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.

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.

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.

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.

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.