Cloning, functional characterization, and mode of action of a novel insecticidal pore-forming toxin, sphaericolysin, produced by Bacillus sphaericus.
An insecticidal protein produced by Bacillus sphaericus A3-2 was purified to elucidate its structure and mode of action. The active principle purified from the culture broth of A3-2 was a protein with a molecular mass of 53 kDa that rapidly intoxicated German cockroaches (Blattela germanica) at a dose of about 100 ng when injected. The insecticidal protein sphaericolysin possessed the undecapeptide motif of cholesterol-dependent cytolysins and had a unique N-terminal sequence. The recombinant protein expressed in Escherichia coli was equally as potent as the native protein. Sphaericolysin-induced hemolysis resulted from the protein's pore-forming action. This activity as well as the insecticidal activity was markedly reduced by a Y159A mutation. Also, coapplication of sphaericolysin with cholesterol abolished the insecticidal action, suggesting that cholesterol binding plays an important role in insecticidal activity. Sphaericolysin-lysed neurons dissociated from the thoracic ganglia of the German cockroaches. In addition, sphaericolysin's activity in ganglia was suppressed by the Y159A mutation. The sphaericolysin-induced damage to the cockroach ganglia was greater than the damage to the ganglia of common cutworms (Spodoptera litura), which accounts, at least in part, for the higher sensitivity to sphaericolysin displayed by the cockroaches than that displayed by cutworms.
Nishiwaki
Hisashi
H
Department of Applied Biological Chemistry, School of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan.
Nakashima
Kenta
K
Ishida
Chiharu
C
Kawamura
Tadayuki
T
Matsuda
Kazuhiko
K
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001407:Bacillus / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics* / Q000302:isolation & purification / Q000633:toxicity*; D001483:Base Sequence; D002784:Cholesterol / Q000378:metabolism; D003001:Cloning, Molecular; D003058:Cockroaches / Q000187:drug effects; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli; D005724:Ganglia / Q000187:drug effects; D007306:Insecticides; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020125:Mutation, Missense; D009474:Neurons / Q000187:drug effects; D052899:Pore Forming Cytotoxic Proteins / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity*; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D018411:Spodoptera / Q000187:drug effects
D001427:Bacterial Toxins; D004269:DNA, Bacterial; D007306:Insecticides; D052899:Pore Forming Cytotoxic Proteins; D002784:Cholesterol