Pseudomycoicidin, a Class II Lantibiotic from Bacillus pseudomycoides

Abstract

Lantibiotics are ribosomally synthesized antimicrobial peptides with substantial posttranslational modifications. They are characterized by the unique amino acids lanthionine and methyllanthionine, which are introduced by dehydration of Ser/Thr residues and linkage of the resulting dehydrated amino acids with Cys residues. BLAST searches using the mersacidin biosynthetic enzyme (MrsM) in the NCBI database revealed a new class II lantibiotic gene cluster in Bacillus pseudomycoides DSM 12442. Production of an antimicrobial substance with activity against Gram-positive bacteria was detectable in a cell wash extract of this strain. The substance was partially purified, and mass spectrometric analysis predicted a peptide of 2,786 Da in the active fraction. In order to characterize the putative lantibiotic further, heterologous expression of the predicted biosynthetic genes was performed in Escherichia coli. Coexpression of the prepeptide (PseA) along with the corresponding modification enzyme (PseM) resulted in the production of a modified peptide with the corresponding mass, carrying four out of eight possible dehydrations and supporting the presence of four thioether and one disulfide bridge. After the proteolytic removal of the leader, the core peptide exhibited antimicrobial activity. In conclusion, pseudomycoicidin is a novel lantibiotic with antimicrobial activity that was heterologously produced in E. coli.

FIG 1

(A) Putative lantibiotic gene cluster in Bacillus pseudomycoides consists of the precursor gene pseA (gray), modification gene pseM (black), the transporter pseT (white), and three immunity genes, pseEFG (diamond pattern; bpmyx0001_45490, pseE; bpmyx0001_45500, pseG; and bpmyx0001_45510, pseF). The numbers indicate the locus tags of the genes. The arrows indicate the relative direction of transcription. (B) Amino acid sequence of PseA. The cysteine residues and possible dehydration sites are shown in gray. The cleavage site is indicated by the box. The predicted core peptide region is underlined. (C) Amino acid sequence alignment of PseA with class II lantibiotics: haloduracin (HalA1), mersacidin (MrsA), and actagardine (ActA). Amino acid identities are highlighted in light gray, while the lipid II binding motif (TxS/TxE/DC) is highlighted in dark gray.

FIG 2

(A) Antibacterial spectrum of the B. pseudomycoides cell wash extract. The cell extract showed activity against most Gram-positive bacteria. (B) Effect of the antimicrobial compound on the growth of M. luteus (■) and S. aureus SG511 (•). The bacterial cultures treated with the antimicrobial substance are indicated by a dashed line, while the untreated controls are indicated by a solid line. The antimicrobial substance was added after 5 h of growth.

FIG 3

(A) MALDI-TOF mass spectrum of the HPLC-purified isopropanol cell wash extract of B. pseudomycoides with a mass signal of m/z 2,786.0. (Inset) Zone of inhibition produced by the HPLC fraction of cell wash extract against M. luteus. (B) Heterologously expressed PseAXa after cleavage of the leader by factor Xa with a mass signal of m/z 2,785.7 for the core peptide and three mass signals (m/z 2,171.3, 3,526.5, and 5,679.8) representing the leader fragments. a.u., arbitrary units.

FIG 4

(A) MALDI-TOF mass spectrum of heterologously produced PseAXa with a mass signal of m/z 10,203.7. (B) Peptide after IAA treatment in the presence of TCEP, with an observed mass of 10,314.3 Da and expected mass of 10,318.6 Da (m − 4H₂O + 2 IAA adducts). (C) PseA after β-ME treatment; it did not show the addition of β-ME (observed mass, 10,205.4 Da), but the mass difference of 2 Da indicates reduction of the disulfide bridge.

FIG 5

Bioactivity assay against the indicator strain M. luteus. Spot 1, Tris buffer; spot 2, heterologously produced PseAXa before treatment with factor Xa; spot 3, modified PseAXa after treatment with factor Xa (the zone of inhibition is shown by an arrow). Spot 4 was not used.