Bacillus thuringiensis (Bt) is a gram-positive, rod-shaped, aerobic, and spore-forming bacterium closely related to the omnipresent soil bacteria Bacillus cereus. The vegetative cells are 1 Î¼m in width, 5 Î¼m long, and have short hair-like flagellae. The species is distinguished from B. cereus by its ability to produce a protein crystal during sporulation. In 1916, Aoki and Chigasaki found that its activity was due to a toxin present in sporulated cultures, but not in young cultures of vegetative cells.
Bt is ubiquitous and the spores exist naturally on the surfaces of living and dead organisms in the environment and can be isolated from soil, foliage, water and air. They can be found on many plant surfaces. Some subspecies of Bt have been collected and raised from pine trees, broadleaved trees and vegetables. Bt can be found in some stored products and growing in soil. Bt subspecies have been taken from dead or dying insects. The Bt spore and crystal protein have a half life of a few hours to 10 days in field conditions. Both the spores and the insecticidal crystals break down in sunlight.
Although the main biocontrol activity of B. thuringiensis is due to its entomopathogenic potential, different strains in this species have been shown to produce many potential factors that could be of great interest in the biocontrol of phytopathogenic bacteria. This makes from B. thuringiensis a polyvalent biocontrol agent that allows a better protection of the plants and could lead to decrease the use of chemical pesticides which have usually harmful impact on the environment. The following enzymes are found in B. thuringiensis as biocontrol properties.
Chitin, is an insoluble linear homopolymer of N-acetylglucosamine (GlcNAc) connected by Î²âˆ’1,4-linkages which is utilised as a structural polysaccharide in nature. Chitin occurs in insects as a major component of the cuticle and is also present (between 3% and 13%) in the peritrophic membrane (PM), a protective sleeve lining the gut of many insects. Hence, it is reasonable to speculate that chitinase activities may play key roles in the virulence of some pathogens that infect insects via the peritrophic membrane since pathogens that infect through the gut must penetrate this chitin-containing barrier. In this view, chitinase has been used together with B. thuringiensis to enhance larvicidal activity since the early 1970s. Initial experiments were conducted by mixing exogenous chitinase produced by Bacillus circulans or Serratia marcescens with B. thuringiensis. Bacillus thuringiensis itself produces chitinases and the role of these endogenous chitinases has recently come under investigation. A synergistic action of chitinase Chi36 from Bt HD-1 along with the vegetative insecticidal protein (Vip) against Spodoptera litura larvae. The presence of endochitinase and exochitinase genes was detected via PCR screening of 16 B. thuringiensis isolates which showed also an important chtinolytic activity on plates containing colloidal chitin as a major or unique carbon source. The observed potentiation of insecticidal activity of Cry and Vip in the presence of chitinase offers another tool to enhance the application of B. thuringiensis proteins and highlights the importance of these synergistic proteins as a tool to delay development of insect resistance to B. thuringiensis.
Proteases and phospholipases:
Bacillus thuringiensis is highly resistant to the humoral defence system of the host, especially to cecropins and attacins, which are the main classes of inducible antibacterial peptides in various lepidopterans and dipterans. An extracellular zinc metalloprotease, termed InhA or InA (immune inhibitor A) which specifically hydrolyzes antibacterial proteins produced by the insect host, in vitro, was suggested to partly explain the success of the bacterium in invading hemocoel. Recently a new B. thuringiensis virulence factor, InhA2 that is highly homologous to InhA, has been characterised and shown to play a major role in potentiating the toxicity of Cry proteins in orally infected insects. inhA2 is a PlcR regulated gene essential for B. thuringiensis virulence. Also, the insect peritrophic membrane is formed by four classes of proteins, in addition to chitin, glycoproteins and proteoglycans, which confers strength and elasticity to the PM and influences its permeability properties. Hence production of proteases among other secreted proteins could have a synergistic effect on the entomopathogenic effect of B. thuringiensis. The expression of several virulence genes, at the end of the exponential growth phase, that encode secreted proteins, including phospholipases C, haemolysins, enterotoxins, and proteases is regulated by PlcR. PlcR-regulated toxins and degradative enzymes may facilitate the spread of the bacterium through host tissues, thus allowing bacterial cells to gain access to alternative sources of nutrients and to cause septicaemia. In addition, previous work suggested that phopholipases C are involved in the entomopathogenic properties of the bacteria.
Autolysins are endogenous peptidoglycan hydrolases that digest cell wall peptidoglycans of the producer bacterium and of other bacteria. According to the chemical bond cleaved in the peptidoglycan molecule, four different specificities have been defined: N-acetylmuramidase, N-acetylglucosaminidase, N-acetylmuramyl-L-alanine amidase and endopeptidase. Such peptidoglycan hydrolases are synthesized during cellular growth and are involved in various fundamental steps of the bacterial life cycle: cell separation, cell wall turnover, peptidoglycan maturation and cell differentiation (mother cell lysis and spore outgrowth) in spore-forming bacteria. The characterisation of the autolytic phenotype of 112 B. thuringiensis strains showed seven major proteins of molecular weights ranging between 25 and 90 kDa which exhibited peptidoglycan hydrolase activity, particularly at alkaline pH. Several of these proteins retained lytic activity against other bacterial species such as Micrococcus lysodeikticus, Listeria monocytogenes and Staphylococcus aureus.
These proteins could be of great interest in field application of B. thuringiensis e.g. for improving bacterial or insect biocontrol by coupling with other antagonistic factors such as bacteriocins or chitinases since the products of chitinase action are degraded to N-acetylglucosamine by N-acetylglucosaminidases. Applications of these enzymes producing BT could lead to decrease the use of chemical pesticides which have usually harmful impact on the environment.
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