In vitro exploration of the Xanthomonas hortorum pv. vitians genome using transposon insertion sequencing and comparative genomics to discriminate between core and contextual essential genes

Abstract

The essential genome of a bacterium encompasses core genes associated with basic cellular processes and conditionally essential genes dependent upon environmental conditions or the genetic context. Comprehensive knowledge of those gene sets allows for a better understanding of fundamental bacterial biology and offers new perspectives for antimicrobial drug research against detrimental bacteria such as pathogens. We investigated the essential genome of Xanthomonas hortorum pv. vitians, a gammaproteobacterial plant pathogen of lettuce (Lactuca sativa L.) which belongs to the plant-pathogen reservoir genus Xanthomonas and is affiliated to the family Xanthomonadaceae. No practical means of disease control or prevention against this pathogen is currently available, and its molecular biology is virtually unknown. To reach a comprehensive overview of the essential genome of X. hortorum pv. vitians LM16734, we developed a mixed approach combining high-quality full genome sequencing, saturated transposon insertion sequencing (Tn-Seq) in optimal growth conditions, and coupled computational analyses such as comparative genomics, synteny assessment and phylogenomics. Among the 370 essential loci identified by Tn-Seq, a majority was bound to critical cell processes conserved across bacteria. The remaining genes were either related to specific ecological features of Xanthomonas or Xanthomonadaceae species, or acquired through horizontal gene transfer of mobile genetic elements and associated with ancestral parasitic gene behaviour and bacterial defence systems. Our study sheds new light on our usual concepts about gene essentiality and is pioneering in the molecular and genomic study of X. hortorum pv. vitians.

Keywords: Tn-Seq; Xanthomonadaceae; Xanthomonas hortorum; bacterial leaf spot of lettuce; essential genome; mobile genetic elements.

Fig. 1.

Complete genome atlas of X. hortorum pv. vitians LM16734. The localizations of essential, growth-defect and growth-advantage loci identified by Tn-Seq are indicated on (a) the circular chromosome (5, 213,310 bp) and (b) plasmid pLM16734 (57, 250 bp). The outermost ring track is a clockwise-arranged base pair (bp) ruler, with ticks every 20,000 bp for (a) and every 200 bp for (b). The next two tracks represent predicted protein-coding gene sequences on the forward and reverse strands, respectively. The chromosome replication origin (oriC) and terminus (dif) are marked as red and black ticks in (a), respectively, and protein-coding genes on the leading and lagging strands of replication are shown in orange and blue, respectively. The third (purple) track shows predicted ncRNA sequences on both strands. After the separation line, the next two tracks represent essential (red), growth-defect (blue) or growth-advantage (green) protein-coding genes on the forward and reverse strands, while the sixth track indicates important RNAs with the same colour code. Finally, the seventh track (gold) shows the mean distribution of read counts between the two technical replicates throughout the entire genome, and the eighth track depicts the GC-skew (positive in gold and negative in black). (c, d) Correlations between the two technical replicates of the Tn-Seq experiment. The log-transformed numbers of reads per TA site are plotted and compared between replicates, and Pearson correlation coefficients are indicated for (a) the circular chromosome (R=0.97) and (b) plasmid pLM16734 (R=0.97).

Fig. 2.

Clustering of essential genes based on Cluster of Orthologous Groups (COG) functional classes. (a) Proportion of each class in the essential gene set, with the number of genes next to the bars; (b) log-fold enrichment of COG classes, calculated as the ratio of the proportion of a given class in the essential gene set to its overall proportion in the genome.

Fig. 3.

Conservation of the essential genes of X. hortorum pv. vitians LM16734 in the family Xanthomonadaceae . Colours on the phylogenetic tree indicate taxonomic levels: yellow = X. hortorum pv. vitians, green = X. hortorum , blue = Xanthomonas clade I, pink = Xanthomonas clade II, purple = Stenotrophomonas maltophilia, and red = Xylella fastidiosa . Taxonomic separations are also indicated by red lines on the gene conservation matrix. The position of the X.hortorum pv. vitians LM16734 genome in the phylogeny is highlighted by a red arrow. In the matrix, green boxes indicate detection with blastp (>50% coverage, >30% identity, e-value >10^-5), yellow boxes detection with tblastn (> 50 % coverage, > 30 % identity, e-value > 10^-5), and blue boxes detection with blastn (>50 % coverage, >70% identity, e-value >10^-5). The numbers of gene loci are shown below the matrix, and gene product annotations are indicated above it. Empty and filled black inverted triangles refer to the two distribution patterns discussed in the text. Filled red asterisks indicate that at least one homologue was detected in the DEG10 v15.2 essential gene database with blastp (>50% coverage, >30% identity, e-value >10^-5), while empty red asterisks indicate lower homology thresholds (>40% coverage and/or >20% identity and e-value >10^-5). Finally, purple asterisks highlight the presence of mobile genetic-element-associated genes (transposase, integrase, reverse transcriptase) in the neighbourhood of the gene (<10 kb distance).

Fig. 4.

Schematic representations of gene essentiality within the genomic context. Representations are shown for (a) the pha gene cluser (574,031…582,547 bp), (b) the gum gene cluster (1,912,494…1,927,020 bp), (c) the transposable element containing the putative OLD family endonuclease XHV734_0172 (180,015…183,519 bp) and (d) the genomic island containing the restriction-modification system composed of restriction endonuclease XHV734_3749 and methyltransferase XHV734_3750 (3,868,330…3,884,140 bp). Essential genes or domains are shown in red, non-essential genes or domains in grey, transposases in black in (c), and duplicated loci in blue in (d). Gene names or simplified loci numbers are indicated at the bottom of each box. Green bars indicate the position of TA sites, and golden bars represent the read count at each TA site. In (c), shadowed TA sites indicate unavailability of read counts because of repeated sequences.