Functional Identification Of ATP-binding Cassette Transporter From Bacillus Thuringiensis

ABC family is one of the largest superfamilies of prokaryotes and eukaryotes.In this study,an ABC gene which located downstream of chiA in Bt(Bacillus thuringiensis)BRC-HZP7 was cloned and named as ABC-1.It closes to chiA.A green fluorescent protein gene(Gfp)was also cloned.Then a fusion expression vector pET32a-Gfp-ABC-1 was constructed with pET32a as a carrier,expressed in E.coli BL21.After induced,pET32a-Gfp-ABC-1 was disrupted.And the components were seperated for microscopic examination.SDS-PAGE was also detected,the result showed that the ABC-1 protein was located in the cell fractions.Further examination was conducted with membrane extraction.It confirmed that the ABC-1 transporter was located on the cell membrane when it functioned.Online softwares were used for ABC-1 bioinformatic analysis.ABC-1 was mainly composed of a-helix,(3-angle,and a small amount of random coils and extend strands,where a helical(47.39%)has the maximum proportion.It suggested that ABC-1 protein was a "half molecule" model,contained only one NBD and a TMD,and the direction may be NBD-TMD.3D structure of ABC-1 displays a plurality of a-helix,which were together,showing a similar"channel" conformation.The formation may be a transmembrane channel of the solute.The results also showed that ABC-1 would form dimers when play a role in the transporting.Four strains including pET32a,pET32a-Gfp,pET32a-ABC-1,pET32a-Gfp-ABC-1,were selected to measure their growth curves.The result was that in LB medium,the growth of the strains containing ABC-1 were improved to 170.81%,compared with the control.Peptone and yeast extract were also used as the main nutrient respectively,showed that the growth trends were the same to LB,the growth increased 58.3%and 61.1%,respectively.Presumably,with the presence of ABC-I.strains may be more conducive to using the nutrients to enhance growth.In the screening of organic carbons,four monosaccharides including glucose,fructose,galactose and arabinose were used.It showed sugars may not be substrate of ABC-1.Meanwhile,four amino acids including glycine,glutamic acid,lysine and arginine were selected for screening of organic nitrogens,The results showed that ABC-1 had a higher affinity to lysine and arginine,compared with the control,the growth were increased to 19.52%and 38.66%,respectively.When analysing the possible functional relevance between ABC-1 and chi A,NAG was selected as the special nutrient to culture the thallus.It showed that ABC-1 had a high affinity to NAG,the growth improved to 85.3%.The result indicated that NAG was likely to be the substrate of ABC-1.With referring to the positional relationship between ABC-1 and chiA,they may have functional correlation.