Isolation and identification of a Paenibacillus polymyxa strain that coproduces a novel lantibiotic and polymyxin

Abstract

A new bacterial strain, displaying potent antimicrobial properties against gram-negative and gram-positive pathogenic bacteria, was isolated from food. Based on its phenotypical and biochemical properties as well as its 16S rRNA gene sequence, the bacterium was identified as Paenibacillus polymyxa and it was designated as strain OSY-DF. The antimicrobials produced by this strain were isolated from the fermentation broth and subsequently analyzed by liquid chromatography-mass spectrometry. Two antimicrobials were found: a known antibiotic, polymyxin E1, which is active against gram-negative bacteria, and an unknown 2,983-Da compound showing activity against gram-positive bacteria. The latter was purified to homogeneity, and its antimicrobial potency and proteinaceous nature were confirmed. The antimicrobial peptide, designated paenibacillin, is active against a broad range of food-borne pathogenic and spoilage bacteria, including Bacillus spp., Clostridium sporogenes, Lactobacillus spp., Lactococcus lactis, Leuconostoc mesenteroides, Listeria spp., Pediococcus cerevisiae, Staphylococcus aureus, and Streptococcus agalactiae. Furthermore, it possesses the physico-chemical properties of an ideal antimicrobial agent in terms of water solubility, thermal resistance, and stability against acid/alkali (pH 2.0 to 9.0) treatment. Edman degradation, mass spectroscopy, and nuclear magnetic resonance were used to sequence native and chemically modified paenibacillin. While details of the tentative sequence need to be elucidated in future work, the peptide was unequivocally characterized as a novel lantibiotic, with a high degree of posttranslational modifications. The coproduction of polymyxin E1 and a lantibiotic is a finding that has not been reported earlier. The new strain and associated peptide are potentially useful in food and medical applications.

FIG. 1.

Scanning electron microscope observation of Paenibacillus polymyxa OSY-DF.

FIG. 2.

High-performance liquid chromatography profile of a crude extract of Paenibacillus polymyxa OSY-DF culture supernatant. Fraction A has activity against Escherichia coli K-12; fraction B has activity against Lactobacillus plantarum ATCC 8014.

FIG. 3.

LC-MS profile of a crude extract of Paenibacillus polymyxa OSY-DF culture supernatant.

FIG. 4.

Verification of molecular mass and purity of compound B, as determined by MALDI-TOF MS analysis (main figure) and SDS-PAGE (figure insert). Lanes 1 and 2, molecular mass marker (in kilodaltons) and purified paenibacillin, stained with Coomassie blue G-250; lane 3, purified paenibacillin, overlaid with soft agar containing Lactobacillus plantarum ATCC 8014 after incubation.

FIG. 5.

MS/MS sequencing of native paenibacillin. The partial sequence was identified as -Leu/Ile-Leu/Ile-Lys/Gln-Dhb-Dhb-Leu/Ile-Lys/Gln-. Leu/Ile and Lys/Gln cannot be differentiated due to the identical and similar molecular masses, respectively.

FIG. 6.

NMR evidence for the existence of two Dhb residues in paenibacillin. The expansions of NMR spectra show the unique quartet peaks of H^β at ∼6.7 ppm in one-dimensional ¹H-NMR spectroscopy (top) and the through-bond cross-peaks between H^β and H^γ (∼1.80 ppm) (left) in the TOCSY experiment (middle).

FIG. 7.

MS/MS sequencing results of the modified paenibacillin; Lys 5, 9, 12, 16, and 30 cannot be differentiated from Gln due to the similar mass, whereas Ile in 3, 4, 8, 18, and 21 cannot be distinguished from Leu due to the identical mass.

FIG. 8.

MALDI-TOF MS analysis of the tryptic digestion product of modified paenibacillin, confirming the presence of Lys5, 9, 12, and 16, whereas Lys/Gln30 remains undetermined; Leu and Ile also remain undistinguishable.