Mutational analysis of the sbo-alb locus of Bacillus subtilis: identification of genes required for subtilosin production and immunity

Abstract

The Bacillus subtilis 168 derivative JH642 produces a bacteriocin, subtilosin, which possesses activity against Listeria monocytogenes. Inspection of the amino acid sequence of the presubtilosin polypeptide encoded by the gene sboA and sequence data from analysis of mature subtilosin indicate that the precursor subtilosin peptide undergoes several unique and unusual chemical modifications during its maturation process. The genes of the sbo-alb operon are believed to function in the synthesis and maturation of subtilosin. Nonpolar mutations introduced into each of the alb genes resulted in loss or reduction of subtilosin production. sboA, albA, and albF mutants showed no antilisterial activity, indicating that the products of these genes are critical for the production of active subtilosin. Mutations in albB, -C, and -D resulted in reduction of antilisterial activity and decreased immunity to subtilosin, particularly under anaerobic conditions. A new gene, sboX, encoding another bacteriocin-like product was discovered residing in a sequence overlapping the coding region of sboA. Construction of an sboX-lacZ translational fusion and analysis of its expression indicate that sboX is induced in stationary phase of anaerobic cultures of JH642. An in-frame deletion of the sboX coding sequence did not affect the antilisterial activity or production of or immunity to subtilosin. The results of this investigation show that the sbo-alb genes are required for the mechanisms of subtilosin synthesis and immunity.

FIG. 1

Organization of the sbo-alb operon and the recombination between the integrative plasmid and alb DNA that gives rise to the nonpolar insertion mutation. The example shown is albF. An internal region of the albF gene is amplified by PCR and inserted into the integration vector pAG58-bleo-1. After recombination, a copy of the plasmid is integrated into the alb locus. albF is disrupted, and transcription from the sbo promoter is blocked by the plasmid DNA. Expression of the downstream genes is driven by the IPTG-inducible Pspac promoter.

FIG. 2

Bioautography of extracts from culture fluid of B. subtilis strain JH642 and the sboA and alb mutants. Supernatant fluid from YG liquid batch cultures collected at 36 h was subjected to (NH₄)SO₄ precipitation, followed by methanol extraction. Methanol extracts were evaporated, and the residue was dissolved in 20 mM Tris-HCl buffer, pH 7.0 (see Materials and Methods). Samples from extracts of wild-type and mutant culture supernatants were applied to Tricine SDS-polyacrylamide gels, and bioautography was performed. A zone of inhibition is observed in the area of the L. monocytogenes overlay corresponding to the position of bacteriocin in the gel (indicated by arrow). Lanes: 1, JH642; 2, sboA::neo; 3, albA; 4, albB; 5, albC; 6, albD; 7, albE; 8, albF; 9, albG.

FIG. 3

Complementation of the albB mutation by the amyE::sboAXalbABC′ construct. (A) Genomic organization of albB/amyE::sboAXalbABC′. The circle indicates the B. subtilis genomic map, and the locations of the replication origin (oriC) and termination site (ter) are shown. The amyE and sbo-alb loci are labeled on the genomic map, and the organization of the sbo-alb operon bearing the albB::cat mutation and the sbo-alb DNA of the amyE::sboAXalbABC′ locus is shown. (B) MICs of subtilosin for the wild-type parent, JH642, the albB::cat mutant ORB3400, and the albB::cat/albB diploid ORB3472. An immunity assay was performed on anaerobically grown lawns of cells on 2×YT medium supplemented with glucose and KNO₃ as described in Materials and Methods. The assay was performed in the presence (+C) or absence (−C) of chloramphenicol. NA, not applicable.

FIG. 4

Organization of the sboAX locus. (A) Diagram of the sboA, sboX, and albA region of the sbo-alb gene cluster. Psbo indicates the location of the sbo-alb operon promoter, and the arrow marks the direction of transcription. (B) Nucleotide sequence of the sboAX genes and amino acid sequence (below the horizontal line) of the products. The restriction enzyme sites introduced into the sboX coding sequence (HindIII and BamHI) are indicated. The segment of the sboX gene that is deleted in the sboXΔ89-139 allele is labeled Δ. The BamHI sequence that replaced this segment is indicated. (C) Expression of sboX::lacZ under anaerobic (−O₂) and aerobic (O₂) conditions. An sboX-lacZ translational fusion was introduced into the amyE locus of B. subtilis JH642 cells. WT, wild type.