 ◽

Cited By ~ 33

Author(s):

O Ploux ◽

A Marquet

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Absorption Band ◽

Specific Activity ◽

Bacillus Sphaericus ◽

Complete Agreement ◽

Amino Acid Residues ◽

Characteristic Absorption Band ◽

Preliminary Characterization

The 8-amino-7-oxopelargonate synthase [6-carboxyhexanoyl-CoA:L-alanine carboxyhexanoyltransferase (decarboxylating); EC 2.3.1.47] from Bacillus sphaericus involved in biotin biosynthesis was purified from an Escherichia coli overproducing strain. The purification afforded an electrophoretically homogeneous enzyme with a specific activity of 0.67 unit/mg. The purified enzyme is a monomer of 41 kDa. N-Terminal sequencing of the first 14 amino acid residues showed complete agreement with the predicted sequence from the bioF gene. The pure enzyme showed the characteristic absorption band (425 nm) of pyridoxal 5′-phosphate-dependent enzymes. Furthermore, the holoenzyme was resolved during an affinity step yielding the inactive apoenzyme, which recovered activity and the 425 nm-absorption band on dialysis against pyridoxal 5′-phosphate. Km values for L-alanine and pimeloyl-CoA were respectively 3 mM and 1 microM.

Download Full-text