Zwittermicin A Biosynthesis And Regulation In Bacillus Thuringiensis Strain YBT-1520

Zwittermicin A (ZmA) is a hybrid polyketide-nonribosomal peptide that is produced by some Bacillus strains, including Bacillus cereus strain UW85and Bacillus thuringiensis strain YBT-1520. It displays broad-spectrum antimicrobial activity and synergistically activity with the crystal proteins from B. thuringiensis. Its unusual structure and diverse biological activities make us interested in identifying its gene cluster and regulation.The biosynthetic pathway of ZmA in B. cereus strain UW85by analyzing the nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules involved in ZmA biosynthesis has been proposed, but there is no any experimental evidence to show that the ZmA biosynthetic gene gluster is complete and sufficient for ZmA biosynthesis.In this study, we constructed a bacterial artificial chromosome (BAC) library from genomic DNA of B. thuringiensis subsp. kurstaki strain YBT-1520and screened for the presence of genes known to be involved in the biosynthesis of ZmA. The library consists of1200clones, with an average insert size around45kb, and would have a10-fold coverage of the strain YBT-1520genome. Nine positive clones were identified. By the BAC clones terminal sequence result and the strain YBT-1520genome sequence result, these nine clones can overlap a103-kb CONTIG.Two of the nine clones (totally covering approximately60-kb region) are found to be able to confer ZmA biosynthetic capability upon B. thuringiensis strain BMB171after simultaneously transferring them into this surrogate host by a series of compatible shuttle BAC vectors constructed in this study (pEMB0557, pEMB0603or pEMB0606). Another gene cluster containing three previously unidentified ORFs (named zmaWXY), downstream the60-kb region, were found contribute to the yield of ZmA after transferring another two BAC clone inserts (totally covering approximately70-kb region, including the60-kb region and zmaWXY) into the host BMB171. They are experimentally defined function as ZmA resistance transporter which expels ZmA from the cells.As we known, bacteria which produces antibiotic always has a corresponding resistance mechanism to protect itself from the antibiotic. According to the earlier reports, the zmaR (the ZmA self-resistance gene which has been identified before) mutant was sensitive to high concentrations of ZmA, but could still produce ZmA at near-wild-type levels without compromising growth compared to the parent strain, suggesting that another mechanism of self-resistance must be present. This additional mechanism of self-resistance has remained undiscovered until now. In this study, the whole mechanism for ZmA self-resistance of the producer strain has been proposed: resistance transporter ZmaWXY recognise ZmA and expel it from the cytoplasmic membrane, thus keeping ZmA molecules out of the cell; if the concentration of ZmA in the culture supernatants is higher than the concentration ZmaWXY can cope with, ZmA molecules could diffuse into the cell; once the ZmA molecules get into the cell, ZmaR acetylates ZmA, thereby inactivating it and rendering the cell resistant to ZmA.Gene knockout experiment showed that a previously unidentified gene orfl23which locates in the60-kb region maybe act as a potential negative regulatory gene of ZmA biosynthesis. On the flanking regions of the two gene clusters are putative transposase genes which implies the mobile nature of these two gene clusters.The ZmA intact gene cluster was validated by heterologous expression and is proposed to comprise three parts:biosynthesis, resistance, and regulation. A resistance mechanism of ZmA complementary to zmaR was revealed and the whole mechanism for ZmA self-resistance of the producer strain has been proposed. This study also develops a straightforward strategy to isolate and identify a huge gene cluster from Bacillus, even from bacteria.