AHL-lactonase (AiiA), ametallo-beta-lactamaseproducedby Bacillus thuringiensis, Bacillus cereus and Bacillus anthracis, specificallyhydrolyzes N-acyl-homoserine lactones (AHLs)secretedbyGram-negativebacteriaandtherebyattenuatesthe symptoms causedbyplantpathogens. Inthisstudy, an aiiA gene wasclonedfrom Bacillus subtilis BS-1 byPCRwithapairofdegenerateprimers. Thededuced250 amino acidsequencecontainedtwosmallconservedregions, 103SHLHFDH109 and 166TPGHTPGH173, whicharecharacteristicofthemetallo-beta-lactamasefamily. Homology comparisonrevealedthatthededucedaminoacidsequencehadahigh degree ofsimilaritywiththoseoftheknownAiiAproteinsinthe B. cereus group. Additionally, the aiiA gene wasexpressedin Escherichia coli BL21 (DE3)pLysSand the expressedAiiAproteincouldattenuatethesoftrotsymptomscausedby Erwinia carotovora var. carotovora.

Aims: To isolate and characterize the novel Bacillus thuringiensis strains frombryophytes collected from Wuyi Mountain, Fujian Province of China, andidentify new B. thuringiensis strains and toxins active against mosquitoes.Methods and Results: Twelve novel B. thuringiensis strains were isolated from76 bryophyte samples. According to the results of this preliminary screening,LLB6 was the most toxic to Aedes albopictus. Then phase-contrast as well asscanning electron microscopy, bioassays, cloning, sequencing and expressionwere performed to characterize the novel isolate LLB6 and its new genecry2Ac5.Conclusions: Bacillus thuringiensis occurred naturally on bryophytes. LLB6 isolatedfrom Physcomitrium japonicum was toxic to A. albopictus. A new cry2Ac5gene of LLB6 was detected, cloned and expressed successfully. Bioassays onA. albopictus showed that the expressed Cry2Ac5 was also toxic to the thirdinstar larvae.Significance and Impact of the Study: This is the first report of B. thuringiensisstrains isolated from bryophytes. It represents a specific source of new B. thuringiensisstrains and is of great importance for the knowledge of the ecologyof B. thuringiensis. Novel LLB6 harboring the new gene cry2Ac5 and itsexpressed Cry2Ac5 protein revealed activity against A. albopictus and became anew member of B. thuringiensis toxins.

InhA, a zinc metalloprotease secreted byBacillus thuringiensis, specifically hydrolyzes antibacterialpeptides produced by insect hosts. In this study, the inhAgene was cloned from B. thuringiensis 8010 using a pair ofdegenerate primers and the deduced 796 amino acid sequenceshowed a high degree of similarity with other InhAproteins in the Bacillus cereus group. The deduced aminoacid sequence contained the zinc-binding motif (HEXXH),which is characteristic of the zinc-metalloprotease family.Additionally, the inhA gene was expressed in Escherichiacoli BL21 (DE3). The expressed InhA protein was shownto be toxic to the third larvae of Plutella xylostella, contraryto preliminary study concerning the effect of InhA onBombyx mori. This study provided insights into the potentialof InhA for the biological control of certain lepidopteraninsects.

The inhibition kinetics of alkylbenzoic acids on the diphenolase activity of mushroom tyrosinase have been investigated. Theresults show that the alkylbenzoic acids assayed can lead to reversible inhibition of the enzyme; furthermore, o-toluic acid andm-toluic acid are mixed-type inhibitors and p-alkylbenzoic acids are uncompetitive inhibitors. The inhibition constants have beendetermined. For these p-alkylbenzoic acids, the inhibition strength follows the order: p-toluic acid < p-ethylbenzoic acid < p-propylbenzoicacid < p-isopropylbenzoic acid < p-tert-butylbenzoic acid < p-butylbenzoic acid < p-pentylbenzoic acid < p-hexylbenzoicacid < p-heptylbenzoic acid < p-octylbenzoic acid, indicating that the hydrophobic p-alkyl group played an important role in theinhibition of the enzyme. The inhibitory effects were potentiated with increasing lengths of the hydrocarbon chains. The inhibitoryeffects of o-toluic acid and p-isopropylbenzoic acid on the monophenolase activity have also been studied. The results show thatboth o-toluic acid and p-isopropylbenzoic acid can lengthen the lag time and decrease the steady-state activity of the enzyme.

An insecticidal protein gene, vip3A, was cloned from Bacillus thuringiensis strainWB50. The nucleotide sequenceof 2,460 bp (GenBank acc. No. AY295778) showed 99% homology with the known vip3A genes. Using specificprimers for vip3A gene, PCR was performed to demonstrate that the gene was not located on the bacterial chromosomeand this was confirmed by Southern blotting using an internal fragment (486 bp) from vip3A gene as aprobe. The gene was carried on a plasmid of 31.8 kb.

A new cry1Ab-type gene, cry1Ab17, was cloned from Bacillus thuringiensis WB9 by PCR. Nucleotide sequenceindicated that the open reading frames (ORFs) consists of 3471 bases and encodes a protein of 1156 residueswith a calculated molecular weight of 130.5 kDa and an pI value of 5.04. Homology comparison revealed thatthe deduced amino acid sequence of Cry1Ab17 had 95.4% to 99.7% identity with those of the known Cry1Abproteins. The Cry1Ab17 was one residue longer than the known Cry1Ab (except for Cry1Ab2). Domain I (Tyr33 toArg253), II (Arg265 to Phe462), III (Asn464 to Thr610) of the Cry1Ab17 were 96.8%, 68.2% and 100% identical tothe corresponding domains of Cry1Aa. Additionally, the cry1Ab17 gene was expressed in Escherichia coli BL21under the control of T7 promoter and the Cry1Ab17 isolated from the culture medium was toxic to 3rd instarPlutella xylostella larvae.

In order to better understand the fundamental biology of Bacillus thuringiensis, a single oligonucleotide primer (50-CATSSCCATCAASYTAAVR-30) was used to investigate the distribution pattern of IS231 elements in B. thuringiensis by PCR. Theresults indicated that IS231 elements appeared in 20 standard strains and 107 of 111 China isolates. Three novel IS231, IS231J,IS231O and IS231Q, five variants and a mobile insertion cassette MICBth4 were cloned from eight standard strains of B. thuringiensis,respectively. Interestingly, BLAST analysis revealed that the 50 end of novel IS231J shared 99% identity in 495-bp with aDNA segment adjacent to the 30 end of B. thuringiensis vip1Ac gene (GenBank Accession No. AY245547). Two phylogenetic treesof IS231 elements were constructed and analyzed by neighbor-joining and UPGMA methods from PHYLIP 3.6b program,respectively.