Genome-wide mapping of gene essentiality in Pseudomonas chlororaphis ATCC 9446 using transposon mutagenesis.

Abstract

Pseudomonas chlororaphis ATCC 9446 is a non-pathogenic rhizobacterium with biotechnological relevance as a biocontrol agent and a promising chassis for synthetic biology. Understanding which genes are strictly required for survival is fundamental to both bacterial physiology and chassis engineering. Here, we generate a genome-wide map of genetic essentiality for P. chlororaphis using high-density Random Barcoded Transposon Mutagenesis (RB-TnSeq). To convert gene-level annotation into biological insight, we layered functional assignments from complementary annotation pipelines, integrating orthology/domain classifiers, ontology mapping, protein export, and delineation of secondary metabolism. These overlays reveal that essentiality concentrates in canonical information processing, envelope biogenesis, and central energy/cofactor and nucleotide metabolism, while large genomic regions are non-essential and therefore represent safe candidates for streamlining and pathway installation. Mapping essentiality onto biosynthetic gene clusters (BGCs) shows that most pathways are dispensable, but some essential genes co-localize, clarifying boundaries for safe editing around BGC loci. Comparison of experimentally determined essential genes with in silico predictions across additional P. chlororaphis genomes show strong overall agreement. Conversely, 32 essential gene orthogroups were found to be conserved across most genomes, yet were classified as non-essential by a prediction algorithm. Together, the resolved essential genome and its integrative functional interpretation provide a durable reference for P. chlororaphis biology and a functional blueprint that can be leveraged for rational streamlining in agricultural, biocontrol and industrial biotechnology. Data summaryThe complete genome sequence of Pseudomonas chlororaphis subsp. chlororaphis ATCC 9446 is publicly available in NCBI under RefSeq accession NZ_CP144767.1 (BioProject PRJNA224116; BioSample: SAMN39889236; assembly GCF_036689615.1). Gene annotation, essentiality calls, insertion mapping outputs, and gene-level insertion statistics are provided in Supplementary Table S1. Predicted biosynthetic gene clusters were identified using antiSMASH v8.0 (bacterial version; https://antismash.secondarymetabolites.org); an annotated GenBank file including BGC coordinates is provided as Supplementary File 3 and a summary of BGC features is provided in Supplementary Table S2. Signal peptides were predicted using SignalP 6.0 (https://services.healthtech.dtu.dk/service.php?SignalP-6.0). Protein-coding sequences were functionally annotated using eggNOG-mapper v2 with the eggNOG 5.0 database (https://github.com/eggnogdb/eggnog-mapper), producing Gene Ontology terms and COG functional classifications. Additional functional annotations, including SEED Subsystems classifications, were obtained from BV-BRC (https://www.bv-brc.org/view/Genome/333.24) and are included in Supplementary Table S1. Transposon insertion processing was performed using the FEBA pipeline (PoolStats.R and associated scripts; https://bitbucket.org/berkeleylab/feba), and essential gene inference was performed using the Bio-Tradis pipeline (https://github.com/sanger-pathogens/Bio-Tradis). Comparative essentiality predictions were obtained using DELEAT (https://github.com/jime-sg/deleat). Raw sequencing reads of transposon-genome junctions are available in the NCBI Sequence Read Archive (SRA) under BioProject accession PRJNA1436027.