Comparative essentialome analysis of six Pectobacteriaceae strains using the TNSEEK pipeline identifies conserved and strain-specific fitness determinants

Transposon sequencing (Tn-seq) is a powerful technique for defining the essential genes required for bacterial survival. However, gene essentiality can vary significantly across taxonomic levels, and comparing large Tn-seq datasets from multiple strains presents considerable analytical challenges. To address this, we developed TNSEEK, a fully automated bioinformatics pipeline for the systematic and comparative analysis of transposon sequencing experiments. We applied TNSEEK to analyze six Soft Rot Pectobacteriaceae (SRP) strains, encompassing species from the Dickeya and Pectobacterium genera, grown in a rich medium. This approach identified a core essentialome of 225 genes, primarily involved in fundamental cellular maintenance, conserved across all six strains, a set comparable in size to that of the broader Enterobacteriaceae family. Only a few genus-specific essential genes were found highlighting interesting distinct metabolic capabilities between Dickeya and Pectobacterium genera. In striking contrast, we discovered a large variable essentialome comprising 181 strain-specific genes, many of which of unknown function. A portion of these strain-specific essential genes are components of defense systems and prophage genomic regions. The unexpected essentiality of these modules suggests they form a constitutively active frontline defense. Furthermore, a comparison with the E. coli essentialome demonstrates that discrepancies in gene essentiality can often be attributed to differences in growth conditions, particularly temperature, as well as variations in genetic redundancy. In conclusion, the TNSEEK pipeline is a robust tool for exploring functional genomics across multiple strains. IMPORTANCEDickeya and Pectobacterium are two genera of the Pectobacteriaceae family that contain mainly plant pathogenic bacteria. To analyze the diversity within bacteria of this family, we performed a Tn-seq analysis on six strains representing a range of ecological niches. To this aim we developed a bioinformatics pipeline, termed TNSEEK which allows the comparative analysis of results across diverse experimental conditions and multiple strains. Applied to growth in rich medium of the tested strains, it allowed the identification of an essentialome of 225 genes at the family level, few genus-specific essential genes but a large essentialome comprising 181 strain-specific genes, many of which of unknown function. Thus, TNSEEK proved its ability to analyze a large data set coming from different Tn-seq experiments and it offers an unparalleled flexibility in handling any number of strains and experiments.