Genome Sequence of the Versatile Fish Pathogen Edwardsiella tarda Provides Insights into its Adaptation to Broad Host Ranges and Intracellular Niches
Qiyao Wang, Minjun Yang, Jingfan Xiao et al.
Research Article — Peer-Reviewed Source
Original research published by Wang et al. in PLoS ONE. Redistributed under Open Access — see publisher for license terms. MedTech Research Group provides these references for informational purposes. We do not conduct original research. All studies are the work of their respective authors and institutions.
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.
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.
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.
Introduction
Edwardsiella tarda , a Gram-negative bacteria belonging to Enterobacteriaceae , is the etiological agent for edwardsiellosis, a devastating fish disease prevailing in worldwide aquaculture industries and accounting for severe economical losses [1] , [2] . The organism commonly affects more than 20 species of freshwater and marine fishes including carp, tilapia, eel, catfish, mullet, salmon, trout, turbot and flounder, causing systemic hemorrhagic septicemia and emphysematous putrefactive disease with swelling skin lesions, as well as ulcer and necrosis in internal organs such as liver, kidney, spleen, and musculature [1] . Besides piscine species, E. tarda also inhabits and infects a broad range of cold or warm -blooded hosts such as reptiles, amphibians, birds, mammals and even humans [2] , [3] , raising a concern about E. tarda being a significant zoonotic pathogen. Edwardsiellae bacterium resides in subgroup 3 in γ-group of Proteobacteria [2] and contains 3 species, E. tarda , E. hoshinae and E. ictaluri , the notorious pathogen relatively strictly inhabiting and causing enteric septicemia in Channel catfish [4] . Like phylogenetically related Enterobacteriaceae bacteria Salmonella spp. [5] , E. tarda possesses the capacity of invading epithelial cells [6] , [7] and macrophages [8] , and multiplies in the cells, which is implicated to be one of the critical steps in its pathogenesis by subverting the fish immune system and causing systemic hemorrhagic septicemia [9] . For the present, the scant knowledge about the genetic basis for the intracellular lifestyle and molecular pathogenesis of E. tarda infection has largely hindered the development of a practical effective vaccine used for combating fish edwardsiellosis. Moreover, the criticized indiscriminate long-term application of antibiotics is marginally effective [10] and raises the increasing concern of multi-drug resistant E. tarda strains [11] , [12] , leaving satisfactory control methods of the disease currently unavailable. To unravel the genetic properties for habitat adaptation, virulence determinants, invasive nature and multi-drug resistance of E. tarda and to facilitate the construction of a practical effective vaccine used for combating fish edwardsiellosis, we utilized the high-throughput pyrosequencing (454 Life Sciences Corporation) together with conventional sequencing method (PCR-based sequencing on ABI3730 automated capillary electrophoresis sequencer, Applied Biosystem Inc.) to quickly determine the complete genome sequence of E. tarda EIB202, a chloramphenicol, tetracycline, rifampicin, and streptomycin-resistant and highly virulent strain isolated from a recent outbreak in farmed turbot in Shandong province of China [12] . E. tarda EIB202 has 50% lethal doses (LD 50 ) of 3.8×10 3 colony forming units (CFU) g −1 for swordtail fish [12] , 5×10 2 CFU g −1 for zebra fish, and 4.5×10 2 CFU g −1 for turbot, and displays fast growth rates in a wide range of sodium chloride concentrations (0.5%–5%) as well as temperature shifts (20°C–37°C) (our unpublished data), presenting as a versatile fish pathogen. Analysis of the complete genome sequence of EIB202 revealed a number of gene hallmarks in E. tarda for adaptation to broad host niches and shed lights on the mechanisms underlying the intracellular colonization of the bacterium in host cells.
Results and Discussion
General features of the complete chromosome sequence E. tarda EIB202 contains a single circular chromosome of 3,760,463 bp with an average G+C content of 59.7% ( Table 1 ). The chromosome is predicted to distinctly harbor 8 rRNA operons, 95 tRNA genes, and 8 stable noncoding RNAs, relatively higher than that of other sequenced enterobacteria ( Table S1 ) and in consistent with the rapid growth of the bacterium [12] . The eight rRNA operons, among which one operon contains a duplication in 5S rRNA gene, are scattered in the circular genome except for two locating in tandem as previously reported [13] ( Figure 1 ). In addition to 77 pseudogenes (including 32 phage and 31 transposase genes), 3,486 coding sequences (CDSs) with an average length of 906 bp were encoded in the chromosome, representing 83.9% of the genome. Among all the protein-coding genes, 79.2% of the CDSs (n = 2,823) were assigned to a functional category of Cluster of Orthologous Groups (COG). Approximately 28% (980/3563) of the chromosomal genes are hypothetical in nature, accounting for the majority of genes (597/852) that are specific to the E. tarda genome among the enterobacterium genome samples. 10.1371/journal.pone.0007646.g001 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. 10.1371/journal.pone.0007646.t001 Table 1 Overall features of the genome of E. tarda EIB202. Chromosome Count or percent Size 3,760,463 bp C+G content (%) 59.7 CDS a 3,486 Coding percentage 83.9% Pseudogenes or gene fragments b 77 IS elements 19 rRNA genes 7*(16S+23S+5S), 1*(16S+23S+5S+5S) tRNA genes 95 Other RNA gene 8 Average CDS length 906 Plasmid Size 43,703 bp CDS 53 C+G content(%) 57.3 IS elements 1 Total not including pseudogenes. Pseudogenes include transposase and phage-related genes.
A conjugative plasmid pEIB202
A circular plasmid (designated as pEIB202) of 43,703 bp was identified from the assembled sequences. The plasmid pEIB202 carries 53 predicted CDSs, among which around 27% encode hypothetical proteins ( Figure 1 ). The open reading frames (ORFs) of putative replication initiator protein (RepA) and plasmid partition proteins (KorA, IncC, KorB, TopA, and ParA) were found on this plasmid, suggesting that this plasmid might belong to IncP plasmid and was capable of replication and stable inheritance in a wide variety of gram-negative bacteria [14] . In the sequence of pEIB202, six genes were probably involved in resistance to antibiotics, including tetA and tetR for tetracycline, strA and strB for streptomycin, sulII for sulfonamide, and catA3 for chloramphenicol resistance, providing genetic properties for previously described multi-drug resistance in EIB202 [12] . A complex transposon IS Sf1 containing IS 4 family transposase [15] and the catA3 gene was identified. The average G+C content of this region was observed to be comparatively extremely low (37.4%) ( Figure 1 ), and differed by above 3σ (standard deviation) from the average G+C content of the plasmid (57.3%) or of the genome (59.7%) (σ = 0.053 for pEIB202; σ = 0.062 for the genome; window length 1.2 kb), suggesting that the chloramphenicol resistance might be recently acquired by the plasmid. Notably, the plasmid encodes an incomplete set of components involved in the type IV A secretion system (T4ASS) ( virB2 , - B4 , - B5 , - B6 , - B8 , - B9 , - B10 , - B11 , - D2 , and - D4 ). The VirB/VirD4 T4ASS was well documented in various pathogens to be involved in horizontal DNA transfer, and in secretion or injection of protein effectors into the medium milieu or into host cells [16] . In addition, several genes associated to plasmid conjugation ( mobC , traC , traD , traL , traN , traX ) are present in the pEIB202 sequence, demonstrating the genetic basis for its capability to transfer between bacteria in the laboratory system with a conjugation frequency of 1.6×10 −6 (data not shown).
Genomic plasticity and genomic islands
As illustrated by Figure 1 , the G+C content of the E. tarda EIB202 genome is highly variable. A large portion (15%) of the genome is composed of mobile genetic elements or related to special genomic islands, displaying a mosaic structure of the genome. EIB202 contains 46 genes which are shown to be phage-like products, integrases or recombinases. In addition, a large quantity (n = 599, a total of 560 kb) of variable number of tandem repeats (VNTRs) or direct repeat sequences were detected in the genome. It also harbors 15 complete and 4 disrupted insertion sequences (IS) including 10 intact IS 100 , 2 truncated IS 100 and a copy of IS 1414I that might lost its transposition activity as a consequence of the nonsense mutation in this insertion sequence (data not shown). Given the reported continued transposition activity of IS 100 [17] , we postulated that the particular IS 100 copies were due to duplicated translocation or multiple integration events of this element occurred within this strain. Interestingly, EIB202 and E. ictaluri 93–146 share an insertion sequence IS Saen1 , which could also be found in S. enterica serovar Enteritidis [18] . All these genes may represent tremendous potential for generating genetic diversity within protein-coding genes over a very short evolutionary time for its adaptation to various niches. In the genome sequence of EIB202, a total of 24 genomic islands (GI) were discerned ( Table 2 ) to scatter throughout the chromosome and contain a total of 852 EIB202-specific genes that were not found in the other Enterobacteriaceae bacteria investigated so far. The previously described type III secretion system (TTSS) [19] and type IV secretion system (T6SS) [19] , [20] are included in the genomic islands (GI7 and GI17). The GI10 contains a mammalian Toll-like/IL-1 receptor (TIR) domain protein, a novel virulence factor implicated in the intracellular survival and lethality of S. enteric [21] , and may also contribute to the intracellular colonization of E. tarda in host cells. In addition to these GIs, the regions, including GI4, GI6, GI9, and GI22 encoding type I secretion system (T1SS), hemagglutinin, O-polysaccharide (OPS) biosynthesis enzymes, and type I restriction-modification system, respectively, maybe consist of the major pathogenicity islands (PAIs) of the bacterium. Some of the GIs are flanked by tRNAs or contain transposases and prophage proteins ( Table 2 ), indicating that these GIs are still involved in the evolution of the bacterium. Among these GIs, GI2 and GI4 are absent in the genome of E. ictaluri 93–146 and might be characteristics of the main difference s between the two species. Interestingly, all of the GIs except for GI7 and GI17, which encodes TTSS and T6SS, are absent in the phylogenetically related Salmonella spp., suggesting that E. tarda , as discussed below, the descendent of a lineage that diverged from the ancestral trunk before Salmonella and Escherichia split, might acquire these genome regions from its evolution events or Salmonella and its predecessors might not have acquired these GIs at the first place. 10.1371/journal.pone.0007646.t002 Table 2 Overview of the genomic islands of EIB202. No. CDS Characteristics or putative functions GI1 ETAE_0032-ETAE_0037 Peroxidase, peptidase GI2 ETAE_0049–ETAE_0058 Hypothetical proteins GI3 ETAE_0252–ETAE_0256 Nitrate/nitrite transporter GI4 ETAE_0315–ETAE_0326 T1SS, invasin GI5 ETAE_0798–ETAE_0805 Oxidoreductase, integrase GI6 ETAE_0808–ETAE_0822 IS, hemagglutinin, haemolysin secretion system GI7 ETAE_0839–ETAE_0892 TTSS GI8 ETAE_1166–ETAE_1177 IS, iron uptake GI9 ETAE_1192–ETAE_1214 OPS , CRISPR GI10 ETAE_1390–ETAE_1396 IS, Toll-like/IL-1 receptor GI11 ETAE_1586–ETAE_1602 IS, hypothetical proteins GI12 ETAE_1608–ETAE_1613 Prophage, O-antigen polymerase protein GI13 ETAE_1759–ETAE_1762 IS, acetyltransferase GI14 ETAE_1811–ETAE_1829 Choline/carnitine/betaine transporter GI15 ETAE_2025–ETAE_2037 Carnitine dehydratase GI16 ETAE_2243–ETAE_2255 P-pilus related proteins GI17 ETAE_2428–ETAE_2443 T6SS GI18 ETAE_2465–ETAE_2476 Prophage Sf6. Flanked by tRNA-Arg GI19 ETAE_2742–ETAE_2748 Integrase, bacteriophage proteins GI20 ETAE_3032–ETAE_3043 Recombinase, invasin. GI21 ETAE_3046–ETAE_3052 Transposase, chorismate mutase. Flanked by IS 100 GI22 ETAE_3069–ETAE_3074 Type I restriction-modification system GI23 ETAE_3078–ETAE_3091 Transposase. Flanked by tRNA-Leu and IS 100 GI24 ETAE_3405–ETAE_3428 Transposase, phage proteins. Flanked by tRNA selC
Relationship of E. tarda to other bacterial taxa
E. tarda shows its specific taxonomic position in Enterobacteriaceae as inferred from the sequence similarities of the housekeeping genes ( Figure 2 ). At variance from the previous description that Trabulsiella guamensis and Enterobacter sakazakii were the closest relatives of Edwardsiella based on analysis of the limited 16S rDNA sequences [2] , E. tarda presents as the sister clad with the phytopathogenic bacterium Erwinia carotovora atroseptica SCRI1043 (branch length value = 0.173), the endophytic bacterium Serratia proteamaculans 568 (value = 0.174), as well as human pathogen Yersinia pestis (value = 0.182). In addition, E. tarda is the most deeply diverging lineage among some notorious enteric pathogenic bacteria such as Escherichia , Salmonella , Shigella , and Klebsiella , but after the divergence of Vibrio cholera and Pseudomonas aeruginosa . The clustering of E. tarda EIB202 is also supported by the previous described biochemical pathways of aromatic amino acid biosynthesis and their regulation in most of the enteric bacteria [22] . Therefore, Edwardsiella species comprise a lineage that diverged from the ancestral trunk before the divergence of some other extensively researched enteric pathogenic bacteria, such as Salmonella and Escherichia . E. tarda adopts both of the intracellular and extracelluar lifestyles as its relatives such as pathogenic S. typhimurium , Y. pestis as well as symbiont Sodalis glossinidius , further suggesting that they experienced independent and divergent evolution driven by their specific hosts and inhabitant niches. 10.1371/journal.pone.0007646.g002 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 .
Comparative genomics analysis with other enterobacteria
Utilizing the COG database [23] , about 64.3% of the E. tarda proteins were grouped into three functional groups ( Table 3 ), and only 14.8% were assigned to the “poorly characterized” group. The differences between E. tarda EIB202 and other Enterobacteriaceae bacteria were overviewed in Table 3 . Among the whole-genome sequenced enterobacteria, E. tarda EIB202 contains a genome of the minimum size ( Table S1 ), which may correspond to the previous suggestion that E. tarda may not be present as a free-living microorganism in natural waters but multiply intracellularly in protozoan and transmission to fish, reptile and other animals or humans [2] . Despite of the minor variations in all areas, the most obvious differences where E. tarda EIB202 consistently varied from all the other Enterobacteriaceae bacteria were discerned with the counts of E. tarda proteins for translation, ribosomal structure and biogenesis (J), cell envelope biogenesis, outer membrane (M), signal transduction mechanisms (T), nucleotide transport and metabolism (F), and coenzyme metabolism (H) as the highest and that for carbohydrate transport and metabolism (G) as the lowest ( Table 3 ). The significant differences of these COG distributions were also statistically supported by the Chi-square tests using pair-wise comparisons with EIB202 (χ 2 >3.84, P <0.05) ( Table 3 ). The relatively high proportion of genes in the J and M group in E. tarda EIB202 is consistent with the high growth rate of the bacterium as previously described [12] . Moreover, the abundance of genes in F and H group as well as the relative paucity of genes in G group may reflect that the organism is well adapted to the aquatic ecosystems and intracellular niches, where may exist relatively mean carbohydrates and wealth of nucleic acid molecules. Again, the comparatively high level of genes in signal transduction mechanisms (T) is a well manifestation of its capacities to cope with various growth conditions and to enhance its survival and persistence under a series of stresses ( Table 3 ). 10.1371/journal.pone.0007646.t003 Table 3 Comparison of COG category distributions of EIB202 with Enterobacteriaceae * . Functional categories E. tarda S. typhimurium E. carotovora E. sakazakii E. coli K. pneumoniae P. luminescens S. proteamaculans S. flexneri Y. pestis Information storage and processing Translation, ribosomal structure and biogenesis (J) 171 (4.80%) 185 (3.98, 3.90%) 176 (4.95, 3.80%) 179 (3.61, 3.91%) 184 (3.03, 4.01%) 199 (5.88, 3.75%) 184 (6.95, 3.65%) 197 (4.24, 3.89%) 173 (3.51, 3.94%) 175 (2.42, 4.08%) Transcription (K) 222 (6.23%) 329 (1.66, 6.94%) 333 (2.95, 7.19%) 290 (0.02, 6.34%) 308 (0.75, 6.71%) 445 (14.22, 8.39%) 279 (1.83, 5.54%) 449 (20.25, 8.87%) 249 (1.11, 5.67%) 237 (1.79, 5.52%) DNA replication, recombination and repair (L) 163 (4.58%) 167 (5.89, 3.52%) 188 (1.30, 4.06%) 156 (6.90, 3.41%) 215 (0.05, 4.68%) 227 (0.45, 4.28%) 260 (1.53, 5.16%) 175 (6.96, 3.46%) 513 (127.79, 11.69%) 346 (39.02, 8.06%) Cellular processes Cell division and chromosome partitioning (D) 34 (0.95%) 37 (0.72, 0.78%) 50 (0.31, 1.08%) 39 (0.13, 0.85%) 36 (0.68, 0.78%) 41 (0.84, 0.77%) 44 (0.15, 0.87%) 35 (1.83, 0.69%) 34 (0.75, 0.77%) 35 (0.43, 0.82%) Defense mechanisms (V) 35 (0.98%) 49 (0.05, 1.03%) 47 (0.02, 1.02%) 43 (0.01, 0.94%) 49 (0.14, 1.07%) 74 (2.98, 1.39%) 69 (2.62, 1.37%) 57 (0.41, 1.13%) 44 (0.01, 1.00%) 43 (0.01, 1.00%) Posttranslational modification, protein turnover, chaperones (O) 119 (3.34%) 161 (0.02, 3.40%) 140 (0.66, 3.02%) 142 (0.29, 3.11%) 138 (0.74, 3.01%) 147 (2.38, 2.77%) 118 (7.76, 2.34%) 150 (0.99, 2.96%) 131 (0.82, 2.98%) 134 (0.30, 3.12%) Cell envelope biogenesis, outer membrane (M) 209 (5.87%) 259 (0.61, 5.47%) 240 (1.81, 5.18%) 221 (4.08, 4.83%) 239 (1.69, 5.21%) 241 (7.77, 4.54%) 203 (15.46, 4.03%) 246 (4.26, 4.86%) 213 (4.04, 4.85%) 205 (4.64, 4.78%) Cell motility and secretion (N) 78 (2.19%) 125 (1.71, 2.64%) 116 (0.87, 2.51%) 117 (1.01, 2.56%) 116 (0.98, 2.53%) 68 (10.86, 1.28%) 105 (0.11, 2.08%) 115 (0.06, 2.27%) 89 (0.25, 2.03%) 137 (7.34, 3.19%) Inorganic ion transport and metabolism (P) 167 (4.69%) 202 (0.86, 4.26%) 244 (1.43, 5.27%) 191 (1.13, 4.18%) 223 (0.13, 4.86%) 317 (6.83, 5.97%) 151 (16.76, 3.00%) 281 (3.15, 5.55%) 194 (0.33, 4.42%) 210 (0.18, 4.89%) Intracellular trafficking and secretion (U) 84 (2.36%) 142 (3.13, 3.00%) 73 (6.53, 1.58%) 109 (0.00, 2.38%) 135 (2.60, 2.94%) 116 (0.29, 2.19%) 128 (0.29, 2.54%) 140 (1.37, 2.77%) 97 (0.20, 2.21%) 157 (11.06, 3.66%) Signal transduction mechanisms (T) 151 (4.24%) 183 (0.75, 3.86%) 121 (16.56, 2.61%) 191 (0.01, 4.18%) 184 (0.27, 4.01%) 200 (1.24, 3.77%) 137 (14.89, 2.72%) 191 (1.20, 3.77%) 151 (3.44, 3.44%) 130 (8.27, 3.03%) Metabolism Energy production and conversion (C) 214 (6.01%) 292 (0.09, 6.16%) 232 (3.88, 5.01%) 203 (9.81, 4.44%) 291 (0.38, 6.34%) 296 (0.72, 5.58%) 169 (34.52, 3.35%) 280 (0.88, 5.53%) 245 (0.66, 5.58%) 183 (12.34, 4.26%) Carbohydrate transport and metab
Predicted metabolic pathways
EIB202 genome encodes the complete sets of enzymes necessary for glycolysis, the tricarboxylic cycle, the pentose phosphate pathway and Entner-Doudoroff pathway ( Figure 3 ). In contrast, the glyoxylate shunt is not complete because isocitrate lyases ( icl1 and icl2 ) and malate synthases are missing. Except for the gene encoding pyruvate carboxylase, the complete set of genes for gluconeogenesis is present in the EIB202 genome ( Figure 3 ). The strain also encodes a putative citrate lyase synthetase complex (ETAE_0223–0228), which may be involved in the lysis of citrate into acetate and oxaloacetate or the reverse reaction. Though genes encoding for oxalate decarboxylase, alanine transaminase and LL-diaminopimelate aminotransferase which are involved in synthesizing L-alanine were not identified in the EIB202 genome, the growth test of the bacterium indicated that it could synthesize L-alanine in an unidentified mechanism (data not shown), suggesting that the bacterium might be highly self-sufficient in amino acid biosynthesis ( Figure 3 and Table S2 ). 10.1371/journal.pone.0007646.g003 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 . EIB202 is able to produce adenosine triphosphate (ATP) through a complete respiratory chain as well as an ATP synthetase complex (ETAE_3528–3534). The genome encodes a variety of dehydrogenases (n = 80, Table 4 ) that enable it to draw on a variety of substrates as electron donors, such as NADH, succinate, formate, isocitrate, proline, acyl-CoA, D-amino acids and so on. The genome also encodes a number of reductases [fumarate reductase (ETAE_0335–0338), nitrate reductase (ETAE_0248–0252), dimethylsulfoxide (DMSO) reductase (ETAE_2192–2195), arsenate reductase (ETAE_1091), anaerobic sulfide reductase (ETAE_1738–1740), thiosulfate reductase (ETAE_1843–1845), anaerobic ribonucleoside triphosphate reductase (ETAE_0422–0423) and tetrathionate reductase (ETAE_1647–1649)], which may contribute to the respiration with alternative electron acceptors to oxygen (fumarate, nitrate, DMSO arsenate, thiofulfate and tetrathionate) under anaerobic conditions, which is in agreement with its facultative anaerobic lifestyle in intracellular niches. 10.1371/journal.pone.0007646.t004 Table 4 Dehydrogenases in EIB202. CDS Gene Annotation ETAE_0085 tdh L-threonine 3-dehydrogenase ETAE_0104 wecC UDP-N-acetyl-D-mannosaminuronate dehydrogenase ETAE_0240 Putative iron-containing alcohol dehydrogenase ETAE_0386 mdh Malate/lactate dehydrogenases ETAE_0565 thrA Bifunctional aspartokinase I/homeserine dehydrogenase I ETAE_0571 gabD Succinate-semialdehyde dehydrogenase I ETAE_0587 xdhC Xanthine dehydrogenase, Fe-S binding subunit ETAE_0588 xdhB Xanthine dehydrogenase, FAD-binding subunit ETAE_0589 xdhA Xanthine dehydrogenase subunit ETAE_0601 pdxA 4-Hydroxythreonine-4-phosphate dehydrogenase ETAE_0620 leuB 3-Isopropylmalate dehydrogenase ETAE_0658 pdhR Pyruvate dehydrogenase complex repressor ETAE_0659 aceE Pyruvate dehydrogenase subunit E1 ETAE_0662 lpdA Dihydrolipoamide dehydrogenase ETAE_0770 Putative alcohol dehydrogenase ETAE_0899 gldA Glycerol dehydrogenase ETAE_0967 sdr Short-chain dehydrogenase/reductase ETAE_0968 gutB L-iditol 2-dehydrogenase ETAE_1048 dld D-lactate dehydrogenase ETAE_1161 sfcA Malate dehydrogenase (oxaloacetate-decarboxylating) ETAE_1202 ugd UDP-glucose 6-dehydrogenase ETAE_1212 gnd 6-Phosphogluconate dehydrogenase ETAE_1248 pyrD Dihydroorotate dehydrogenase 2 ETAE_1334 Iron-containing alcohol dehydrogenase ETAE_1364 Pyruvate/2-Oxoglutarate dehydrogenase complex ETAE_1416 D-isomer specific 2-hydroxyacid dehydrogenase NAD-binding ETAE_1449 zwf Glucose-6-phosphate 1-dehydrogenase ETAE_1474 dadA D-amino-acid dehydrogenase ETAE_1483 gapA Glyceraldehyde-3-phosphate dehydrogenase ETAE_1508 adhE Aldehyde-alcohol dehydrogenase ETAE_1549 Short chain dehydrogenase ETAE_1658 putA Proline dehydrogenase ETAE_1724 Short-chain alcohol dehydrogenase of unknown specificity ETAE_1753 Short-chain dehydrogenase/reductase ETAE_1771 ldhA D-lactate dehydrogenase ETAE_1899 kduD 2-Deoxy-D-gluconate 3-dehydrogenase ETAE_1922 3-Hydroxyisobutyrate dehydrogenase and related beta-hydroxyacid dehydrogenases ETAE_2050 Isocitrate dehydrogenase, specific for NADP+ ETAE_2070 ndh NADH dehydrogenase,
Stress adaptation and signal transduction
E. tarda has been implicated to inhabit diverse host niches [2] , where it encounters and responds to ecological changes, such as temperature change, osmolarity variation, UV/oxidative stress, pH shift, famine as well as the responsive reactions of hosts, before and during survival, invasion and cause diseases in the hosts. In E. tarda EIB202, an array of sigma factor (σ 70 ), alternative sigma factors or extracytoplasmic function (ECF) sigma factors (σ 54 , −28, −24, −32, −38, −54) as well as anti-sigma factors were identified ( Table 5 ), illuminating its basis to respond to various environmental or host stimuli and drive the expression of related functional genes for cellular fitness. 10.1371/journal.pone.0007646.t005 Table 5 Sigma factors and anti-simga factors in EIB202. Type CDS Gene Annotation Sigma factor ETAE_0454 rpoD DNA-directed RNA polymerase, subunit sigma-70 RpoD ETAE_2728 rpoE RNA polymerase factor sigma-24 RpoE ETAE_2126 rpoF Flagellar biosynthesis factor sigma-28 FliA ETAE_3326 rpoH RNA polymerase factor sigma-32 RpoH ETAE_0497 rpoN RNA polymerase factor sigma-54 RpoN ETAE_2873 rpoS RNA polymerase factor sigma-38 RpoS Anti-sigma factor ETAE_0508 Putative anti-sigma B factor antagonist ETAE_0576 dnaK Molecular chaperone ETAE_1223 flgM Anti-sigma 28 factor ETAE_1867 pspA Phage shock protein A, suppresses σ 54 -dependent transcription ETAE_2684 Anti-sigma regulatory factor (Ser/Thr protein kinase) The organism is well equipped to cope with the first main obstacle, temperature fluctuations, in aquatic ecosystems. Six homologues of cold shock proteins (CspA, −B, C, D, G, H, I), among which two copies of cspC were included, were discerned to represent one of the largest paralogue gene family in EIB202. Closer investigation indicated that the established cold adaptation-related proteins RpoE (ETAE_2728), RseA (ETAE_2727), Rnr (ETAE_0360), DeaD (ETAE_0411), RbfA (ETAT_0406), NusA (ETAE_0404), and PNP (ETAE_0409) were all encoded in the EIB202 genome, consisting a reservoir to cope with the physically extreme cold in the environment, and may help the organism to persist in a previously described dormant state known as viable but not culturable state (VBNC) [24] . In line with the versatility in coping with the cold scenarios, EIB202 genome also has an arsenal of 34 shock proteins (GroEL, GroES, IbpAB, GrpE, etc.) or chaperons for other environmental or host changes ( Table 6 ). One operon, sspAB , as well as another conserved ORF (ETAE_2419) which was known to play essential roles in acid tolerance response, were found in the chromosome, as was the operon pspFABCD , pspE and pspG genes known as encoding phage shock proteins responding to various membrane stimuli other than phage induction. 10.1371/journal.pone.0007646.t006 Table 6 Shock proteins in EIB202. Shock protein Gene Function ETAE_0008 ibpA Molecular chaperone (small heat shock protein) ETAE_0009 ibpB Molecular chaperone (small heat shock protein) ETAE_0086 Putative ATPase with chaperone activity ETAE_0235 pspG Phage shock protein G ETAE_0297 torD Chaperone protein ETAE_0313 groES Co-chaperonin GroES (HSP10) ETAE_0314 groEL Chaperonin GroEL (HSP60 family) ETAE_0576 dnaK Molecular chaperone ETAE_0577 dnaJ DnaJ-class molecular chaperone with C-terminal Zn finger domain ETAE_0746 ompH Periplasmic chaperone ETAE_0867 escB Type III secretion system chaperone protein B ETAE_0871 escA Type III secretion low calcium response chaperone ETAE_0945 yegD Putative chaperone ETAE_1031 Fimbrial chaperon protein ETAE_1420 torD Cytoplasmic chaperone TorD family protein ETAE_1657 htpX Heat shock protein ETAE_1774 hslJ Heat shock protein HslJ ETAE_1864 pspD Phage shock protein D ETAE_1865 pspB Phage shock protein B ETAE_1867 pspA Phage shock protein A (IM30), suppresses sigma54-dependent transcription ETAE_1868 pspF Phage shock protein operon transcriptional activator ETAE_2048 hchA Molecular chaperone ETAE_2146 fliJ Flagellar biosynthesis chaperone ETAE_2218 fimC Periplasmic chaperone ETAE_2251 papD Chaperone protein ETAE_2362 Hydrogenase 2-specific chaperone ETAE_2419 Acid shock protein precursor ETAE_2466 Phage tail assembly chaperone gp38 ETAE_2735 grpE Heat shock protein ETAE_2811 hscA Chaperone protein ETAE_2812 hscB Co-chaperone Hsc20 ETAE_2829 clpB Protein disaggregation chaperone ETAE_3271 Heat shock protein 15 ETAE_3272 Disulfide bond chaperones of the HSP33 family Regarding to osmotic stress, EIB202 has developed the ability to tolerate high concentrations of sodium chloride (up to 5%) [12] . The genes responsible for the synthesis and uptake of several compatible solutes (osmolytes), such as ectABC for ectoine biosynthesis, bccT and betABI for the transport of betaine, as well as proVWX for the uptake and transport of proline and glycine betaine, which often reside on the genome of halophilic bacteria, are absent in EIB202. In contrast, the caiTABCDC (ETAE_2658–2664) and caiF (ETAE_2672) genes involved in carnitine/betaine uptake and metabolism are
Surface structures and putative virulence factors
Previous studies have determined that E. tarda infects fish via the following three principle entry sites: skin, gill and intestine [6] . A variety of surface structures mediating motility, adherence and pathogen-host recognition seem to be the most important properties for the initiation of infection process in E. tarda . The gene clusters for P pilus ( pap genes), type 1 fimbriae ( fim genes) as well as several genes for other nonfimbrial adhesins, invasins and hemagglutinins ( Table 9 ) are present in the EIB202 genome, suggesting its ability to bind to specific receptors distributed in its various hosts and therefore defining the site of entry and colonization. Interestingly, dozens of these surface structure related proteins are encoded in the EIB202 GIs such as GI4 (invasion), GI6 (hemagglutinin), GI9 (OPS biosynthesis cluster), GI12 (OPS biosynthesis protein) and GI16 (P pilus related proteins) ( Table 2 ), further suggesting that its surface structures might be shaped by the evolution events to acquire colonization and fitness when approaching various hosts. These observations also underlie the previous descriptions regarding the various mannose-resistant hemagglutination (MRHA) and mannose-sensitive hemagglutination (MSHA) phenotypes as well as serotypes in E. tarda strains [2] , [32] , [33] . 10.1371/journal.pone.0007646.t009 Table 9 Partial of surface structures and virulence related genes in EIB202. CDS Characteristics ETAE_0315 Hypothetical protein, putative BAP type adhesins ETAE_0323 Putative invasin, shdA, non-fimbrial adhesin ETAE_0613 Putative hemolysin secretion ATP-binding protein ETAE_0817 Filamentous haemagglutinin family outer membrane protein ETAE_0818 Putative adhesin/hemagglutinin/hemolysin ETAE_0821 ShlB/FhaC/HecB family haemolysin secretion/activation protein ETAE_0910 Hemolysin transporter protein ETAE_0911 Putative hemolysin precursor ETAE_1008 Hemolysin expression modulating family protein ETAE_1267 OmpA, outer membrane protein A ETAE_1528 OmpW, outer membrane protein W ETAE_2089 Pic serine protease precursor, FhaB filamentous heamagglutinin ETAE_2842 Putative adhesin ETAE_2937 Hemolysin III family ETAE_3034 Putative invasin ETAE_3045 Temperature sensitive hemagglutinin EIB202 was observed to be of non-motile and deficient in flagellar biosynthesis ( Figure 4A ). A set of early, middle and late flagellar genes displaying high similarities to S. enterica Serovar Typhimurium were found to be scattered present in the EIB202 genome sequence, encoding components required for flagellar hook basal body and hook-filament junction structures [34] . The main dissimilarities between the two organisms seem to lie in the late stage genes for flagella assembly. In EIB202, though two homologues of S. enterica phase-1 flagellin fliC gene were identified (ETAE_2128 and ETAE_2130) ( Figure 4B ), genes for S. enterica phase-2 flagellin ( fljB ), flagellin methylase ( fliB ), flagellin repressor ( fljA ), and methyl accepting chemotaxis component ( aer ) were absent in the EIB202 genome, which might account for the incapacity of flagellar biogenesis and weak motility in EIB202. However, the inability of flagellar biogenesis may enhance its invasion capacity by avoiding the pro-inflammatory responses and escaping the recognition by Toll-like receptor 5 [35] and the attack by caspase-1 and interleukin 1β secreted by host cells which recognize the flagellin of the bacteria mounting the host cells [36] . 10.1371/journal.pone.0007646.g004 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. tard a EIB202 (ETAE_2128, ETAE_2130), E. tard a 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. Surface-exposed and secreted proteins are of significance for the niche adaptations and pathogenesis of pathogens. There are various secretion pathways generally including type I to VI secretion systems and other specific protein transduction systems to fulfill the functions of protein secretion in Gram negative bacteria [37] . The Sec-dependent transport system, the components of the main terminal branch of the general secretory pathway (GSP), the signal recognition particle (SRP) and the Sec-independent twin arginine transport (Tat), T1SS, TTSS and T6SS were all identified in the geno
Gene properties for intracellular colonization
It has been demonstrated that EIB202 and other virulent isolates of E. tarda are capable of living and persisting inside the phagocytes before leading a systemic infection [44] . In the facultative intracellular pathogens such as Mycobacterium tuberculosis and Salmonella , fatty acids metabolism and the glyoxylate shunt play important roles in their long-term persistence and infection in hosts [45] . In contrast, as above-mentioned, the genes fadD , fadF , fadE and icl , which are required for fatty acids metabolism and the glyoxylate shunt, are absent in the genome sequence of EIB202, indicating that the bacterium might adopt an unusual intracellular persistent strategy to fulfill colonization and infection in various hosts. In E. tarda , the ability to produce enzymes including catalase, peroxidase and superoxide dismutase (SOD) to detoxify various reactive oxygen species (ROS) has been implicated to be essential for counteracting phagocyte-mediated killing. The EIB202 genome contains genes putatively for a copper-zinc SOD (ETAE_0247) and an iron-cofactored SOD (ETAE_1676), as well as catalases (ETAE_0889 and ETAE_1368), which were believed to be the genetic marker for the E. tarda virulent strains [46] . Moreover, several genes (n = 9) encoding functions for protecting the cells from ROS damages with peroxidase activities (7) or repairing functions (2) are found, intriguingly including a non-haem peroxidase AhpC (ETAE_0956) for alkyl hydroperoxide reductase and a Dyp-type haem-dependent peroxidase (ETAE_1129) ( Table 11 ). These genes confer the organism broader capacities to cope with the oxidative stresses and may necessarily contribute to the abilities to multiply inside the host cells (e.g. macrophage cells), and further the virulence of the bacterium. Actually, when the alternative sigma factor RpoS is deleted to significantly decrease the expression of SOD and catalase, the bacterium shows deficiency in the internalization and colonization of fish cells [47] . 10.1371/journal.pone.0007646.t011 Table 11 ROS related proteins in EIB202. CDS gene Function ETAE_0034 Putative iron-dependent peroxidase ETAE_0099 trxA Thioredoxin domain-containing protein ETAE_0247 sodC Copper-zinc superoxide dismutase ETAE_0889 katG Putative catalase/peroxidase ETAE_0956 ahpC Alkyl hydroperoxide reductase, small subunit ETAE_1094 bcp Thioredoxin-dependent thiol peroxidase ETAE_1129 Dyp-type peroxidase family ETAE_1368 katE Putative catalase B ETAE_1484 msrB Methionine-R-sulfoxide reductase ETAE_1496 xthA Exonuclease III ETAE_1676 sodB Superoxide dismutase ETAE_1715 yhjA Probable cytochrome C peroxidase ETAE_1859 tpx Thiol peroxidase In E. tarda , the TTSS and T6SS have been demonstrated to be essential for resistance of phagocytic killing and replicating within the cells, thus important for the full virulence of the organism [19] , [48] , [49] . The TTSS and T6SS have been suggested to be the genetic hallmarks for the differentiation of virulent and avirulent strains of E. tarda [19] , [20] , [50] . The preservation of intact genomic islands for TTSS and T6SS in EIB202 genome will definitely potentiate it to live an intracellular life after invading hosts. As the circumstances in Salmonella [51] , [52] , the DnaK/DnaJ chaperone machinery and the type I restriction-modification system in GI22 in EIB202 may also contribute to its invasion and survival within macrophages and avoid perturbations from host immune cells for a cosy intracellular life. Interestingly, EIB202 genome harbors two separated genes, mgtB (ETAE_3346) and mgtC (ETAE_1776). Their Salmonella homologues are located on the selC locus as a mgtCB operon and are required for its survival within macrophages and growth in low Mg 2+ environment [53] , while the selC locus on EIB202 is flanked by GI24 containing prophage/transposase/integrase genes. These genetic properties provided strong evidence that EIB202 possesses the capacity of invading macrophages and subverting the fish immune systems, maybe in a manner different from that of extensively studied Salmonella . Further experiments are required to unravel their exact roles in the edwardsiellosis pathogenesis.
Conclusions
E. tarda is well established to be one of the leading fish pathogens haunting the aquaculture industries throughout the world, and its association with high value fish species such as turbot has impelled the attempts for vaccine development against this organism. In this study, we have determined the complete genome sequence of EIB202, a highly virulent and multi-drug resistant isolate. The comprehensive analysis of the genome sequence provides evidences that the bacterium harbors an array of antibiotics-resistance determinants and well prepares for the antibiotics cocktail that might be present in the aquaculture ecosystem, similarly to that described in another E. tarda strain TX01 isolated from moribund turbot ( Scophthalmus maximus ) in Shandong, China [11] . The self-transmissibility of the plasmid pEIB202 further intensifies the concern that the genome contents of E. tarda are partly shaped by its life in various aquatic ecological niches. The findings of stress responding genes as well as signal transduction systems also confirmed the jack of all trade nature of the bacterium which could survive in a variety of hosts and growth conditions, including intracellular niches. Moreover, analysis of the genome sequence also revealed a virulence arsenal in the bacterium, confirming special pathogenic mechanisms of the organism. The determination of genome sequence of the bacterium will undoubtedly facilitate our understanding of this organism and will set the basis for vaccine development using the “reverse genetics” approach.
Materials and Methods
Bacterial growth and DNA extraction E. tarda EIB202 (previously referred to as isolate EH202 [12] with a CCTCC No. M208068 and available upon request) was recently isolated from diseased turbot ( Scophthalmus maximus ) in a mariculture farm in Yantai, Shandong province of China and was routinely cultured on Luria-Bertani (LB) medium at 28°C. Genomic DNA was isolated from 10 ml overnight culture using the TIANamp Bacteria DNA Kit (TIANGEN Biotech, Beijing, China). Genomic DNA was quantified on 0.7% agarose gel stained with ethidium bromide and spectrophotometrically assessed. The stock DNA solution was separated into two aliquots, one for sequencing via pyrosequencing and the other stored at −80°C for further gap closing.
High-density pyrosequencing and sequence assembly of the genome
The complete sequencing work was conducted using Roche GS FLX system [54] . A total of 286,550 reads counting up to 64,706,315 bases (averaged read length as 225 bp), were obtained resulting in a 17-fold coverage of the genome. Assembly was performed using the GS de novo Assembler software ( http://www.454.com/ ) and produced 64 contigs ranging from 500 bp to 337,284 bp (the N50 contig size is 116,367 bp). Relationship of the contigs was determined by multiplex PCR [55] . Gaps were then filled in by sequencing the PCR products using ABI 3730xl capillary sequencers. Phred, Phrap and Consed software packages ( http://www.genome.washington.edu ) were used for the final assembly and edition, and low quality regions of the genome were resequenced. The assembly of the genome was verified by digestion of the genomic DNA with restriction enzymes and then running the products with pulsed-field gel electrophoresis (PFGE).
Sequence analysis and annotation
Putative CDSs were identified by GeneMark [56] and Glimmer3 [57] , and peptides shorter than 30 aa were eliminated. Sequences from the intergenic regions were compared to GenBank's non-redundant (nr) protein database [58] to identify genes missed by the Glimmer or GeneMark prediction and to detect pseudogenes. Insert sequences were first detected using IS Finder database ( http://www-is.biotoul.fr/is.html ) with default parameters and selected manually. Transfer RNA genes were predicted by tRNAScan-SE [59] , while ribosomal DNAs (rDNAs) and other RNA genes were identified by comparing the genome sequence to the rRNA database [60] , [61] and by using Infernal program [62] . Functional annotation of CDSs was performed through searching against nr protein database using BLASTP [63] . The protein set was also searched against COG ( http://www.ncbi.nlm.nih.gov/COG/ ; [64] ) and the KEGG (Kyoto encyclopedia of genes and genomes; http://www.genome.jp/kegg/ ) [65] for further function assignment. The criteria used to assign function to a CDS were (1) a minimum cutoff of 40% identity and 60% coverage of the protein length and (2) at least two best hits among the COG, KEGG, or nr protein database. A search for gene families in the genome was performed by BLASTCLUST. Subcellular localization of the proteins was predicted by PSORTb program (v2.0.1) [66] . TatP 1.0 server (v2.0) [67] and TATFIND 1.2 program [39] were used to detect the potential substrates of the Tat secretion system. Pathogenicity islands and anomalous genes were detected by PAI-IDA [68] and SIGI-HMM [69] , respectively.
Construction of phylogenetic tree
Phylogenetic position of E. tarda EIB202 within the Enterobacteriaceae was determined based on the protein sequences of 44 housekeeping genes ( adk , aroC , dnaA , dnaK , frr , fusA , gapA , gyrA , gryB , infC , nusA , pgk , phoB , phoR , pyrG , recC , rplA , rplB , rplC , rplD , rplE , rplF , rplK , rplL , rplM , rplN , rplP , rplS , rplT , rpmA , rpoA , rpoB , rpoC , rpoE , rpsB , rpsC , rpsE , rpsI , rpsJ , rpsK , rpsM , rpsS , smpB , and tsf ) [70] . BLAST algorithm was used when needed and ambiguous regions were trimmed according to an embedded mask. Concatenated protein sequences were aligned by ClustalW [71] . Maximum likelihood tree based on the aligned protein sequences was constructed by using PhyML [72] with 100 bootstrap iterations.
Genome comparison
Orthologs between E. tarda EIB202 and other Enterobacteriaceae bacteria ( Escherichia coli K-12 substr MG1655, Erwinia carotovora atrosepticum SCRI1043, Klebsiella pneumoniae subsp. pneumoniae MGH 78578, Salmonella typhimurium LT2, Serratia proteamaculans 568, Shigella flexneri 5 str. 8401, Yersinia pestis CO92, Enterobacter sakazakii ATCC BAA-894 and Photorhabdus luminescens subsp. laumondii TTO1) were detected by all-vs-all reciprocal_BLASTP search against the protein sets of these strains ( http://www.ncbi.nlm.nih.gov/RefSeq ), respectively. Criteria were as following: (1) E-value = e −20 or less and (2) >40% amino acid sequence identity, then the best hit was selected. Predicted E. tarda EIB202-specific genes were detected by screening EIB202 protein set against orthologs.
Statistical analysis
The contingency Chi-square tests were performed to detect the significant differences between the counts of ORFs in each COG category for EIB202 and other Enterobacteriaceae bacterium ( http://img.jgi.doe.gov ). In the significant difference test, in which a , b were the observed numbers of ORFs in in each COG category for EIB202 and other Enterobacteriaceae bacterium, and c , d were the counts of the rest of all ORFs in each COG category for EIB202 and other Enterobacteriaceae bacterium, respectively. Significant differences were determined at P <0.05 (critical value ) [73] .
Data Availability
The nucleotide sequence of the E. tarda EIB202 chromosome and the plasmid pEIB202 were submitted to the GenBank database under accession numbers CP001135 and CP001136 , respectively.
Supporting Information
Table S1 Genomic features of E. tarda EIB202 and other sequenced enterobacteria (0.04 MB DOC) Click here for additional data file. Table S2 Amino acid biosynthesis genes in E. tarda EIB202 (0.17 MB DOC) Click here for additional data file. Table S3 The predicted Tat substrates in EIB202 (0.07 MB DOC) Click here for additional data file.
| DOI | 10.1371/journal.pone.0007646 |
| PubMed ID | 19865481 |
| PMC ID | PMC2764856 |
| Journal | PLoS ONE |
| Year | 2009 |
| Authors | Qiyao Wang, Minjun Yang, Jingfan Xiao, Haizhen Wu, Xin Wang, Yuanzhi Lv, Lili Xu, Huajun Zheng, Shengyue Wang, Guoping Zhao, Qin Liu, Yuanxing Zhang |
| License | Open Access — see publisher for license terms |
| Citations | 239 |