Cloning, Sequencing, and Expression of the Chitinase Gene chiA74 from Bacillus thuringiensis

Abstract

The endochitinase gene chiA74 from Bacillus thuringiensis serovar kenyae strain LBIT-82 was cloned in Escherichia coli DH5␣F. A sequence of 676 amino acids was deduced when the gene was completely sequenced. A molecular mass of 74 kDa was estimated for the preprotein, which includes a putative 4-kDa signal sequence located at the N terminus. The deduced amino acid sequence showed high degree of identity with other chitinases such as ChiB from Bacillus cereus (98%) and ChiA71 from Bacillus thuringiensis serovar pakistani (70%). Additionally, ChiA74 showed a modular structure comprised of three domains: a catalytic domain, a fibronectin-like domain, and a chitin-binding domain. All three domains showed conserved sequences when compared to other bacterial chitinase sequences. A ca. 70-kDa mature protein expressed by the cloned gene was detected in zymograms, comigrating with a chitinase produced by the LBIT-82 wild-type strain. ChiA74 is active within a wide pH range (4 to 9), although a bimodal activity was shown at pH 4.79 and 6.34. The optimal temperature was estimated at 57.2°C when tested at pH 6. The potential use of ChiA74 as a synergistic agent, along with the B. thuringiensis insecticidal Cry proteins, is discussed.Bacillus thuringiensis is an insecticidal bacterium whose activity is based on the effect of single or mixed Cry or Cyt proteins, acting additively or synergistically, although antagonism has also been reported (9). Despite reports of more than 3,000 insect species, within 16 orders and susceptible to different B. thuringiensis toxins, important pests are highly susceptible to only a few toxins (14). Additionally, development of resistance to Cry proteins, their slow mode of action, and the requirement of ingestion, along with some other limitations, justify the search for new approaches to improve the conventional use of B. thuringiensis.The insect midgut is internally coated by the peritrophic membrane, whose structure is basically composed of proteins and reinforced with chitin fibers. This is a physical barrier to bacterial or viral infections but allows the flow of digested nutrients, minerals, and water towards the midgut epithelium. It is known that the approach of ␦-endotoxins of B. thuringiensis to the microvillus receptor is highly facilitated when the peritrophic membrane is damaged or degraded, causing an increase in its insecticidal activity (25). Factors that damage the peritrophic membrane, such as the enhancin of granuloviruses, promote the insecticidal activity of commercial formulations of B. thuringiensis, especially on those pests with relatively low natural susceptibility, such as Helicoverpa zea (Boddie) and Spodoptera exigua (Hübner) (16). Likewise, the same effect is observed when Cry proteins are tested along with wild-type (31) or recombinant (11) chitinolytic bacteria, as well as with unpurified chitinase (Chi) (25,40).On the other hand, chitinases from B. thuringiensis are poorly studied (2, 7, 15), although interest has slowly increased due to their potential role as...

A Chitinase Indispensable for Formation of Protoplast ofSchizophyllum communein Basidiomycete-lytic Enzyme Preparation Produced byBacillus circulansKA-304

“…[18][19][20][21][22] However, N-terminal amino acid sequence of chitinase I, A L A T P T L N V S A S S G M (15 residues), had no identity to those of known chitinases.…”

Cloning and expression of chitinase A, B, and C (chiA, chiB, chiC) genes from Serratia marcescens originating from Helicoverpa armigera and determining their activities

“…Bacterial chitinases have also been shown to be potential insecticides when coupled with other suitable proteins such as Cry (Kramer and Koga, 1986;Regev et al, 1996;Barboza-Corona et al, 1999;Barboza-Corona et al, 2003). Recently, a number of heterologous chi genes have been engineered from wide bacterial sources into B. thuringiensis with the main interest to increase the insecticidal activity of entomopathogenic bacteria (Bhattacharya et al, 2007).…”

Cloning of the Bacillus thuringiensis serovar sotto chitinase (Schi) gene and characterization of its protein

“…Insecticidal proteins, mainly Cry (crystal) proteins, play the leading role in controlling of insect pests. It has been reported that chitinases are widely distributed in Bt strains and that some of the chitinase-producing strains can enhance the insecticidal activity of Bt (Liu et al, 2002), although, as far as we know, only a few chitinase genes have yet been cloned (Thamthiankul et al, 2001;Barboza-Corona et al, 2003;Zhong et al, 2003;Lin and Guan, 2004) and furthermore, the synergistic effect of purified Bt chitinase and Cry proteins has still not been quantitatively demonstrated (Barboza-Corona et al, 2003).…”