ABSTRACT
Both the bacterium Photorhabdus luminescens alone and its symbiotic Photorhabdus-nematode complex are known to be highly pathogenic to insects. The nature of the insecticidal activity of Photorhabdus bacteria was investigated for its potential application as an insect control agent. It was found that in the fermentation broth of P. luminescens strain W-14, at least two proteins, toxin A and toxin B, independently contributed to the oral insecticidal activity against Southern corn rootworm. Purified toxin A and toxin B exhibited single bands on native polyacrylamide gel electrophoresis and two peptides of 208 and 63 kDa on SDS-polyacrylamide gel electrophoresis. The native molecular weight of both the toxin A and toxin B was determined to be approximately 860 kDa, suggesting that they are tetrameric. NH2-terminal amino acid sequencing and Western analysis using monospecific antibodies to each toxin demonstrated that the two toxins were distinct but homologous. The oral potency (LD50) of toxin A and toxin B against Southern corn rootworm larvae was determined to be similar to that observed with highly potent Bt toxins against lepidopteran pests. In addition, it was found that the two peptides present in toxin B could be processed in vitro from a 281-kDa protoxin by endogenous P. luminescens proteases. Proteolytic processing was shown to enhance insecticidal activity.
Subject(s)
Bacterial Toxins/isolation & purification , Enterobacteriaceae/chemistry , Insecticides , Plants , Amino Acid Sequence , Animals , Bacterial Toxins/chemistry , Bacterial Toxins/pharmacology , Blotting, Western , Coleoptera/drug effects , Electrophoresis, Polyacrylamide Gel , Fermentation , Larva/drug effects , Molecular Sequence Data , Molecular Weight , Pest Control, Biological , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
High affinity [3H]GABA (gamma-aminobutyric acid) binding sensitive to muscimol and bicuculline was detected in membranes derived from rat brain and brine shrimp. Avermectin stimulated this GABA binding with maximum stimulation seen in these membranes at 400 and 40-80 ng/ml, respectively. This avermectin stimulation of GABA binding was Cl--dependent, bicuculline and picrotoxin-sensitive and was associated with an increase in Bm but not Kd of the systems. The membranes from Mucor miehei also exhibited high affinity [3H]GABA binding that was insensitive to classical neuronal GABA receptor agonists/antagonists and other agents. This novel GABA receptor was sensitive to Na+ and extremely sensitive to low levels of avermectin (apparent Ki 20-40 ng/ml). This inhibition of GABA binding by avermectin was associated with a decrease in affinity (increase in Kd) and an increase in concentration of receptors (Bm). It is possible that these GABA receptors might play crucial roles in control of cell metabolism and that avermectin can prevent growth of this organism via interference in the receptor activity.
Subject(s)
Brain/metabolism , Decapoda/metabolism , Ivermectin/analogs & derivatives , Lactones/pharmacology , Mucor/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Bicuculline/pharmacology , Chlorides/pharmacology , Kinetics , Male , Picrotoxin/pharmacology , Rats , Rats, Inbred Strains , Receptors, Cell Surface/metabolism , Receptors, GABA-A , Sodium/metabolismABSTRACT
Avermectin inhibits Mucor miehei and Artemia salina chitin synthesis and to a degree DNA synthesis in the former. The antibiotic interferes with chitin turnover in brine shrimp and inhibits Streptomyces antibioticus chitinase activity in vitro. In light of the proposed mode of action of avermectin and the anomolies in the literature, it is proposed that avermectin can kill susceptible organisms not only by a neurotoxic mechanism but also by inhibiting chitin turnover and synthesis at low concentration and thus the molting/ecdysis process.
