ApnI, a transmembrane protein responsible for subtilomycin immunity, unveils a novel model for lantibiotic immunity.

Subtilomycin was detected from the plant endophytic strain Bacillus subtilis BSn5 and was first reported from B. subtilis strain MMA7. In this study, a gene cluster that has been proposed to be related to subtilomycin biosynthesis was isolated from the BSn5 genome and was experimentally validated by gene inactivation and heterologous expression. Comparison of the subtilomycin gene cluster with other verified related lantibiotic gene clusters revealed a particular organization of the genes apnI and apnT downstream of apnAPBC, which may be involved in subtilomycin immunity. Through analysis of expression of the apnI and/or apnT genes in the subtilomycin-sensitive strain CU1065 and inactivation of apnI and apnT in the producer strain BSn5, we showed that the single gene apnI, encoding a putative transmembrane protein, was responsible for subtilomycin immunity. To our knowledge, evidence for lantibiotic immunity that is solely dependent on a transmembrane protein is quite rare. Further bioinformatic analysis revealed the abundant presence of ApnI-like proteins that may be responsible for lantibiotic immunity in Bacillus and Paenibacillus. We cloned the paeI gene, encoding one such ApnI-like protein, into CU1065 and showed that it confers resistance to paenibacillin. However, no cross-resistance was detected between ApnI and PaeI, even though subtilomycin and paenibacillin share similar structures, suggesting that the protection provided by ApnI/ApnI-like proteins involves a specific-sequence recognition mechanism. Peptide release/binding assays indicated that the recombinant B. subtilis expressing apnI interacted with subtilomycin. Thus, ApnI represents a novel model for lantibiotic immunity that appears to be common.

[Cloning of a large plasmid pBMB28 in Bacillus thuringiensis].

Bacillus thuringiensis serovar. finitimus strain YBT-020 is a typical strain with the spore-crystal association (SCA) phenotype. In our previous studies, plasmid curing experiment suggested that native plasmid pBMB28 of strain YBT-020 might contribute to the SCA phenotype. Thus, plasmid pBMB28 was cloned in order to isolate the genes related to SCA on pBMB28. Using shuttle vector pEMB0557, a shuttle genomic bacterial artificial chromosome (BAC) library of B. thuringiensis strain YBT-020 was constructed. The plasmid pBMB231 containing crystal protein gene cry28Aa, which was located on plasmid pBMB28, was screened out. By SDS-PAGE analysis and microscopic observation, we discovered the recombinant strain BMB231 that originated from the electrotransfer strain BMB171 with pBMB231 could produce Cry28Aa protein. With the chromosome walking strategy and terminal sequencing of pBMB231, four clones covering the full length of plasmid pBMB28 were screened out from this BAC library. With pulsed gel analysis of the four BAC clones and terminal sequencing, the size of the plasmid was calculated to be 140 kb. This study additionally revealed that we could clone a large plasmid from B. thuringiensis by genomic BAC library construction and overlaping fragment screening.