Ericin A

Five antibacterial peptides produced by Bacillus subtilis NCIB 3610 were purified, quantified, characterized, and identified in the present study. Cell-free extracts were subjected to three purification protocols employing ammonium sulfate or organic solvent precipitation and their combination, followed by ion-exchange chromatography, solid-phase extraction, and preparative high-performance liquid chromatography (HPLC). The combined ammonium sulfate and organic solvent precipitation extraction protocol presented the best results for peptide purification. In the five fractions that presented antimicrobial activity, antibacterial peptides were quantified by the turbidometric method and by HPLC using nisin for external calibration, with the second providing more accurate results. All peptides were pH- and temperature-resistant and their sensitivity to proteases treatment indicated their proteinic nature. The five peptides were subjected to microwave-assisted acid hydrolysis (MAAH) and following derivatization were analyzed using norleucine as the internal standard, to determine their amino acid content. The identification of the isolated peptides using the UniProt and PubChem databases indicated that the four peptides correspond to UniProt entries of the bacteriocins Subtilosin-A (Q1W152) Subtilosin-SbOX (H6D9P4), Ericin B (Q93GH3), Subtilin (P10946), and the fifth to the non-ribosomal antibacterial lipopeptide surfactin (CID:443592). The amino acid content determination and computational analyses, applied in the present work on the antimicrobial peptides of B. subtilis, proved an efficient screening and quantification method of bacteriocins that could potentially be applied in other bacterial strains. The constructed phylogenetic trees heterogeneity observed across the five peptides investigated might be indicative of competitive advantage of the strain.

Bacillus subtilis RC 218 was originally isolated from wheat anthers as a potential antagonist of Fusarium graminearum, the causal agent of Fusarium head blight (FHB). It was demonstrated to have antagonist activity against the plant pathogen under in vitro and greenhouse assays. The current study extends characterizing B. subtilis RC 218 with a field study and genome sequencing. The field study demonstrated that B. subtilis RC 218 could reduce disease severity and the associated mycotoxin (deoxynivalenol) accumulation, under field conditions. The genome sequencing allowed us to accurately determine the taxonomy of the strain using a phylogenomic approach, which places it in the Bacillus velezensis clade. In addition, the draft genome allowed us to use bioinformatics to mine the genome for potential metabolites. The genome mining allowed us to identify 9 active secondary metabolites conserved by all B. velezensis strains and one additional secondary metabolite, the lantibiotic ericin, which is unique to this strain. This study represents the first confirmed production of ericin by a B. velezensis strain. The genome also allowed us to do a comparative genomics with its closest relatives and compare the secondary metabolite production of the publically available B. velezensis genomes. The results showed that the diversity in secondary metabolites of strains in the B. velezensis clade is driven by strains making different antibacterials.

A lantibiotic gene cluster was identified in Bacillus subtilis A1/3 showing a high degree of homology to the subtilin gene cluster and occupying the same genetic locus as the spa genes in B. subtilis ATCC 6633. The gene cluster exhibits diversity with respect to duplication of two subtilin-like genes which are separated by a sequence similar to a portion of a lanC gene. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analyses of B. subtilis A1/3 culture extracts confirmed the presence of two lantibiotic-like peptides, ericin S (3,442 Da) and ericin A (2,986 Da). Disruption of the lanB-homologous gene eriB resulted in loss of production of both peptides, demonstrating that they are processed in an eriB-dependent manner. Although precursors of ericins S and A show only 75% of identity, the matured lantibiotic-like peptides reveal highly similar physical properties; separation was only achieved after multistep, reversed-phase high-performance liquid chromatography. Based on Edman and peptidase degradation in combination with MALDI-TOF MS, for ericin S a subtilin-like, lanthionine-bridging pattern is supposed. For ericin A two C-terminal rings are different from the lanthionine pattern of subtilin. Due to only four amino acid exchanges, ericin S and subtilin revealed similar antibiotic activities as well as similar properties in response to heat and protease treatment. For ericin A only minor antibiotic activity was found.