Ca. Endozoicomonas cretensis: A Novel Fish Pathogen Characterized by Genome Plasticity

Abstract

Endozoicomonas bacteria are generally beneficial symbionts of diverse marine invertebrates including reef-building corals, sponges, sea squirts, sea slugs, molluscs, and Bryozoans. In contrast, the recently reported Ca. Endozoicomonas cretensis was identified as a vertebrate pathogen, causing epitheliocystis in fish larvae resulting in massive mortality. Here, we described the Ca. E. cretensis draft genome, currently undergoing genome decay as evidenced by massive insertion sequence (IS element) expansion and pseudogene formation. Many of the insertion sequences are also predicted to carry outward-directed promoters, implying that they may be able to modulate the expression of neighbouring coding sequences (CDSs). Comparative genomic analysis has revealed many Ca. E. cretensis-specific CDSs, phage integration and novel gene families. Potential virulence related CDSs and machineries were identified in the genome, including secretion systems and related effector proteins, and systems related to biofilm formation and directed cell movement. Mucin degradation would be of importance to a fish pathogen, and many candidate CDSs associated with this pathway have been identified. The genome may reflect a bacterium in the process of changing niche from symbiont to pathogen, through expansion of virulence genes and some loss of metabolic capacity.

Fig. 1.

—Circular representation of the genome of Ca. Endozoicomonas cretensis. Scaffolds were ordered against the genome of E. elysicola DSM 22380 (Neave et al. 2014). Scaffolds not aligned to Endozoicomonas elysicola were appended to the ordered scaffolds after 495520 bp. The tracks from the outside in represent: (1) the scaffolds (n = 638); (2) ISIR located at the ends of the scaffolds, colored by IS families; (3) forward CDSs; (4) reverse CDSs; (5) pseudogenes; (6) species-specific genes in enriched COG categories: replication, recombination and repair (cyan), cell wall/membrane/envelope biogenesis (blue), cell motility (magenta) and Mobilome: prophages, transposons (red); (7) phages (red), unordered phage genes (yellow) and newly expanded families of pathogenic genes (green); (8) virulence factors including T3SS (cyan), flagella (purple), chemotaxis (green), Tfp (red), T2SS (blue), mucin degradation genes (yellow), invasion biofilm formation genes (grey), invasin (orange), and effectors nucleomodulin (black) and E3 ubiquitin ligases (pink).

Fig. 2.

—Phylogenetic relationship of Ca. Endozoicomonas cretensis to other Endozoicomonas species. Maximum-likelihood tree based on concatenated aligned protein sequences of 43 conserved single-copy marker genes, extracted from the Ca. E. cretensis genome draft, and 11 publically available Endozoicomonas genomes (table 4). The tree was rooted using the Gammaproteobacterium Pseudomonas aeruginosa PA01 (GCF_000006765.1). In total 6,120 sites were used, which were extracted from the 12,791 sites in the original protein alignment by Gblocks after eliminating poorly aligned and divergent regions. The scale bar indicates the number of substitutions per site.

Fig. 3.

—Functional categories enriched with Ca. Endozoicomonas cretensis Dpd28tailN specific genes. COG (Clusters of Orthologous Groups) functional categories where the numbers of species-specific genes (pink horizontal bars) are more (+) than expected by Fisher’s exactly test (red: P value < 0.01; black: 0.01 < P value < 0.05) are marked. The numbers of “all genes” in each category are shown as the neighboring blue horizontal bars. For visualization purpose, only categories with more than two genes are shown.

Fig. 4.

—Location of outward-directed promoters predicted within Ca. Endozoicomonas cretensis IS elements. “-10 box” and “-35 box” represent the two short conserved sequence elements in the bacterial promoters, which are, respectively, approximately 10 and 35 nucleotides upstream of the transcription start site (TSS). Each CDS is shown as a blue arrow, with the flanking yellow arrows representing the ISIRs.