Oct 29, 2025
Computational workflow for the study “PBP2 as the Predominant Siderophore Recognizer in Bacillus”
- Linlong Yu1
- 1Peking University
- LinlongYu
Protocol Citation: Linlong Yu 2025. Computational workflow for the study “PBP2 as the Predominant Siderophore Recognizer in Bacillus”. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly45dmlx9/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 29, 2025
Last Modified: October 29, 2025
Protocol Integer ID: 231023
Keywords: siderophore biosynthetic gene cluster, pbp2 as the predominant siderophore recognizer, biosynthetic gene cluster, predominant siderophore recognizer, bacillus, genome retrieval, genome mining, sequence clustering, siderophore, phylogenetic reconstruction, pbp2, motif scanning, taxonomic classification, computational workflow for the study
Abstract
This protocol describes the complete computational workflow used to identify, classify, and analyze siderophore biosynthetic gene clusters (BGCs) and their cognate receptors. All analyses were performed using version-controlled scripts and publicly available databases to ensure full reproducibility. The workflow includes genome retrieval, taxonomic classification, genome mining, phylogenetic reconstruction, sequence clustering, mutual information analysis, and motif scanning.
Guidelines
4. Define feature sites as residues with MI values greater than 85% of the theoretical maximum MI in Bacillus.
Step 7: Motif scanning
1. Construct a Fur-binding PWM using 19 validated Bacillus subtilis motifs via MEME Suite. Obtain the DmdR1 PWM from the LogoMotif database.
2. Scan upstream regions (150 bp) of all genes using FIMO (p < 0.001).
- Fur PWM applied to Bacillus genomes
- DmdR1 PWM applied to Streptomyces genomes
Materials
Category | Tool / Database | RRID
--- | --- | ---
Genome database | NCBI RefSeq | SCR_003496
Taxonomy assignment | GTDB-Tk (release R220) | SCR_019136
Phylogenetic reconstruction | PhyloPhlAn3 | SCR_013082
Genome mining | antiSMASH v7.0.0 | SCR_022060
Known cluster comparison | MIBiG database | SCR_023660
Domain identification | HMMER v3.0 / Pfam | SCR005305 / SCR004726
Sequence alignment | Clustal Omega | SCR_001591
Statistical analysis | MATLAB R2024a | SCR_001622
Motif discovery / scanning | MEME Suite v5.5.7 | SCR_001783
Structure annotation | DSSP | SCR_016067
Troubleshooting
Procedure
Retrieve complete genomes from the NCBI RefSeq database (as of January 21, 2025).
Assign taxonomy using GTDB-Tk (release R220).
Run antiSMASH v7.0.0 on all annotated genomes.
Identify BGCs annotated as NRP-metallophore or NIS-siderophore.
Compare predicted clusters against the MIBiG database (similarity ≥80%) using the knownclusterblast module.
Use PhyloPhlAn3 to extract and align 400 universal marker genes.
Build a phylogenetic tree and visualize it with the R package ggtree.
Extract all core biosynthetic genes from siderophore BGCs.
Compute pairwise p-distance values.
Apply hierarchical clustering; determine the optimal threshold using silhouette scores.
Compute pairwise distance matrices for biosynthetic and candidate genes.
Calculate Pearson correlation coefficients in MATLAB using the corr function.
Define feature sites as residues with MI values 3e85% of the theoretical maximum in Bacillus.
Collect six experimentally validated siderophore receptors from Bacillus species based on published literature.
Use BLASTP (RRID:SCR_001010) to identify homologous receptor sequences across all Bacillus genomes.
Perform multiple sequence alignment and calculate mutual information (MI) between alignment positions and receptor identity labels.
Define feature sites as residues with MI values greater than 85% of the theoretical maximum MI in Bacillus.
Construct a Fur-binding PWM using 19 validated Bacillus subtilis motifs via MEME Suite. Obtain the DmdR1 PWM from the LogoMotif database.
Scan upstream regions (150 bp) of all genes using FIMO (p 3c 0.001).
Fur PWM applied to Bacillus genomes
DmdR1 PWM applied to Streptomyces genomes
Acknowledgements
Data and Code Availability
All genomes are available from NCBI RefSeq (RRID:SCR_003496).
All scripts and analysis pipelines are publicly available on GitHub: https://github.com/Linlong-Yu/PBP2-as-the-Predominant-Siderophore-Recognizer
Expected Output
- Annotated siderophore BGCs
- Hierarchical clustering dendrograms of biosynthetic genes
- Coevolutionary correlation matrices
- Predicted Fur- and DmdR1-binding site lists and motif logos
Version Control
All analyses were executed using version-controlled scripts with fixed random seeds to ensure reproducibility.