1. Purification and characterization of two novel antimicrobial peptides Subpeptin JM4-A and Subpeptin JM4-B produced by Bacillus subtilis JM4
Shimei Wu, Shifang Jia, Dandan Sun, Meiling Chen, Xiuzhu Chen, Jin Zhong, Liandong Huan Curr Microbiol. 2005 Nov;51(5):292-6. doi: 10.1007/s00284-005-0004-3. Epub 2005 Oct 5.
An antimicrobial peptides-producing strain was isolated from soil and identified as Bacillus subtilis JM4 according to biochemical tests and 16S rDNA sequence analysis. The corresponding antimicrobial peptides were purified to homogeneity by ammonium sulfate precipitation, sequential SP-Sepharose Fast Flow, Sephadex G-25 and C18 reverse-phase chromatography, and in the final purification step, two active fractions were harvested, designated as Subpeptin JM4-A and Subpeptin JM4-B. The molecular weights, determined by mass spectrometry, were 1422.71 Da for Subpeptin JM4-A and 1422.65 Da for Subpeptin JM4-B, respectively. Amino acid sequencing showed that they differed from each other only at the seventh amino acid except for three unidentified residues, and the two peptides had no significant sequence homology to the known peptides in the database, indicating that they are two novel antimicrobial peptides. In addition, characteristic measurements indicated that both peptides had a relatively broad inhibitory spectrum and remained active over a wide pH and temperature range.
2. Genetics of subpeptin JM4-A and subpeptin JM4-B production by Bacillus subtilis JM4
Shimei Wu, Jin Zhong, Liandong Huan Biochem Biophys Res Commun. 2006 Jun 16;344(4):1147-54. doi: 10.1016/j.bbrc.2006.04.022.
Subpeptin JM4-A and subpeptin JM4-B are two novel antimicrobial peptides produced by Bacillus subtilis JM4. To identify putative genes involved in their production, degenerate PCR primers targeted to conserved motifs of nonribosomal peptide synthetases (NRPSs) were used. A resulting 1.2 kb PCR product had high sequence similarity to genes of NRPSs, and then a 2.8 kb DNA fragment flanking it was cloned subsequently. Gene disruption of the resulting 4 kb DNA fragment produced subpeptin-deficient mutant, suggesting that subpeptin JM4-A and subpeptin JM4-B were biosynthesized by NRPSs. Based on this result, a 48 kb gene cluster was cloned, which consisted of nine coding sequences (CDSs) involved in antimicrobial peptide biosynthesis, regulation, and resistance. Disruption of two relatively large CDSs subA and subC led to subpeptin-deficient mutants, which supported the involvement of the cloned gene cluster in subpeptin biosynthesis.
