Two different lantibiotic-like peptides originate from the ericin gene cluster of Bacillus subtilis A1/3

Abstract

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.

FIG. 1.

Positive-mode DE-MALDI-TOF mass spectra of butanolic extracts from B. subtilis A1/3 culture supernatants of wild-type (A) and mutant GB787 (lanB::pESB1) (B). Peak clusters at m/z 1050 to 1150 and 1450 to 1600 are in the range of cyclic lipopeptides. Signals observed at higher molecular masses (m/z 2989 and 3445) were postulated to be lantibiotic-like peptides because they were not found in the culture of a mutant lacking the lanB-type gene.

FIG. 2.

Antibiotic activity of B. subtilis A1/3 wild type (WT) and of mutant strain GB787 (lanB::pESB1). Thirty-microliter aliquots of the sterilized supernatants taken from 48 h-cultures were tested for growth inhibition of S. carnosus (A), C. michiganensis (B), and M. luteus (C).

FIG. 3.

(A) Map of the opuBD-yvaQPO region of B. subtilis strains 168. Arrows indicate open reading frames deduced by the B. subtilis genome project (38). In the ericin- and subtilin-producing strains, the greater part of the yvaQ gene is replaced by a lan gene cluster. (B) The eri gene cluster of B. subtilis A1/3 comprises 12 genes and about 12.5 kb (GenBank accession no. AF233755). Ericin A- and S-encoding genes eriA and eriS are separated by orf1, which exhibits 96% identity to a gray portion of eriC. (C) The spa gene cluster of B. subtilis ATCC 6633 comprises 10 genes and about 12 kb. The gene order spaIFEG is given according to the revised sequence of spa genes (EMBL Gene Bank accession no. U09819).

FIG. 4.

(A) Alignment of eriA and -S and spaS of B. subtilis A1/3 and 6633. The promoter −10 box of spaS (T. Stein, S. Borchert, P. Kiesau, S. Heinzmann, S. Klöß, C. Klein, M. Helfrich, and K.-D. Entian, unpublished data), the ribosome binding site (R.B.S.), translation initiation (marked by an arrow), and stop codons are indicated in boldface. Identical nucleotides in two sequences are shaded. (B) Alignment of the deduced ericin A and ericin S precursor peptides EriA and -S with precursor peptides of subtilin (SpaS) and nisin (NisA). Conserved residues are shaded, and the cleavage site of the processing protease is symbolized by a vertical line, separating N-terminal leader peptides from C-terminal propeptides. (C) Alignment of matured lantibiotics. Thioether bridging of subtilin and nisin and proposed bridges of ericin S and ericin A are shown. Posttranslational modified residues of the lantibiotics are given in boldface.

FIG. 5.

MALDI-TOF mass spectra of reversed-phase HPLC-purified ericins A and S. (A) The most prominent peak corresponds to protonated ericin A; a molecular mass of 2,986.7 Da was determined using the monoisotopic signal (inset). (B) The prominent signal corresponds to the protonated species of ericin S; a molecular mass of 3,342.8 Da was determined using the monoisotopic signal (inset). Two minor signals can be attributed to traces of ericin A (black dot) and degraded ericin S species at m/z 3142.2 (star), from which pyruvyl-lysine was removed. Protonated ericin clusters were measured with a resolution of about 12,500.

FIG. 6.

Antibiotic activity of reversed-phase HPLC- purified ericin S (A), ericin A (B), and subtilin (C). An amount (0.4 μg) of purified peptides (in water, pH 2, with HCl) was poured per well and tested for growth inhibition of S. carnosus, B. megaterium, M. luteus, L. sake, and C. michiganensis. The inhibition zones are from one homologous series of indicator plates.