Discovery of a Novel Antimicrobial Peptide from Paenibacillus sp. Na14 with Potent Activity Against Gram-Negative Bacteria and Genomic Insights into Its Biosynthetic Pathway

Abstract

Background/objectives: Antimicrobial resistance (AMR) contributes to millions of deaths globally each year, creating an urgent need for new therapeutic agents. Antimicrobial peptides (AMPs) have emerged as promising candidates due to their potential to combat AMR pathogens. This study aimed to evaluate the antimicrobial activity of an AMP from a soil-derived bacterial isolate against Gram-negative bacteria.

Method: Soil bacteria were isolated and screened for antimicrobial activity. The bioactive peptide was purified and determined its structure and antimicrobial efficacy. Genomic analysis was conducted to predict the biosynthetic gene clusters (BGCs) responsible for AMP production.

Results: Genomic analysis identified the isolate as Paenibacillus sp. Na14, which exhibited low genomic similarity (61.0%) to other known Paenibacillus species, suggesting it may represent a novel species. The AMP from the Na14 strain exhibited heat stability up to 90 °C for 3 h and retained its activity across a broad pH range from 3 to 11. Structural analysis revealed that the Na14 peptide consisted of 14 amino acid residues, adopting an α-helical structure. This peptide exhibited bactericidal activity at concentrations of 2-4 µg/mL within 6-12 h, and its killing rate was concentration-dependent. The peptide was found to disrupt the bacterial membranes. The Na14 peptide shared 64.29% sequence similarity with brevibacillin 2V, an AMP from Brevibacillus sp., which also belongs to the Paenibacillaceae family. Genomic annotation identified BGCs associated with secondary metabolism, with a particular focus on non-ribosomal peptide synthetase (NRPS) gene clusters. Structural modeling of the predicted NRPS enzymes showed high similarity to known NRPS modules in Brevibacillus species. These genomic findings provide evidence supporting the similarity between the Na14 peptide and brevibacillin 2V.

Conclusions: This study highlights the discovery of a novel AMP with potent activity against Gram-negative pathogens and provides new insight into conserved AMP biosynthetic enzymes within the Paenibacillaceae family.

Keywords: Paenibacillus sp. Na14; antimicrobial peptides; biosynthetic gene clusters; non-ribosomal peptide synthetase; soil bacteria.

Figure 1

Bacterial growth curve and kinetics of antibacterial substance production by bacterial isolate Na14.

Figure 2

(a) Reversed-phase HPLC chromatogram of bioactive compounds obtained from the Na14 isolate. The purified fraction was obtained by RPC. Each peak fraction was evaluated for antimicrobial activity against E. coli TISTR 887 using the agar well diffusion method. An asterisk (*) indicates the peak fractions containing the active compounds. (b) SDS-PAGE and gel overlay inhibition assay of the purified Na14 peptide. (Left) Peptides obtained from RPC (L1) were analyzed using 15% SDS-PAGE and visualized by Coomassie blue staining. A protein marker (M) was included for molecular weight comparison. (Right) The SDS-PAGE gel was overlaid with soft agar containing E. coli TISTR 887 and incubated at 37 °C for 18 h. An inhibition zone corresponding to the Na14 peptide was observed.

Figure 3

The de novo sequencing of the Na14 peptide by LC-MS/MS. The fragmentation patterns corresponding to b- and y-ion series were observed, and the resulting mass differences were analyzed to reconstruct the amino acid sequence.

Figure 4

(a) Circular dichroism spectra of the Na14 peptide. The peptide was dissolved in either deionized water (blue) or a 50 mM SDS solution (red). (b) Structural modeling of the Na14 peptide using AlphaFold. The peptide structure is shown in cartoon style with side chains displayed as stick-and-ball models and overlaid with the molecular surface. The surface is color-coded to indicate polarity: blue for positively charged regions, red for negatively charged regions, and green for non-polar areas. (c) Predicted α-helical conformation of the Na14 peptide, generated by HeliQuest. The helical wheel projection illustrates the amphipathic nature of the peptide using single-letter amino acid codes and color shading: blue for polar residues, yellow for non-polar residues, and grey for small residues.

Figure 5

Time–kill kinetics of the Na14 peptide. The Na14 peptides at different concentrations (0.5×, 1×, and 2× MIC) were tested against (a) E. coli TISTR 887, (b) P. aeruginosa TISTR 357, and (c) K. pneumoniae TISTR 1383. Untreated cells served as controls. At specified time intervals, either undiluted or diluted bacterial suspensions were plated on MH agar. Viable cell counts were determined and expressed on a logarithmic scale. The limit of detection (LOD) of the assay was 10 CFU/mL, indicated by a dashed line.

Figure 6

SEM images showing morphological changes in bacterial cells following treatment with the Na14 peptide and colistin at 1× MIC level, compared to the untreated control. Images were captured at 20,000× magnification.

Figure 7

Genomic identification and taxonomic classification of Paenibacillus sp. Na14. (a) The phylogenetic tree illustrates the relationship of the Na14 strain to other members of the Paenibacillus genus, showing the closest taxonomic affiliation to Paenibacillus oleatilyticus SM69. (b) The circular genome map displays annotated genes along with predicted biosynthetic gene clusters associated with secondary metabolite production. NRPS-encoding regions and their domains are visualized in detail.