Calculation of essential genes.

(A) Comparison of the insertion index scores of two technical replicates of the transposon mutant library. (B) Bi-modal distribution of the total insertion index scores for the transposon library. The exponential distribution fit to the left mode includes the essential genes (red), and the gamma distribution fit to the right mode captures the nonessential genes (blue). (TIF)</p

Bi-modal essential gene analysis.

Diphtheria is a respiratory disease caused by Corynebacterium diphtheriae. While the toxin-based vaccine has helped control outbreaks of the disease since the mid-20th century there has been an increase in cases in recent years, including systemic infections caused by non-toxigenic C. diphtheriae strains. Here we describe the first study of gene essentiality in C. diphtheriae, providing the most-dense Transposon Directed Insertion Sequencing (TraDIS) library in the phylum Actinobacteriota. This high-density library has allowed the identification of conserved genes across the genus and phylum with essential function and enabled the elucidation of essential domains within the resulting proteins including those involved in cell envelope biogenesis. Validation of these data through protein mass spectrometry identified hypothetical and uncharacterized proteins in the proteome which are also represented in the vaccine. These data are an important benchmark and useful resource for the Corynebacterium, Mycobacterium, Nocardia and Rhodococcus research community. It enables the identification of novel antimicrobial and vaccine targets and provides a basis for future studies of Actinobacterial biology.</div

Essential protein predicted localisation.

Diphtheria is a respiratory disease caused by Corynebacterium diphtheriae. While the toxin-based vaccine has helped control outbreaks of the disease since the mid-20th century there has been an increase in cases in recent years, including systemic infections caused by non-toxigenic C. diphtheriae strains. Here we describe the first study of gene essentiality in C. diphtheriae, providing the most-dense Transposon Directed Insertion Sequencing (TraDIS) library in the phylum Actinobacteriota. This high-density library has allowed the identification of conserved genes across the genus and phylum with essential function and enabled the elucidation of essential domains within the resulting proteins including those involved in cell envelope biogenesis. Validation of these data through protein mass spectrometry identified hypothetical and uncharacterized proteins in the proteome which are also represented in the vaccine. These data are an important benchmark and useful resource for the Corynebacterium, Mycobacterium, Nocardia and Rhodococcus research community. It enables the identification of novel antimicrobial and vaccine targets and provides a basis for future studies of Actinobacterial biology.</div

Conserved essential genes restricted to actinomycetes.

(A) and (B) Transposon insertion data for diphtheriae_00818 and diphtheriae_01854 genes with the respective domains of unknown function (DUF) DUF501 (PF04417) and DUF3073 (PF11273) displayed beneath. Transposon insertion sites are represented by vertical black bars, capped at a frequency of 1. (C) Distribution of homologs of diphtheriae_00818 and diphtheriae_01854 within representative genomes of the Actinobacteria phylum. Bacillus subtilis was used as an outgroup for construction of the tree; only bootstrap values under 100 are shown on the tree. Species are shaded by Class. The presence of a homolog, identified by BLASTP, is indicated by a coloured circle. (TIF)</p

Protein function and abundance plot of the whole and secreted proteome detected by mass spectrometry analysis.

The abundance of the total proteome (A) or secreted proteome (B) detected by mass spectrometry analysis grouped and coloured according to function, with block size corresponding with protein abundance. Essential proteins are highlighted in green.</p

Protein function and predicted localization.

(A) The number of proteins within each functional category for both the total genome and the list of essential genes. (B) The predicted localization of essential proteins. (C) Functional characterization of essential proteins by localization. Abbreviations: Cyt, cytoplasmic; TM, transmembrane; Sec, secreted; Amb, ambiguous.</p

Genes with essential regions.

Diphtheria is a respiratory disease caused by Corynebacterium diphtheriae. While the toxin-based vaccine has helped control outbreaks of the disease since the mid-20th century there has been an increase in cases in recent years, including systemic infections caused by non-toxigenic C. diphtheriae strains. Here we describe the first study of gene essentiality in C. diphtheriae, providing the most-dense Transposon Directed Insertion Sequencing (TraDIS) library in the phylum Actinobacteriota. This high-density library has allowed the identification of conserved genes across the genus and phylum with essential function and enabled the elucidation of essential domains within the resulting proteins including those involved in cell envelope biogenesis. Validation of these data through protein mass spectrometry identified hypothetical and uncharacterized proteins in the proteome which are also represented in the vaccine. These data are an important benchmark and useful resource for the Corynebacterium, Mycobacterium, Nocardia and Rhodococcus research community. It enables the identification of novel antimicrobial and vaccine targets and provides a basis for future studies of Actinobacterial biology.</div

Conservation of essential genes across the <i>Corynebacterium</i> genus.

Heatmap showing the presence/absence of protein orthologs of the 358 essential genes (x-axis) identified in C. diphtheriae in the genomes of 140 representative Corynebacterium species (y-axis) adapted from Dover et al. (2021). Blocks are coloured blue according to percentage identity, above a threshold of 30%, with darker shading corresponding with a higher percentage identity shared with the C. diphtheriae query gene. (PDF)</p

Essential domains of MviN.

(A) Schematic alignment of MurJ orthologs from Corynebacterium diphtheriae, Mycobacterium tuberculosis, Mycobacterium smegmatis, Streptomyces coelicolor, Bacillus subtilis and Escherichia coli. (B) Transposon insertion data of mviN. Transposon insertion sites represented by black vertical bars, capped at a frequency of 1. Essential regions of the coding sequence that coincide with insertion-free regions are highlighted in green within the gene arrow. Protein domain boundaries are plotted below: MurJ lipid-II flippase domain (green), pseudokinase domain (purple). (C) Pairwise sequence comparison of the cytoplasmic pseudokinase domains of Mtb-MviN with Cdip-MviN. Conserved residues are highlighted in red, P-Thr947 indicated by an asterisk (*), with the secondary structure of Mtb-MviN displayed above. (D) Comparison of the crystal structure of the Mtb-MviN pseudokinase domain (grey) and the predicted model of Cdip-MviN pseudokinase domain (purple) from Phyre2.</p

Parameters for TraDIS data set derived from the non-toxigenic <i>C</i>. <i>diphtheriae</i> strain ISS 3319 transposon library.

Parameters for TraDIS data set derived from the non-toxigenic C. diphtheriae strain ISS 3319 transposon library.</p