Genome Sequence of the Versatile Fish Pathogen Edwardsiella tarda Provides Insights into its Adaptation to Broad Host Ranges and Intracellular Niches
et al. (2009) Genome Sequence of the Versatile Fish Pathogen Edwardsiella tarda Provides Insights into its
Adaptation to Broad Host Ranges and Intracellular Niches. PLoS ONE 4(10): e7646. doi:10.1371/journal.pone.0007646
Genome Sequence of the Versatile Fish Pathogen Edwardsiella tarda Provides Insights into its Adaptation to Broad Host Ranges and Intracellular Niches
Qiyao Wang 0
Minjun Yang 0
Jingfan Xiao 0
Haizhen Wu 0
Xin Wang 0
Yuanzhi Lv 0
Lili Xu 0
Huajun Zheng 0
Shengyue Wang 0
Guoping Zhao 0
Qin Liu 0
Yuanxing Zhang 0
Niyaz Ahmed, University of Hyderabad, India
0 1 State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology , Shanghai , People's Republic of China, 2 Shanghai - MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai , Shanghai , China
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, drugresistance, 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.
Funding: This work was supported by grants from the National High Technology Research and Development Program of China (2008AA092501), Ministry of
Agriculture of China (Nos. nyhyzx07-046 and nycytx-50-G08), Shanghai Science and Technology Development Funds (No. 08QA14024) and Shanghai Leading
Academic Discipline Project (No. B505) as well as the National Special Fund for State Key Laboratory of Bioreactor Engineering (No. 2060204). The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
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 . 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 c-group of
Proteobacteria  and contains 3 species, E. tarda, E. hoshinae and
E. ictaluri, the notorious pathogen relatively strictly inhabiting and
causing enteric septicemia in Channel catfish . Like
phylogenetically related Enterobacteriaceae bacteria Salmonella spp. , E.
tarda possesses the capacity of invading epithelial cells [6,7] and
macrophages , 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 .
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  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 . E. tarda EIB202 has 50% lethal
doses (LD50) of 3.86103 colony forming units (CFU) g21 for
swordtail fish , 56102 CFU g21 for zebra fish, and
4.56102 CFU g21 for turbot, and displays fast growth rates in a
wide range of sodium chloride concentrations (0.5%5%) as well
as temperature shifts (20uC37uC) (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 . 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  (Figure 1). In
Pseudogenes or gene fragmentsb
Average CDS length
a Total not including pseudogenes.
b Pseudogenes include transposase and phage-related genes.
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.
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 .
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 . A
complex transposon ISSf1 containing IS4 family transposase 
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 3s (standard deviation) from the
average G+C content of the plasmid (57.3%) or of the genome
(59.7%) (s = 0.053 for pEIB202; s = 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 . 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 1.661026 (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
phagelike 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 IS100, 2 truncated IS100 and a
copy of IS1414I 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 IS100 , we postulated that the particular IS100
copies were due to duplicated translocation or multiple integration
events of this element occurred within this strain. Interestingly,
EIB202 and E. ictaluri 93146 share an insertion sequence ISSaen1,
which could also be found in S. enterica serovar Enteritidis . 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)
 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 , 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,
Opolysaccharide (OPS) biosynthesis enzymes, and type I
restrictionmodification 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 93146 and might be
characteristics of the main differences 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.
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
Characteristics or putative functions
GI4 ETAE_0315ETAE_0326 T1SS, invasin
GI6 ETAE_0808ETAE_0822 IS, hemagglutinin, haemolysin secretion system
GI7 ETAE_0839ETAE_0892 TTSS
GI8 ETAE_1166ETAE_1177 IS, iron uptake
GI10 ETAE_1390ETAE_1396 IS, Toll-like/IL-1 receptor
GI11 ETAE_1586ETAE_1602 IS, hypothetical proteins
Prophage, O-antigen polymerase protein
GI13 ETAE_1759ETAE_1762 IS, acetyltransferase
P-pilus related proteins
GI17 ETAE_2428ETAE_2443 T6SS
Prophage Sf6. Flanked by tRNA-Arg
GI19 ETAE_2742ETAE_2748 Integrase, bacteriophage proteins
GI21 ETAE_3046ETAE_3052 Transposase, chorismate mutase. Flanked by IS100
GI22 ETAE_3069ETAE_3074 Type I restriction-modification system
GI23 ETAE_3078ETAE_3091 Transposase. Flanked by tRNA-Leu and IS100
GI24 ETAE_3405ETAE_3428 Transposase, phage proteins. Flanked by tRNA selC
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 , 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 . 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
Comparative genomics analysis with other
Utilizing the COG database , 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
wholegenome 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
freeliving microorganism in natural waters but multiply intracellularly
in protozoan and transmission to fish, reptile and other animals or
humans . 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 (x2.3.84, 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 . 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).
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_02230228), 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).
EIB202 is able to produce adenosine triphosphate (ATP) through
a complete respiratory chain as well as an ATP synthetase complex
(ETAE_35283534). 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_03350338), nitrate reductase (ETAE_02480252),
dimethylsulfoxide (DMSO) reductase (ETAE_21922195), arsenate
reductase (ETAE_1091), anaerobic sulfide reductase (ETAE_1738
1740), thiosulfate reductase (ETAE_18431845), anaerobic
ribonucleoside triphosphate reductase (ETAE_04220423) and
tetrathionate reductase (ETAE_16471649)], 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.
Stress adaptation and signal transduction
E. tarda has been implicated to inhabit diverse host niches ,
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 (s70), alternative sigma
factors or extracytoplasmic function (ECF) sigma factors (s54,
228, 224, 232, 238, 254) 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.
The organism is well equipped to cope with the first main
obstacle, temperature fluctuations, in aquatic ecosystems. Six
homologues of cold shock proteins (CspA, 2B, 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
adaptationrelated 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) . 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
Regarding to osmotic stress, EIB202 has developed the ability to
tolerate high concentrations of sodium chloride (up to 5%) .
The genes responsible for the synthesis and uptake of several
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
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
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
ETAE_2050 Isocitrate dehydrogenase, specific for NADP+
ETAE_2070 ndh NADH dehydrogenase, FAD-containing subunit
ETAE_2118 D-beta-hydroxybutyrate dehydrogenase
ETAE_2276 hisD Histidinol dehydrogenase
ETAE_2380 nuoG NADH dehydrogenase/NADH:ubiquinone oxidoreductase
75 kD subunit (chain G)
ETAE_2416 asd Aspartate-semialdehyde dehydrogenase
ETAE_2417 pdxB Erythronate-4-phosphate dehydrogenase
ETAE_2583 sucB 2-Oxoglutarate dehydrogenase, E2 subunit,
ETAE_2584 sucA Component of the 2-oxoglutarate dehydrogenase
ETAE_2585 sdhB Succinate dehydrogenase iron-sulfur subunit
ETAE_2586 sdhA Succinate dehydrogenase catalytic subunit
ETAE_2587 sdhD Succinate dehydrogenase cytochrome b556 small
ETAE_2588 sdhC Succinate dehydrogenase cytochrome b556 large
ETAE_2663 caiA Crotonobetainyl-CoA dehydrogenase
ETAE_2695 folD Methylenetetrahydrofolate dehydrogenase (NADP(+))
ETAE_2705 dltE Short chain dehydrogenase
ETAE_2787 guaB Inositol-5-monophosphate dehydrogenase
ETAE_2836 tyrA Bifunctional chorismate mutase/prephenate
ETAE_2856 sdhA Succinate dehydrogenase flavoprotein subunit
ETAE_2857 sdhD Succinate dehydrogenase hydrophobic membrane
ETAE_2858 sdhC Succinate dehydrogenase cytochrome b-556 subunit
ETAE_2934 aroE Shikimate 5-dehydrogenase
ETAE_2939 gcvP Glycine dehydrogenase
ETAE_2949 serA D-3-phosphoglycerate dehydrogenase
ETAE_2958 epd D-erythrose 4-phosphate dehydrogenase
ETAE_2986 Short chain dehydrogenase
ETAE_3113 glpC Sn-glycerol-3-phosphate dehydrogenase subunit C
ETAE_3114 glpB Anaerobic glycerol-3-phosphate dehydrogenase subunit B
ETAE_3115 glpA Sn-glycerol-3-phosphate dehydrogenase subunit A
ETAE_3141 Probable zinc-binding dehydrogenase
ETAE_3173 xdhC Xanthine dehydrogenase accessory factor, putative
ETAE_3194 aroE Shikimate 5-dehydrogenase
ETAE_3312 glpD Glycerol-3-phosphate dehydrogenase
ETAE_3336 fdhD Formate dehydrogenase accessory protein
ETAE_3337 Formate dehydrogenase-O, major subunit
ETAE_3338 Anaerobic dehydrogenases, typically
ETAE_3339 fdnH Formate dehydrogenase-N beta subunit
ETAE_3340 fdnI Formate dehydrogenase-N subunit gamma
ETAE_3341 fdhE Formate dehydrogenase accessory protein
ETAE_3344 asd Aspartate-semialdehyde dehydrogenase
ETAE_3353 gdhA Glutamate dehydrogenase/leucine dehydrogenase
ETAE_3429 thrA Homoserine dehydrogenase
ETAE_3457 gpsA Glycerol-3-phosphate dehydrogenase (NAD(P)+)
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 present in the genome. Carnitine is a
ubiquitous substance in eukaryotes and, in coupling with its
metabolic intermediates crotononbetaine and c-butyrobetaine,
may be served as osmoprotectant and stimulate growth in
anaerobic and starvation conditions. Spermidine and putrescine
uptake system potABCD (ETAE_18811886) and biosynthesis
Anti-sigma factor ETAE_0508
Putative anti-sigma B factor antagonist
RNA polymerase factor sigma-24 RpoE
Flagellar biosynthesis factor sigma-28 FliA
RNA polymerase factor sigma-32 RpoH
RNA polymerase factor sigma-54 RpoN
RNA polymerase factor sigma-38 RpoS
Anti-sigma 28 factor
Phage shock protein A, suppresses
related genes (ETAE_29622963) (Figure 3) are also present in the
genome which may be involved in acid resistance and biofilm
formation and can act as a free radical ion scavenger . The
lacking of genes encoding osmolarity responding proteins OmpC
and OmpF also supports the idea that EIB202 might utilize
unusual mechanisms to cope with the osmosis challenges.
Two-component signal transduction system (TCS), comprising
of a sensor histidine kinase (HK) protein and a response regulator
(RR) protein, is well documented to regulate various biophysical
processes as well as virulence in bacteria . EIB202 harbors
dozens of TCSs including 31 HK genes and 33 RR genes
(including ETAE_1502 as an orphan RR protein) (Table 7). Most
of the HK genes reside adjacent to RR genes on the chromosome
and are likely to be functional pairs involved in responses to
environmental changes. However, the order of these gene pairs
(59-HK/39-RR or 59-RR/39-HK) and the transcriptional direction
relative to the chromosome (direct or complementary) appear to
be random. In this respect, four particular pairs (i.e., ArcB/ArcA,
CheA/CheB/CheY, BarA/UvrY and YehU/YehT) are
exceptional in the sense that each corresponding partner resides at a
different location of the chromosome, although each pair is known
to function together in a certain signaling pathway. In E. tarda,
TCSs may mediate adaptive responses to a broad range of
environmental stimuli, including phosphate/Mg2+ limitation
(Pho), anaerobic condition (Cit and Arc), heavy metal overload
(Cus), osmosis change (EnvZ/OmpR), and motility/chemotaxis
(Che), etc. It is probably safe to conclude that most
twocomponent regulation is used for enhancing the versatility of the
response of the organism to environmental stimuli by the
regulation of normally unexpressed genes, while some TCSs, such
as the previously described EsrA/EsrB  and PhoP/PhoQ ,
may also contribute to the virulence in EIB202.
Quorum sensing (QS) is the signal transduction system that
responds to cell density for inter- and intra- species communication
under various conditions or stresses . E. tarda EIB202 carries
AI1/AHL dependent EdwI/EdwR (ETAE_2593/2594) system ,
AI-2/LuxS (ETAE_2854) system  and a putative AI-3/
epinephrine/norepinephrine system which might be sensed by the
QseB/QseC (ETAE_0447/0448) TCS system to activate the
expression of flagellar operons and virulence related genes, the
way as that in E. coli . Also, EIB202 contains dozens of proteins
that may be involved in the c-di-GMP mediated signal transduction
system, including the effector protein with a PilZ domain
(ETAE_3384), and 10 proteins with c-di-GMP biosynthesis related
GGDEF or degradation associated EAL domain (Table 8). It is
intriguing to detect another 5 proteins carrying both GGDEF and
EAL domain (Table 8). These proteins may be involved in the signal
transduction networks controlling the make and break of the
second messenger c-di-GMP, which in turn binds to an
unprecedented range of effector components and controls diverse targets
necessary for virulence and different bacterial lifestyles in various
niches . In all, the abundant repertoire (5% of all the EIB202
CDS) of signal transduction related genes in terms of their numbers
and families (Table 3) fundamentally contribute to the survival of
EIB202 in various hosts.
Molecular chaperone (small heat shock protein)
Molecular chaperone (small heat shock protein)
Putative ATPase with chaperone activity
Phage shock protein G
Co-chaperonin GroES (HSP10)
Chaperonin GroEL (HSP60 family)
Type III secretion system chaperone protein B
Type III secretion low calcium response chaperone
Fimbrial chaperon protein
Heat shock protein HslJ
Phage shock protein D
Phage shock protein B
Cytoplasmic chaperone TorD family protein
Flagellar biosynthesis chaperone
Phage shock protein A (IM30), suppresses
Phage shock protein operon transcriptional activator
Hydrogenase 2-specific chaperone
Acid shock protein precursor
Phage tail assembly chaperone gp38
Protein disaggregation chaperone
Heat shock protein 15
Disulfide bond chaperones of the HSP33 family
Histidine protein kinase (HK)
Response regulator (RR)
Citrate uptake and metabolism
Trimethylamine N-oxide respiration
Modification of lipopolysaccharide
Flagellar biogenesis and motility
Protein secretion and virulence
Hexose phosphate transport
Short chain fatty acid metabolism
Mg2+ starvation and virulence
Hexose phosphate uptake
Cell envelop protein folding and degradation
Carbon storage regulation
Unknown function, orphan RR protein
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 . 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].
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
Response regulator receiver modulated diguanylate cyclase
Cellulose synthase catalytic subunit
* Y indicates presence of EAL, GGDEF or PilZ domain in the listed proteins.
genome sequence, encoding components required for flagellar
hook basal body and hook-filament junction structures . 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
Hypothetical protein, putative BAP type adhesins
Putative invasin, shdA, non-fimbrial adhesin
Putative hemolysin secretion ATP-binding protein
Filamentous haemagglutinin family outer membrane protein
ShlB/FhaC/HecB family haemolysin secretion/activation protein
Hemolysin transporter protein
Putative hemolysin precursor
Hemolysin expression modulating family protein
OmpA, outer membrane protein A
OmpW, outer membrane protein W
Pic serine protease precursor, FhaB filamentous heamagglutinin
Hemolysin III family
Temperature sensitive hemagglutinin
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  and the attack
by caspase-1 and interleukin 1b secreted by host cells which
recognize the flagellin of the bacteria mounting the host cells .
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 . 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
Secindependent twin arginine transport (Tat), T1SS, TTSS and T6SS
were all identified in the genome of E. tarda EIB202 (Figure 3). Tat
mediated protein translocation, which allows for the secretion of
folded proteins such as redox proteins and their chaperones, has
been demonstrated to mediate various stress responses and
virulence in pathogenic bacteria, and is prevalent among
halophiles and helps in transport of proteins in their folded state
[38,39]. In EIB202, a total of 33 CDSs harboring the Tat specific
N-terminus signal peptide were identified to be the potential
substrates of the secretion system (Table S3). These proteins, in
combination with their co-translocated substrates, might also
contribute to its adaptation to high salt conditions (up to 5%) and
other environmental stresses. Unlike Vibrio spp. or other
pathogens, EIB202 contains just one copy of TTSS and T6SS,
and the components and genetic spatial organizations of the two
types of secretion systems in EIB202 were of the same with that
described in E. tarda strain PPD130/91 [19,20]. The various
secretion systems in EIB202 may be also involved in the
transmembrane transduction of fimbrial or non-fimbrial
adhesins/invasions/haemagglutinins to enrich the surface structures of
Survival inside the mucus and fish body seems to be attributed
to a vast arsenal of proteins active against the fish tissues,
epithelial/endothelial cells, fragile cells and vascular system. In
spite of previous description as a mean producer of extracellular
proteases [32,33], EIB202 harbors the genes (n = 22) that encode
putative extracellular proteins which may take part in the
utilization of extracellular matrix such as nucleotides, and
phospholipids for survival and fitness (Table 10). The genes
ETAE_2918 and ETAE_2529 encoding putative chondroitinases
are present in the genome and may be involved in the formation of
the chronic hole-in-the-head lesions due to cartilage
degradation . With the exception of hlyA encoding b-hemolysin, 6
hemolysins and related function genes are found in the genome,
possibly accounting for the usually observed red skin or
hemorrhage symptoms in affected fish . The gene encoding
collagenase (ETAE_0416) may mediate vascular injury and
hemorrhage symptoms during infection. Interestingly, EIB202
genome encodes a protein carrying homology to von Willebrand
factor type A domain of humans (ETAE_3520, vwA), which is
involved in the normal hemostasis by adhering to the
subendothelial matrix following vascular damage .
E. tarda has developed abilities to utilize hemin, hemoglobin and
hematin as iron source as well as siderophore-mediated iron
uptake mechanism . The finding of the clustered genes
(ETAE_1793-ETAE_1801) which encode a coproporphyrinogen
III oxidase (hemN), heme iron utilization protein, hemin receptor
and related ABC transporter proteins underlies its capacities to use
hemin related iron sources (Figure 3) . It is interesting to discern
a gene cluster (ETAE_1968-ETAE_1974) sharing high similarities
to the pvsABCDE-psuA-pvuA operon (Figure 3) which encodes the
proteins for the synthesis and utilization of vibrioferrin, an unusual
type of siderophore requiring nonribosomal peptide synthetase
(NRPS) independent synthetases (NIS) and usually mediating the
iron uptake systems in V. parahaemolyticus and V. alginolyticu, normal
marine flora as well as opportunistic pathogens for sea animals and
humans [42,43]. In EIB202 genome, genes for ferric uptake
regulator, ferric reductase, ferrous iron utilization, ferritin protein
and TonB systems are also present (Figure 3), implicating an
elaborate iron homeostasis system priming for the inhabitation and
invasion of various hosts.
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 . 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
. 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 .
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
nonPutative xanthine/uracil permeases
Outer membrane phospholipase A
Trypsin-like serine proteases
Protease III precursor
Putative membrane-associated zinc metalloprotease
Pyridine nucleotide transhydrogenase
Acyl-CoA thioesterase I
Glycerophosphoryl diester phosphodiesterase
Predicted intramembrane serine protease
Xanthine/uracil permeases family protein
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 .
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 Mg2+ environment , 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
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
Putative iron-dependent peroxidase
Thioredoxin domain-containing protein
Copper-zinc superoxide dismutase
Alkyl hydroperoxide reductase, small subunit
Thioredoxin-dependent thiol peroxidase
Dyp-type peroxidase family
Probable cytochrome C peroxidase
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
antibioticsresistance 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
. 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
Materials and Methods
Bacterial growth and DNA extraction
E. tarda EIB202 (previously referred to as isolate EH202 
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 28uC.
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
280uC for further gap closing.
High-density pyrosequencing and sequence assembly of
The complete sequencing work was conducted using Roche GS
FLX system . 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 . 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  and
Glimmer3 , and peptides shorter than 30 aa were eliminated.
Sequences from the intergenic regions were compared to
GenBanks non-redundant (nr) protein database  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 , 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 . Functional annotation of CDSs was performed
through searching against nr protein database using BLASTP
. The protein set was also searched against COG (http://
www.ncbi.nlm.nih.gov/COG/; ) and the KEGG (Kyoto
encyclopedia of genes and genomes; http://www.genome.jp/
kegg/)  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) . TatP 1.0 server
(v2.0)  and TATFIND 1.2 program  were used to detect
the potential substrates of the Tat secretion system. Pathogenicity
islands and anomalous genes were detected by PAI-IDA  and
SIGI-HMM , 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) .
BLAST algorithm was used when needed and ambiguous regions
were trimmed according to an embedded mask. Concatenated
protein sequences were aligned by ClustalW . Maximum
likelihood tree based on the aligned protein sequences was
constructed by using PhyML  with 100 bootstrap iterations.
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 = e220
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.
The 2|2 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
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,
Table S2 Amino acid biosynthesis genes in E. tarda EIB202
Found at: doi:10.1371/journal.pone.0007646.s002 (0.17
Table S3 The predicted Tat substrates in EIB202
Found at: doi:10.1371/journal.pone.0007646.s003 (0.07
We thank Professor Dr. Xiaohua Zhang, Prof. Dr. Li Sun and Dr. Hui
Gong for providing E. tarda strains.
Conceived and designed the experiments: QW MY QL YZ. Performed the
experiments: QW MY JX XW YL LX HZ SW GZ. Analyzed the data:
QW MY JX HW QL YZ. Wrote the paper: QW MY QL YZ.
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