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...

Bacteriocins are antimicrobial peptides synthesized and secreted by bacteria and could potentially be used as natural food preservatives. Here, we report the production of bacteriocin-like inhibitor substances (Bt-BLIS) by five Mexican strains of Bacillus thuringiensis. Bacillus thuringiensis subsp. morrisoni (LBIT 269), B. thuringiensis subsp. kurstaki (LBIT 287), B. thuringiensis subsp kenyae (LBIT 404), B. thuringiensis subsp. entomocidus (LBIT 420) and B. thuringiensis subsp. tolworthi (LBIT 524) produced proteinaceous Bt-BLIS with high levels of activity against Bacillus cereus and other gram-positive bacteria. Although none was active against the gram-negative bacteria, Escherichia coli, Shigella species and Pseudomonas aeruginosa, the five Bt-BLIS demonstrated antimicrobial activity against Vibrio cholerae, the etiologic agent of cholera. Biochemical and biophysical studies demonstrated that the five Bt-BLIS could be categorized into two groups, those produced by LBIT 269 and 287 (Group A) and LBIT 404, 420, 524 (Group B), based on relative time of peptide synthesis, distinctive bacterial target specificity and stability in a wide range of temperatures and pH. Because of their stability and bactericidal activities against B. cereus and V. cholerae agents of emetic, diarrheal and lethal syndromes in humans, these Bt-BLIS could potentially be used as biodegradable preservatives in the food industry.

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Bacillus thuringiensis subsp. kurstaki HD-73 was transformed with the homologous endochitinase gene chiA74 of B. thuringiensis subsp. kenyae LBIT-82 under the regulation of its own promoter and Shine-Dalgarno sequence. The plasmid, pEHchiA74, which harbors chiA74, was detected by southern blot analysis and showed high segregational stability when the recombinant strain was grown in a medium without antibiotic. The recombinant bacterium transformed with pEHchiA74 showed an improvement in chitinolytic activity three times that of the wild-type strain. Expression of ChiA74 did not have any deleterious effect on the crystal morphology and size, but sporulation and Cry1Ac production in rich medium (nutrient broth with glucose) was reduced by approximately 30%. No significant increase in the toxicity of the transformant bacterium toward Plutella xylostella was detected using the same amount of total protein. However, it is possible that ChiA74 synthesis compensated for the decrease in net Cry1Ac synthesis and toxicity observed with the recombinant strain.