The lantibiotic mersacidin is an autoinducing peptide

Abstract

The lantibiotic (lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide consisting of 20 amino acids and is produced by Bacillus sp. strain HIL Y-85,54728. The structural gene (mrsA) and the genes for producer self-protection, modification enzymes, transport proteins, and regulator proteins are organized in a 12.3-kb biosynthetic gene cluster on the chromosome of the producer strain. Mersacidin is produced in stationary phase in a synthetic medium (K. Altena, A. Guder, C. Cramer, and G. Bierbaum, Appl. Environ. Microbiol. 66:2565-2571, 2000). To investigate the influence of the alternative sigma factor H on mersacidin biosynthesis, a SigH knockout was constructed. The knockout mutant was asporogenous, and a comparison to the wild-type strain indicated no significant differences concerning mersacidin production and immunity. Characterization of the mrsA promoter showed that the gene is transcribed by the housekeeping sigma factor A. The biosynthesis of some lantibiotic peptides like nisin or subtilin is regulated in a cell-density-dependent manner (M. Kleerebezem, Peptides 25:1405-1414, 2004). When mersacidin was added at a concentration of 2 mg/liter to an exponentially growing culture, an earlier production of antibacterial activity against Micrococcus luteus ATCC 4698 in comparison to that of the control culture was observed, suggesting that mersacidin itself functions as an autoinducer. In real-time PCR experiments, the expression of mrsA was remarkably increased in the induced culture compared to the control. In conclusion, mersacidin is yet another lantibiotic peptide whose biosynthesis can be regulated by an autoinducing mechanism.

FIG. 1.

(A) Mersacidin biosynthesis gene cluster. Organization of the biosynthetic gene cluster of mersacidin, which is located on the chromosome of the producer strain Bacillus sp. strain HIL Y-85,54728. Shown are the structural gene (striped arrow), genes necessary for modification and export of mersacidin (white arrows), genes involved in regulation (checkered arrows), and genes for producer self-protection (black arrows). (B) Plasmids pOPAR1 and pPAR1. The plasmids differ in the presence of the putative operator region between the EcoRV and EcoRI sites in plasmid pOPAR1. O stands for operator, P for promoter, A for the structural gene mrsA (striped arrows), and R1 for mrsR1 (checkered arrows). (C) Nucleotide sequence of the promoter region of mrsA. Total RNA of the producer strain was isolated, and the 5′ end of the RNA was identified by a 5′ RACE. The start codon, −10 Pribnow box, −35 region, EcoRI and EcoRV restriction sites, and the ribosome binding site (rbs) are underlined. The 5′ end of the mRNA is shown in bold letters.

FIG. 2.

Agar diffusion assay. (A) Mersacidin production of the wild-type producer strain Bacillus sp. strain HIL Y-85,54728 (1), Bacillus sp. strain HIL Y-85,54728 Rec1 (2), Bacillus sp. strain TT ΔSIGH1.1 (3), Bacillus sp. strain TT ΔSIGH1.2 (4), Bacillus sp. strain TT ΔSIGH1.3 (5), and Bacillus sp. strain TT ΔSIGH1.4 (6) in synthetic medium was analyzed by agar diffusion assay using Staphylococcus carnosus TM 300(pTV0MCS) as the indicator strain. (B) Production of bacteriocins other than mersacidin by Bacillus sp. strain HIL Y-85,54728 (1), Bacillus subtilis 168 1S20 (2), Bacillus sp. strain TT ΔSIGH1.1 (3), Bacillus sp. strain TT ΔSIGH1.2 (4), Bacillus sp. strain TT ΔSIGH1.3 (5), and Bacillus sp. strain TT ΔSIGH1.4 (6) in LB using Micrococcus luteus ATCC 4698 as the indicator organism.

FIG. 3.

Induction of the production of antibacterial activity by mersacidin. Growth and production of antibacterial activity by Bacillus sp. strain HIL Y-85,54728 (A, C) or Bacillus sp. strain TT ΔSIGH1.1 (B). In panels A and B, the antibacterial activity produced by the cells after the addition of 2 ml of sterilized 16-h supernatant (▪), or pure mersacidin at a concentration of 2 mg/liter (▴), and the control (no addition) (⧫) are plotted against the growth of the cultures. In panel C, production of antibacterial activity by Bacillus sp. strain HIL Y-85,54728 after addition of vancomycin (0.5× MIC) (□), mersacidin (0.25 mg/liter) (×), mersacidin (0.5 mg/liter) (⋄), 16-h supernatant of Bacillus sp. strain TTEX (Δ), and the control (no addition) (⧫) are plotted against the optical density (OD₆₀₀) of the cultures. Micrococcus luteus ATCC 4698 was used as the indicator strain in an agar diffusion assay. The addition of mersacidin/vancomycin or supernatant is marked by an arrow.

FIG. 4.

Influence of an autoinducing mechanism in mersacidin biosynthesis. Percentage of copies of mrsA per 10⁶ copies of 16S rRNA after addition of mersacidin (2 mg/liter) to the wild-type producer (Bacillus sp. strain HIL Y-85,54728) and knockout mutants (Bacillus sp. strain TT ΔmrsR2/K2 and Bacillus sp. strain TT ΔmrsR1). The strains were grown to an optical density at 600 nm of 0.5, mersacidin was added, and aliquots were withdrawn after 15 min (white bars) and after 60 min (black bars) for real-time PCR. Controls show the transcript levels of cultures grown in the absence of mersacidin. The transcript levels of the controls (no addition) measured 15 min after addition of mersacidin to the test cultures were set to 100% transcription.