ApnI, a Transmembrane Protein Responsible for Subtilomycin Immunity, Unveils a Novel Model for Lantibiotic Immunity

Abstract

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.

Endophyte Bacillus subtilis evade plant defense by producing lantibiotic subtilomycin to mask self-produced flagellin

“…4), and this result linked the activity to our previously identified lantibiotic subtilomycin, which has been determined with an exact molecular weight of 3234.36 Da 27 . The Apn5 analog sample from subtilomycin mutant ∆apnB (an essential gene encoding subtilomycin biosynthetical enzyme for dehydration reaction was inactivated 27 ) lost antibacterial activity (Fig. 1c).…”

“…The bacterial strains used in this study included wild-type Bacillus subtilis BSn5 isolated from A. konjac 35 , BSn5 ΔapnB (subtilomycin deficiency, a subtilomycin biosynthetical enzyme for essential dehydration reaction was inactivated) 27 , BSn5 Δhag , BSn5 ΔymdB , and E. coli strains BL21 and DH5α. B. subtilis strains were cultured in Luria-Bertani (LB) medium at 28 °C, and Escherichia coli was routinely cultured in LB medium at 37 °C.…”

Differentiation of Bacillus thuringiensis From Bacilluscereus Group Using a Unique Marker Based on Real-Time PCR

“…The HTH proteins, together with sigma factors, participate in a wide range of signaling pathways, for example, as repressors that inhibit sporulation (Murawska et al, 2014), biofilm formation (Colledge et al, 2011), and protease secretion (Pflughoeft et al, 2011). Apart from Cry1Ab21 and δ-endotoxin encoded in the toxigenic plasmid pIS56-63, which are responsible for conjugation and sporulation (Martínez-Núñez et al, 2010; Deng et al, 2014) in addition with 53 different encoded putative proteins.…”

Structural insights into DNA recognition by AimR of the arbitrium communication system in the SPbeta phage

“…Wild-type and mutant aimR genes with their own promoters were cloned into the pDG1730 vector. These plasmids were transformed into B. subtilis CU1065 and integrated into the amyE locus of the B. subtilis genome, as previously described 25 . Phage SPbeta was induced from B. subtilis strain 168 using 0.5 μg/ml mitomycin C (MedChemExpress, HY-13316) for 3 h and was then purified and amplified in B. subtilis strain CU1065 26 .…”