Role of the single regulator MrsR1 and the two-component system MrsR2/K2 in the regulation of mersacidin production and immunity

Abstract

The lantibiotic mersacidin is an antimicrobial peptide of 20 amino acids which inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II and which is produced by Bacillus sp. strain HIL Y-85,54728. The structural gene of mersacidin as well as accessory genes is organized in a biosynthetic gene cluster which is located on the chromosome and contains three open reading frames with similarities to regulatory proteins: mrsR2 and mrsK2 encode two proteins with homology to bacterial two-component systems, and mrsR1 shows similarity to a response regulator. Both mrsR2/K2 and mrsR1 were inactivated by insertion of an antibiotic resistance marker. Disruption of mrsR1 resulted in loss of mersacidin production; however, producer self-protection was not impaired. In contrast, inactivation of mrsR2/K2 led to an increased susceptibility to mersacidin whereas biosynthesis of the lantibiotic remained unaffected. Binding of mersacidin to intact cells was significantly enhanced in the mrsR2/K2 knockout mutant. Reverse transcription-PCR analysis from total RNA preparations showed that in contrast to the wild-type strain, the structural genes of the ABC transporter MrsFGE were not transcribed in the knockout mutant. It was therefore concluded that producer self-protection against mersacidin is conferred by the ABC transporter MrsFGE and that the transcription of mrsFGE is regulated by MrsR2/K2, whereas production of the antibacterial peptide is solely activated by MrsR1.

FIG. 1.

Organization of the biosynthetic gene cluster of mersacidin, which is located between the genes yxdJ and iolJ (broken lines) on the chromosome of the producer strain: 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).

FIG. 2.

RP-HPLC of culture supernatants from the wild-type producer strain (A), Bacillus sp. strain TTΔmrsR1 (B), and Bacillus sp. strain TTΔmrsR2/K2 (C). The peak that displayed antibacterial activity and coeluted with mersacidin is marked in black.

FIG. 3.

Growth of the wild-type producer strain (A), Bacillus sp. strain TTΔmrsR1 (B), and Bacillus sp. strain TTΔmrsR2/K2 (C) in the presence of mersacidin at concentrations of 7.5 (▵), 10 (▴), 20 (○), and 25 (•) μg/ml; ⧫, control.

FIG. 4.

Binding of [¹⁴C]dimethyl-mersacidin (10 μg/ml) to cells of Bacillus sp. strain TTΔmrsR2/K2 (white bars) and the wild-type strain (black bars) and growth of Bacillus sp. strain TTΔmrsR2/K2 (•) and the wild-type strain (▴) in the presence of [¹⁴C]dimethyl-mersacidin (10 μg/ml).

FIG. 5.

RT-PCR of total RNA of Bacillus sp. strain TTΔmrsR2/K2 and the wild-type strain. Cells were harvested after 8 (A) or 16 (B) h of incubation. Slots 3, 5, 8, and 10 contain control reactions for the samples presented in slots 2, 4, 7, and 9, respectively. These controls were performed to rule out contamination with chromosomal DNA by introducing parallel samples into the thermal cycler during the 95°C step, thereby ensuring immediate denaturation of the reverse transcriptase enzyme. Slots 1 and 11, GeneRuler DNA ladder mix; slot 2, RT-PCR of the mrsEFG transcript in the wild-type Bacillus sp. strain HIL Y-85,54728 with RT1/RT2; slot 3, control; slot 4, RT-PCR of the mrsEFG-transcript in Bacillus sp. strain TTΔmrsR2/K2; slot 5, control; 6, GeneRuler 100-bp DNA ladder mix (1.031, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, and 0.2 kb); slot 7, RT-PCR of the mrsA transcript in Bacillus sp. strain HIL Y-85,54728 using RT4/RT5; slot 8, control; slot 9, RT-PCR of the mrsA-transcript in Bacillus sp. strain TTΔmrsR2/K2; slot 10, control.