Article Figures & Data
Figures
- 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.
Tables
- TABLE 1.
Strains used in this study
Strain Description Reference or source Bacillus sp. strain HIL Y-85,54728 Wild-type producer strain of mersacidin 5 Bacillus sp. strain TT Wild-type producer strain that has been transformed and cured again twice This study Bacillus sp. strain TT ΔSIGH1.1 spo0H knockout mutants of the mersacidin producer strain This study ΔSIGH1.2 ΔSIGH1.3 ΔSIGH1.4 Bacillus subtilis 168 IS20 spo0H mutant; trpC2 25 Bacillus sp. strain TT ΔmrsR2/K2 ΔmrsR2/K2 knockout mutant 11 Bacillus sp. strain TT ΔmrsR1 ΔmrsR1 knockout mutant 11 Bacillus sp. strain HIL Y-85,54728 Rec1 Inactivation of mrsA by an erythromycin resistance cassette by double crossover; no mersacidin production 1 Bacillus sp. strain TTEX Inactivation of mrsA by a stop codon in the leader peptide This study Staphylococcus carnosus TM 300 Transformation host 28 Staphylococcus carnosus TM 300(pTV0MCS) Indicator strain 11 Micrococcus luteus ATCC 4698 Indicator strain ATCC, Manassas, Va. - TABLE 2.
Primers used in this study
Primer Sequence 5′mrsAR1 5′AGAAATATGAATTCATCTTAAGACTCTTTATTTAAC3′ 3′mrsAR1 5′TTGGGTCAAGCTTTTTACACGAC3′ SigmaH5′ 5′TATGGTACCATAGGGGCGCACAGAGAGGATA3′ SigmaH3′ 5′CTTTCTAGATCTCCCATTTTCATTTCAAT3′ SigH1 5′GTGAATCTACAGAACAAC3′ SigH2 5′GTACTTCTCCAGCTTGCG3′ pTV0mcsIns-1 5′GATTTACATATGAGTTATGCAG3′ pTV0mcsIns-2 5′CGCTCATGGTCAATATCATC3′ RT-5 5′ATTAACAAATACATTCAGAAGTTAGAGTAC3′ oligo-dT-anchor primer 5′GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTV3′a PCR anchor primer 5′GACCACGCGTATCGATGTCGAC3′ RT-12 5′GAGTACAAACACCGCCGCCA3′ RT-4 5′TGAGTCAAGAAGCTATCATTCGTTC3′ 5′16S rRNA lang 5′GGGAGCTTGCTCCGATGTTAG3′ 3′16S rRNA lang rev 5′CGGCTGGCTCCTAAAAGGTTAC3′ 5′16S rRNA 5′TCCGCAATGGACGAAAGTCTGAC3′ 3′16S rRNA 5′CCCCAGTTTCCAATGACCCTCC3′ E4stopmersacidinb 5′GAATACAATGAGTCAATAAGCTATCATTCGTT3′
