Genome sequence of the versatile fish pathogen Edwardsiella tarda provides insights into its adaptation to broad host ranges and intracellular niches

Abstract

Background: Edwardsiella tarda is the etiologic agent of edwardsiellosis, a devastating fish disease prevailing in worldwide aquaculture industries. Here we describe the complete genome of E. tarda, EIB202, a highly virulent and multi-drug resistant isolate in China.

Methodology/principal findings: E. tarda EIB202 possesses a single chromosome of 3,760,463 base pairs containing 3,486 predicted protein coding sequences, 8 ribosomal rRNA operons, and 95 tRNA genes, and a 43,703 bp conjugative plasmid harboring multi-drug resistant determinants and encoding type IV A secretion system components. We identified a full spectrum of genetic properties related to its genome plasticity such as repeated sequences, insertion sequences, phage-like proteins, integrases, recombinases and genomic islands. In addition, analysis also indicated that a substantial proportion of the E. tarda genome might be devoted to the growth and survival under diverse conditions including intracellular niches, with a large number of aerobic or anaerobic respiration-associated proteins, signal transduction proteins as well as proteins involved in various stress adaptations. A pool of genes for secretion systems, pili formation, nonfimbrial adhesions, invasions and hemagglutinins, chondroitinases, hemolysins, iron scavenging systems as well as the incomplete flagellar biogenesis might feature its surface structures and pathogenesis in a fish body.

Conclusion/significance: Genomic analysis of the bacterium offered insights into the phylogeny, metabolism, drug-resistance, stress adaptation, and virulence characteristics of this versatile pathogen, which constitutes an important first step in understanding the pathogenesis of E. tarda to facilitate construction of a practical effective vaccine used for combating fish edwardsiellosis.

Figure 1. Circular atlas of E. tarda EIB202 genome and plasmid pEIB202.

Left, chromosome; Right, plasmid. Circles range from 1 (outer circle) to 9 (inner circle) for chromosome and I (outer circle) to III (inner circle) for plasmid, respectively. Circle 1, genomic islands; Circles 2/I and 3/II, predicted coding sequences on the plus and minus strands, respectively; Circle 4, variable number of tandem repeats (VNTRs) (black) and direct repeat sequences (DRs) (orange); Circles 5/III, G+C percentage content: above median GC content (red), less than or equal to the median (blue); Circle 6, potential horizontally transferred genes and EIB202-specific genes with respect to Enterobacteriaceae bacteria; Circle 7, stable RNA molecules: tRNA (black), rRNA (yellow); Circle 8, phage-like genes and transposases; Circle 9, GC skew (G−C)/(G+C): values>0 (red), values<0 (blue). All genes are colored by functional categories according to COG classification: gold for translation, ribosomal structure and biogenesis; orange for RNA processing and modification; light orange for transcription; dark orange for DNA replication, recombination and repair; antique white for cell division and chromosome partitioning; pink for defense mechanisms; tomato for signal transduction mechanisms; peach for cell envelope biogenesis and outer membrane; deep pink for intracellular trafficking, secretion and vesicular transport; pale green for posttranslational modification, protein turnover and chaperones; royal blue energy production and conversion; blue for carbohydrate transport and metabolism; dodger blue for amino acid transport and metabolism; sky blue for nucleotide transport and metabolism; light blue for coenzyme metabolism; cyan for lipid metabolism; medium purple for inorganic ion transport and metabolism; aquamarine for secondary metabolites biosynthesis, transport and catabolism; gray for function unknown.

Figure 2. Phylogenetic relationship of EIB202.

Phylogenies of Enterobacteriaceae species inferred from concatenated alignments of the protein sequences encoded by 44 housekeeping genes as described in the Materials and Methods. Bacillus cereus ATCC 14579 was used as the outgroup. Accession numbers for the selected bacterial genome sequences are as following: Bacillus cereus ATCC 14579, NC_004722; Burkholderia mallei ATCC 23344, NC_006348; Citrobacter koseri ATCC BAA-895, NC_009792; E. ictaluri 93-146, NC_012779; E. sakazakii ATCC BAA-894, NC_009778; Enterobacter sp. 638, NC_009436; E. carotovora atrosepticum SCRI1043, NC_004547; E. tasmaniensis Et1/99, NC_010694; E. coli K-12 substr MG1655, NC_000913; E. coli O157:H7 str. Sakai O157:H7, NC_002695; E. fergusonii ATCC 35469, NC_011740; K. pneumoniae 342, NC_011283; K. pneumoniae subsp. pneumoniae MGH 78578, NC_009648; P. luminescens subsp. laumondii TTO1, NC_005126; Proteus mirabilis HI4320, NC_010554; Pseudomonas aeruginosa PAO1, NC_002516; S. enterica subsp. enterica serovar Typhi str. Ty2, NC_004631; S. typhimurium LT2, NC_003197; S. proteamaculans 568, NC_009832; Shewanella oneidensis MR-1, NC_004347; S. flexneri 2a str. 2457T, NC_004741; S. flexneri 5 str. 8401, NC_008258; Sodalis glossinidius str. morsitans, NC_007712; Vibrio cholera O1 biovar eltor str. N16961, NC_002505; Wigglesworthia brevipalpis endosymbiont of Glossina brevipalpis, NC_004344; Xanthomonas campestris pv. campestris str. ATCC 33913, NC_003902; Y. pestis CO92, NC_003143 ; and Y. pestis KIM, NC_004088.

Figure 3. Overview of metabolism and transport in EIB202.

Different transport families are distinguished by different colors and shapes. From top left going clockwise: ABC-2 and other transporters (yellow); phosphate and amino acid transporters (green); Siderophore-iron (III) receptors (brick red); TonB-dependent receptors (rosybrown); P-type ATPase (chocolate); mineral and organic ion transporters (violet); ion efflux (green); secretion systems (pink); drug/metabolite efflux (red); nucleotides transporters (orange); the major facilitator superfamily (MFS) (purple); the resistance-nodulation-cell division family (RND) (blue); phosphotransferase system (PTS) (black). Arrows indicate the direction of transport. All the amino acid biosynthesis genes are listed in Table S2.

Figure 4. Flagella and flagellin genes of Edwardsiella strains.

A. E. tarda strains WY37 (isolated from turbot), ATCC15947 (isolated from human feces) and ETV (isolated from human) were overnight cultured on LB liquid medium. Cells were collected by centrifugation at 1,000 rpm for 2 min following removing supernatant and then fixed with 2.5% glutaraldehyde. Scale bars represent 1 µm. B. The aligned putative flagellin sequences from E. tarda EIB202 (ETAE_2128, ETAE_2130), E. tarda PPD130/91 (AAN52540), E. ictaluri str. 93-146 (gi|238920295|, gi|238920297|, gi|238920300|) and S. typhimurium LT2 (NP_461698, NP_460912). Protein sequences are typically highly conserved at their C-terminal and N-terminal ends encoding the flagellar filament backbone while the middle region is generally quite variable, representing the surface-exposed and antigenically variable portion of the filament.