Whole-Genome Sequence Analysis and Genome-Wide Virulence GeneIdentification of Riemerella anatipestifer Strain Yb2

Xiaolan Wang, Chan Ding, Shaohui Wang, Xiangan Han, Shengqing Yu

Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Shanghai, People’s Republic of China

Riemerella anatipestifer is a well-described pathogen of waterfowl and other avian species that can cause septicemic and exuda-tive diseases. In this study, we sequenced the complete genome of R. anatipestifer strain Yb2 and analyzed it against the pub-lished genomic sequences of R. anatipestifer strains DSM15868, RA-GD, RA-CH-1, and RA-CH-2. The Yb2 genome contains onecircular chromosome of 2,184,066 bp with a 35.73% GC content and no plasmid. The genome has 2,021 open reading frames thatoccupy 90.88% of the genome. A comparative genomic analysis revealed that genome organization is highly conserved among R.anatipestifer strains, except for four inversions of a sequence segment in Yb2. A phylogenetic analysis found that the closestneighbor of Yb2 is RA-GD. Furthermore, we constructed a library of 3,175 mutants by random transposon mutagenesis, and 100mutants exhibiting more than 100-fold-attenuated virulence were obtained by animal screening experiments. Southern blotanalysis and genetic characterization of the mutants led to the identification of 49 virulence genes. Of these, 25 encode cytoplas-mic proteins, 6 encode cytoplasmic membrane proteins, 4 encode outer membrane proteins, and the subcellular localization ofthe remaining 14 gene products is unknown. The functional classification of orthologous-group clusters revealed that 16 genesare associated with metabolism, 6 are associated with cellular processing and signaling, and 4 are associated with informationstorage and processing. The functions of the other 23 genes are poorly characterized or unknown. This genome-wide study iden-tified genes important to the virulence of R. anatipestifer.

Riemerella anatipestifer is a Gram-negative, non-spore-form-ing, nonmotile, capsule-like, rod-shaped bacterium. It is re-

ported worldwide as the cause of epizootic infectious polyserositisin domestic ducks (1); it is also pathogenic for geese, turkeys,chickens, and other birds (2, 3). R. anatipestifer infection occurs inacute form in ducks less than about 8 weeks of age and in chronicform in older birds. It causes major economic losses in the duckindustry by causing a high mortality rate, poor feed conversion,increased condemnations, and high treatment costs (4, 5).

Currently, 21 serotypes of R. anatipestifer have been identifiedby slide and tube agglutination tests using antisera (6). There is alarge variation in virulence between different serotypes andstrains, as assessed by mortality and morbidity rates (7). Infectionswith R. anatipestifer serotypes 1, 2, 3, 5, 6, 7, 8, 10, 11, 13, 14, and15 have been reported in China, with serotypes 1, 2, and 10 beingresponsible for most of the major outbreaks (8). There is very littleknowledge about the molecular bases of R. anatipestifer virulence,except for the virulence factors VapD, the Christie-Atkins-Munch-Peterson (CAMP) cohemolysin, and OmpA. VapD showshomology to virulence-associated proteins in other bacteria (9).The CAMP cohemolysin is a sialoglycoprotease produced duringnatural infection under certain intracellular conditions, andtherefore, it is able to damage the host and facilitate the infectionprocess (10). OmpA is a 42-kDa outer membrane protein thatseems to be not only a predominant specific antigen (11) but alsoan adhesin that plays a critical role in colonization (12). Addition-ally, biofilm formation by R. anatipestifer may contribute to per-sistent infections in duck farms, as biofilm-producing isolates aremore resistant to antibiotic and detergent treatments than plank-tonic isolates are (13).

Thus far, only limited genomic resources are available for R.anatipestifer, given that 21 serotypes have been identified. Thecomplete genome sequences of strains DSM15868 (ATCC 11845),RA-GD, RA-CH-1, and RA-CH-2 have been released (14, 15). The

established genomes are very similar in size and gene number. Tocomprehensively and systematically explore the genetic diversityand evolution of the virulence of R. anatipestifer strains, genome-wide profiling is needed. Here, we report the complete genomicsequence of R. anatipestifer serotype 2 strain Yb2, which was iso-lated in Jiangsu Province, China (8). Analysis of the completegenomic sequence revealed potential virulence factors and meta-bolic pathways in Yb2. Subsequently, a random transposon librarycontaining 3,175 mutants was constructed and screened forTn4351-induced virulence attenuation in animal experiments.Genes involved in bacterial virulence were identified and charac-terized. Further studies of virulence factors are invaluable for un-derstanding R. anatipestifer pathogenesis, with the ultimate goal ofdisease prevention and control.

MATERIALS AND METHODSPlasmids, bacterial strains, and growth conditions. For the bacterialstrains, plasmids, and primers used in this study, see Table S1 in thesupplemental material. R. anatipestifer Yb2 is the wild-type strain used inthis study (8). It was cultured in tryptic soy agar (TSA; Becton Dickinson,Franklin Lakes, NJ, USA) at 37°C for 24 h in 5% CO2 or tryptic soy broth

Received 18 March 2015 Accepted 13 May 2015

Accepted manuscript posted online 22 May 2015

Citation Wang X, Ding C, Wang S, Han X, Yu S. 2015. Whole-genome sequenceanalysis and genome-wide virulence gene identification of Riemerella anatipestiferstrain Yb2. Appl Environ Microbiol 81:5093–5102. doi:10.1128/AEM.00828-15.

Editor: P. D. Schloss

Address correspondence to Shengqing Yu, yus@shvri.ac.cn.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.00828-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.00828-15

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(TSB; Becton Dickinson) at 37°C with shaking at 200 rpm for 8 to 12 h.Escherichia coli strain BW19851 containing plasmid pEP4351 was grownin Luria broth (LB; Becton Dickinson) or on LB agar containing 30 �g/mlchloramphenicol. To select for Tn4351-disrupted Yb2 mutants, erythro-mycin (0.5 �g/ml) and kanamycin (50 �g/ml) were added to TSA.

Animals. One-day-old Cherry Valley ducklings were obtained fromthe ZhuangHang Duck Farm (Shanghai, China), housed in cages, andprovided water and food ad libitum during the study. The animal exper-iments were conducted in strict accordance with the recommendations inthe Guide for the Care and Use of Laboratory Animals of the InstitutionalAnimal Care and Use Committee guidelines set by the Shanghai Veteri-nary Research Institute, Chinese Academy of Agricultural Sciences(CAAS). The protocol was approved by the Committee on the Ethics ofAnimal Experiments of the Shanghai Veterinary Research Institute, CAAS(permit no. 12-12). All surgeries were performed under sodium pento-barbital anesthesia, and all efforts were made to minimize suffering.

Genomic DNA extraction. R. anatipestifer strain Yb2 was cultured inTSB at 37°C for 8 h with shaking until it reached the mid-logarithmicphase. Genomic DNA was extracted from bacterial cell pellets with theTIANamp Bacteria DNA kit (Tiangen, Beijing, China) in accordance withthe manufacturer’s instructions. DNA was resuspended in nuclease-freewater and quantified with a NanoVue spectrophotometer (GE Health-care, Little Chalfont, United Kingdom). DNA quality and RNA contam-ination were assessed by electrophoresis with 0.8% agarose gels.

Genome sequencing and assembly. Genomic DNA of R. anatipestiferstrain Yb2 was sequenced with the MiSeq Sequencing platform (Illumina,San Diego, CA, USA). A shotgun sequencing approach was adopted byemploying two independent genomic libraries. A total of 804,600 reads,totaling 371,017,848 bases (average read length, 346 bp), or about 186-fold coverage of the genome, was obtained from a paired-end 400-bp(PE400) library. A sum of 142,150 reads, totaling 42,577,127 bases (aver-age read length, 422 bp), was obtained from a mate-paired 8-kbp (MP8k)library, resulting in 22-fold genome coverage. Newbler software (version2.8) (Roche, Indianapolis, IN, USA) was used for sequence assembly. Theorder of contigs was determined by multiplex PCR. Gaps were filled infirst with GapCloser software and then by sequencing the PCR products(16). Low-quality regions of the genome sequence were resequenced tocomplete the sequencing.

Gene prediction and annotation. Putative coding sequences (CDSs)were identified by Glimmer 3.0 (17). tRNA genes were predicted bytRNAscan-SE (18), while rRNA genes were predicted by RNAmmer1.2(19). Other noncoding RNA (ncRNA) genes were obtained by comparingthe genome sequences to the Rfam database (http://rfam.sanger.ac.uk)(20). The clustered regularly interspaced short palindromic repeats(CRISPR) recognition tool was used to predict direct repeat sequencesand spacers (21). Functional annotation of CDSs was achieved by search-ing protein databases and a nonredundant protein database from theNational Center for Biotechnology Information (NCBI). Clusters of or-thologous groups (COGs) of proteins were used for functional classifica-tions performed with the eggNOG (version 3) database (http://eggnog.embl.de/version_3.0/). Orthologs and paralogs were defined as proteinswith �30% similarity (22). Metabolic pathways were constructed with theKyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) (23, 24). Genomic islands (GIs) were detectedby IslandViewer (25). The genome atlas was drawn with CGView (26).

Evolutionary analysis. (i) SNP calling. To study the polymorphismand evolutionary rate of R. anatipestifer genes, we identified single nu-cleotide polymorphisms (SNPs) in the Yb2 genome against referencegenomes of the DSM15868 (accession no. CP002346.1), RA-GD(CP002562.1), RA-CH-1 (CP003787.1), and RA-CH-2 (CP004020.1)strains. Genomic sequences with adjusted start positions were submittedto the NUCmer system (NUCleotide MUMmer version 3.07) for align-ment (27, 28), and nonspecific records were filtered by delta-filter. Afteralignment of the sequenced reads with a reference genome, SNP callingwas performed with show-snps software.

(ii) Colinearity analysis. The reference genomes of R. anatipestiferstrains DSM15868 (CP002346.1), RA-GD (CP002562.1), RA-CH-1(CP003787.1), and RA-CH-2 (CP004020.1) were downloaded from theNCBI to identify conserved genomic regions in the presence of rearrange-ments and horizontal transfers. Prior to alignment, the genomic se-quences were adjusted to a common start position. Mauve (version 2.3.1)was used to align the Yb2 genome with the four reference genomes and todraw a colinear atlas (29, 30).

(iii) Homology analysis and phylogenetic tree construction. A localdatabase was created and managed with formatdb software based on thefour reference genomes (31). The query sequence was compared to thenewly created database with the blastall (version 2.2.22) service (32). Forphylogenetic tree construction, 16S rRNA gene sequences were aligned byusing the default settings of Muscle (version 3.8.31) (33). The conservedalignment blocks were then extracted by Gblocks (version 0.91b) (34). Amaximum-likelihood tree was built with Mega6 (version 6.05) by usingthe Kimura two-parameter module. The reliability of the phylogenetictree branches was verified via bootstrap analysis (1,000 replications) (35).

Random transposon mutagenesis and isolation of mutants withattenuated virulence. Tn4351 was introduced into R. anatipestifer wild-type strain Yb2 by conjugation from E. coli BW19851 as previously de-scribed (13). Briefly, donor and recipient cells were cultured to mid-log-arithmic phase, mixed at a ratio of 4:5 (based on optical density at 600nm), and filtered onto a 0.22-�m membrane (EMD Millipore, Billerica,MA, USA). The filter were placed on a TSA plate and incubated at 30°C for8 h. After incubation, the cells was scraped off the filter, resuspended in 10mM MgSO4, and plated onto TSA supplemented with erythromycin (0.5�g/ml) and kanamycin (50 �g/ml) to select for potential transconjugants.Pairs of primers specific for R. anatipestifer (primers RA 16S rRNA-F/RA16S rRNA-R) or transposon Tn4351 (primers Erm-F/Erm-R) were usedto identify mutants by PCR amplification. PCRs were conducted with 2�Taq MasterMix (CWBIO, Beijing, China) with the following parameters:94°C for 4 min; 30 cycles of 94°C for 40 s, 52°C for 40 s, and 72°C for 1 min;and 1 cycle of 72°C for 10 min. The mutants from which the R. anatipes-tifer 16S rRNA and Tn4351 were amplified were deemed to be Tn4351-disrupted mutants.

To obtain mutants with attenuated virulence, 18-day-old Cherry Val-ley ducklings were infected intramuscularly with 1 � 107 CFU of themutants, which is equal to 100 median lethal doses (LD50s) of wild-typestrain Yb2 (36). The infected ducklings were housed in separate cages witha 12-h light-dark cycle and free access to food and water during the study.The clinical symptoms and deaths of ducklings were recorded daily for 7days, and mutants that failed to kill the susceptible ducklings were selectedfor further study.

Identification, sequencing, and bioinformatic analysis of the mu-tated genes. Southern blotting was used to confirm a single Tn4351 inser-tion in the R. anatipestifer genome. The region of transposon Tn4351 wasamplified as a 626-bp PCR product from pEP4351 with primers Tn4351-F/Tn4351-R. The probe was prepared with DIG-High Prime DNA label-ing and detection starter kit I (Roche). Genomic DNA of the attenuatedmutants was purified with the TIANamp Bacteria DNA kit (Tiangen),digested with XbaI, separated by gel electrophoresis, and transferred to anylon membrane. Hybridization procedures were conducted by usingstandard molecular biology protocols.

The sites of transposon insertions in the attenuated mutants weredetermined by cloning the Tn4351-disrupted genes by genomic walkingor inverse PCR. Genomic walking was performed with a genome walkingkit (TaKaRa, Dalian, China) according to the manufacturer’s instruc-tions. Arbitrary primers (AP1, AP2, AP3, and AP4) provided in the kit, aswell as specific primers (SP1, SP2, and SP3), were used to amplify the DNAadjacent to the site of insertion (13). For inverse PCR, genomic DNA wasdigested with HindIII and religated to form closed circles (37). The result-ing circular molecules were used as templates to amplify the Tn4351 in-sertion region with the TN-1/IS4351-F or 340/341 primer pair (38).

After purifying the PCR products from agarose gels with the GeneJET

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gel extraction kit (Thermo Fisher Scientific, Waltham, MA, USA), thePCR products were sequenced by HuaGene Biotech Co., Ltd. (HuaGene,Shanghai, China). Sequences were compared to those in the existingNCBI database with BLAST (http://www.ncbi.nlm.nih.gov/BLAST/).Predictions regarding subcellular localization were made by using thePSORTb (version 3.0) server (http://www.psort.org/). Functional charac-terization of the proteins was predicted by searching against the eggNOG(version 3) database with BLASTP (http://eggnog.embl.de/version_3.0/).

Determination of LD50s. The virulence of the attenuated mutants wasevaluated by determining their LD50s. The attenuated mutants weregrown for 8 to 10 h to mid-logarithmic phase and concentrated by cen-trifugation (1,500 � g, 10 min). The bacterial pellet was washed twice withphosphate-buffered saline (PBS, pH 7.4) and serially diluted to obtaindifferent bacterial concentrations (CFU counts). Eighteen-day-old duck-lings were injected intramuscularly with 107 to 1011 CFU of the attenuatedmutants in 0.5 ml of PBS. The ducklings were monitored daily for clinicalsymptoms, and deaths were recorded until 7 days postinfection. The LD50

was calculated by an improved version of Karber’s method (39).Nucleotide sequence accession number. The full genomic sequence

of R. anatipestifer strain Yb2 has been submitted to the GenBankdatabase (http://www.ncbi.nlm.nih.gov/GenBank/) under accessionnumber CP007204.

RESULTSGenome features of R. anatipestifer Yb2. R. anatipestifer strainYb2 has a single circular chromosome of 2,184,066 bp with a35.73% GC content. There was no evidence of plasmids (Fig. 1; seeTable S2 in the supplemental material). The genome encodes sixrRNA operons, 38 tRNAs that represent all 20 amino acids, and 1other ncRNA. CRISPR is a unique family of DNA direct repeatsequence that widely exist in prokaryotic genomes. There is onepredicted CRISPR array in the Yb2 genome. In addition to sevenpseudogenes, 2,021 open reading frames (ORFs) were identifiedin the Yb2 genome, and they had an average length of 976 bp andconstituted 90.88% of the genome. Among these ORFs, 1,214(60.1%) genes were classified into COG families comprising 20functional categories (see Table S3 in the supplemental material).A total of 127 ORFs were classified into the “translation, ribosomalstructure, and biogenesis” category, 119 ORFs were related to “cellwall/membrane/envelope biogenesis,” 478 ORFs belonged to“metabolism,” and most of the ORFs were related to “amino acidtransport and metabolism,” “energy production and conversion,”and “coenzyme transport and metabolism.” With KEGG, 1,217

FIG 1 Summary of gene annotations and GC skew analysis of the Yb2 genome. From outer to inner, circles 1 and 2 show the CDSs (blue), rRNA operon (lightpurple), and tRNA information (reddish brown); circle 3 shows the GC contents; circle 4 shows the GC skew [(G � C)/(G � C); green, �0; purple, �0]; and circle5 shows the coordinates of the Yb2 genome. The artificial start site is at 0 kbp.

Genome Sequence and Virulence Genes of RA Strain Yb2

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ORFs (60.2%) were assigned to 30 pathways (Fig. 2). Of theseORFs, 948 (77.9%) belonged to “metabolism,” and the majority ofthese were involved in the metabolism of amino acids, carbohy-drates, cofactors, and vitamins. Other ORFs were assigned topathways in “genetic information processing” (13.6%), “environ-mental information processing” (3.7%), and “human diseases”(2.1%).

GIs. GIs are relatively large segments of DNA (usually 10 to 200kb) that are present in the genomes of many bacterial species (40).A total of six GIs were predicted to be scattered throughout thesingle chromosome that makes up the Yb2 genome (see Table S4in the supplemental material). Putative GI-1 contained mostlyORFs that encode hypothetical proteins, as well as two ORFs thatencode a DNA-binding protein and a phage head morphogenesisprotein. Additionally, there was one ORF that encodes each of thefollowing: an N-acetylmuramoyl-L-alanine amidase, a phage headprotein, and an aminopeptidase. Putative GI-2 included ORFsthat encode hypothetical proteins, an integrase, an AAA (ATPasesassociated with diverse cellular activities) ATPase, restriction en-donuclease EcoRI subunit R, and a transcriptional regulator. Pu-tative GI-3 included only two ORFs for hypothetical proteins.Putative GI-4 included ORFs that encode peptidase M23, a trans-posase, a membrane protein, and kinase enzymes, such as a serine/

threonine protein kinase, suggesting that this cluster may play aregulatory role in cellular processing. Putative GI-5 consisted ofORFs that encode a DNA-binding protein, restriction endonu-clease subunit R, and hypothetical proteins. These two clustersmay be involved in transcription and DNA recombination. ORFsin putative GI-6 encode multidrug resistance proteins such asLinF, chloramphenicol acetyltransferase, and a tetracycline resis-tance protein; metal transporters such as a mercury transporter, amulticopper oxidase, and a cation transporter; toxins such as RelEand YoeB; AraC and xenobiotic response element family tran-scriptional regulators; and two proteins involved in twitching mo-tility.

Evolutionary analysis. We performed SNP calling to deter-mine polymorphic sites between Yb2 and the reference genomesof strains DSM15868, RA-GD, RA-CH-1, and RA-CH-2 (see Ta-ble S5 in the supplemental material). A comparison of Yb2 andRA-GD revealed 4,548 SNPs, 4,252 (93.5%) of which are locatedin coding regions. Similarly, of the 5,448 SNPs between Yb2 andRA-CH-2, 5,119 (94.0%) are located in coding regions. There are78,163 SNPs between Yb2 and RA-CH-1 and 11,905 SNPs be-tween Yb2 and DSM15868.

All five R. anatipestifer genomes were aligned by Mauve to in-vestigate the degree of colinearity between them. The alignment

FIG 2 Summary of KEGG pathway classifications. The metabolic pathways were constructed by using the KEGG database. Values are the numbers of respectiveclassifications.

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revealed 50, 29, 203, and 42 colinearity blocks between Yb2 andDSM15868, RA-GD, RA-CH-1, and RA-CH-2, respectively. Ge-nome structure rearrangements, which are shown in Fig. 3, illus-trate a high level of synteny, with the exception of four inversionsin the Yb2 genome.

To investigate the phylogenetic relationships among the five R.anatipestifer strains, we analyzed their 16S rRNA gene sequences.The resulting phylogenetic tree revealed a strikingly short geneticdistance between Yb2 and RA-GD (Fig. 4), indicative of very re-cent divergence, and a much greater distance between Yb2 andDSM15868.

Isolation of Tn4351-induced mutants with reduced viru-lence. We introduced Tn4351 from E. coli BW19851 into R. anati-pestifer via the delivery vector pEP4351 and obtained 3,175 eryth-romycin-resistant Yb2 colonies. Tn4351 insertion mutants weredeemed defective in virulence on the basis of the loss of pathoge-nicity in our animal model. We identified 100 mutants with dif-ferent virulence levels in the initial screening for transconjugants.Southern blot analysis revealed that 62% (62/100) of the attenua-tion mutants had a single Tn4351 insertion, while 30% (30/100)had two and 8% (8/100) had three insertions (data not shown).

Identification of genes disrupted by transposon insertion. Aset of 62 R. anatipestifer Yb2 mutants with virulence defects wasobtained by Tn4351 mutagenesis. The DNA sequences flankingthe transposon insertions were obtained by inverse PCR orgenomic walking. The flanking sequences were then BLASTsearched against the Yb2 genome (accession no. CP007204) andfour other R. anatipestifer genome sequences in the NCBI databaseto determine which genes were interrupted by the transposon.We identified 49 mutated genes (Table 1), 4 of which had multiplemutations. The AS87_06670 gene, which encodes a hypotheti-cal protein, was mutated in mutants RA712 and RA791; theAS87_01210 gene, which encodes a phosphoribosyl formylglyci-namidine synthase, was mutated in mutants RA848, RA908, andRA1333; the AS87_09295 gene, which encodes a pyruvate dehy-drogenase, was mutated in mutants RA1323 and RA2067; and theAS87_06745 gene, which encodes a hypothetical protein, was mu-tated in mutants RA1476 and RA2279. Tn4351 was located out-side ORFs in mutants RA512, RA723, RA750, RA783, RA885,RA2330, RA2792, and RA3096 (data not shown).

Bioinformatic analysis of the proteins encoded by the mu-tated genes. We predicted the subcellular locations of the proteinproducts of these 49 identified genes with PSORTb (version 3.0)software. Twenty-five proteins (51%) were predicted to be cyto-plasmic, six (12%) were predicted to be cytoplasmic membraneproteins, and four (8%) were predicted to be outer membraneproteins. Fourteen proteins (29%) had unknown locations.

These proteins were further categorized on the basis of theirputative functions (Table 1). Sixteen proteins (33%) were classi-fied into the “metabolism” (C, G, E, F, H, P, and Q) category, ofwhich 11 were related to “nucleotide transport and metabolism,”“coenzyme transport and metabolism,” and “energy productionand conversion.” Cellular processes and signaling-related catego-

FIG 3 Schematic representations of R. anatipestifer genomic sequences. Each contiguously colored region is a colinear block, a region without rearrangement ofthe homologous backbone sequence. Lines between genomes trace each orthologous colinear block through every genome. The colinear blocks below a genome’scenter line represent segments that are inverted relative to the reference genome. The image shown was generated by the Mauve rearrangement viewer.

FIG 4 Phylogenetic relationships among R. anatipestifer strains DSM15868,RA-GD, RA-CH-1, RA-CH-2, and Yb2. The maximum-likelihood tree shownwas generated with Mega6 (version 6.05) and is based on 16S rRNA genesequences. Values are branch lengths.

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TABLE 1 Characterization of R. anatipestifer Yb2 mutants with attenuated virulence

Mutant(s) Locus taga Description of gene

Gene product

Subcellular locationb

Function group(COG)c

RA38 AS87_03285 Phosphoribosylaminoimidazole carboxylase (NCAIR synthetase) Cytoplasmic membrane COG0026 FRA55 AS87_10600 Phosphoadenosine phosphosulfate reductase Cytoplasmic COG0175EHRA253 AS87_06385 Transcriptional regulator, LuxR family Unknown —d

RA256 AS87_08785 Gliding-motility protein Gldk Unknown COG1262 SRA514 AS87_04980 Antitermination protein NusB Cytoplasmic COG0781 KRA625 AS87_01735 PncA Cytoplasmic COG1335 QRA702 AS87_04750 Inosine-5-monophosphate dehydrogenase Cytoplasmic COG0517 RRA712, RA791 AS87_06670 Hypothetical protein Cytoplasmic COG0457 RRA756 AS87_01090 Hypothetical protein Cytoplasmic membrane —RA772 AS87_10710 Hypothetical protein Cytoplasmic —RA799 AS87_00910 Phosphoribosylaminoimidazole (AIR) synthetase Cytoplasmic COG0150 FRA835 AS87_00505 Gliding-motility protein GldG Unknown COG3225 NRA848, RA908, RA1333 AS87_01210 Phosphoribosylformylglycinamidine (FGAM) synthase Cytoplasmic COG0047 FRA1072 AS87_09335 Membrane protein Outer membrane —RA1323, RA2067 AS87_09295 Pyruvate dehydrogenase Cytoplasmic COG0508 CRA1340 AS87_04850 Hypothetical protein Unknown —RA1436 AS87_10665 Hypothetical protein Unknown —RA1450 AS87_04765 Beta-carotene 15,15=-monooxygenase Cytoplasmic membrane —RA1476, RA2279 AS87_06745 Hypothetical protein Unknown —RA1633 AS87_01220 Amidophosphoribosyltransferase Cytoplasmic COG0034 FRA1824 AS87_05545 Biotin synthase Cytoplasmic COG0502 HRA1893 AS87_09530 8-Amino-7-oxononanoate synthase Cytoplasmic COG0156 HRA2041 AS87_08840 Holliday junction resolvase Cytoplasmic COG0816 L

RA2101 AS87_02660 DNA-binding protein Cytoplasmic COG0776 LRA2145 AS87_09440 ATPase Cytoplasmic COG0714 RRA2148 AS87_09345 Hypothetical protein Unknown —RA2238 AS87_08115 Gliding-motility protein Unknown —RA2241 AS87_10760 Amino acid transporter Cytoplasmic membrane COG0531 ERA2257 AS87_05585 Beta-lactamase Cytoplasmic COG0607 PRA2281 AS87_08795 Gliding-motility protein GldM Unknown —RA2442 AS87_04755 Membrane protein Cytoplasmic membrane —RA2607 AS87_07960 Dihydroorotase Cytoplasmic COG0044 FRA2634 AS87_01015 Glycosyltransferase family 2 Unknown COG0463 MRA2640 AS87_04050 Vi polysaccharide biosynthesis protein VipB/TviC Cytoplasmic COG0451 MGRA2671 AS87_00250 PorT protein Unknown —RA2672 AS87_07135 Hypothetical protein Unknown —RA2673 AS87_09985 Hypothetical protein Cytoplasmic —RA2742 AS87_04845 Hypothetical protein Cytoplasmic COG0807 HRA2922 AS87_10150 Phosphoesterase Cytoplasmic membrane —RA2928 AS87_05075 ATPase AAA Cytoplasmic COG0507LRA3008 AS87_09245 Restriction endonuclease Cytoplasmic COG0732VRA3034 AS87_09000 Ankyrin Unknown COG0666RRA3052 AS87_07625 Fructose-bisphosphate aldolase Cytoplasmic COG0191 GRA3059 AS87_10020 Hypothetical protein Cytoplasmic —RA3094 AS87_05990 Hypothetical protein Unknown COG1002 VRA3107 AS87_08340 TonB-dependent receptor Outer membrane COG4206HRA3151 AS87_05195 Hypothetical protein Outer membrane —RA3174 AS87_00480 Hypothetical protein Outer membrane COG1843Na Based on the R. anatipestifer Yb2 genome (accession no. CP007204).b Subcellular locations were predicted by the PSORTb (version 3.0) server (http://www.psort.org/).c Functional characterization of the proteins was predicted by searching against the eggNOG (version 3) database with BLASTP.d —, no related COG. Functional categories: (i) information storage and processing (J, translation, ribosomal structure, and biogenesis; K, transcription; L, DNA replication,recombination, and repair); (ii) cellular processes and signaling (D, cell division and chromosome partitioning; V, defense mechanisms; O, posttranslational modification, proteinturnover, chaperones; M, cell envelope biogenesis, outer membrane; N, cell motility; T, signal transduction mechanisms); (iii) metabolism (C, energy production and conversion;G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; P, inorganic ion transport and metabolism; E, amino acid transport and metabolism; F,nucleotide transport and metabolism; Q, secondary metabolite biosynthesis, transport, and catabolism); (iv) poorly characterized (R, general function prediction only; S, functionunknown).

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ries (D, V, O, M, N, and T) contained six proteins (12%). Infor-mation storage and processing categories (J, K, and L) includedfour proteins (8%), and poorly characterized COGs (R and S)contained five proteins (10%). Eighteen proteins (37%) could notbe categorized.

LD50 determination. To elucidate the effects of the mutationson Yb2 virulence, the LD50 of the attenuation mutants was as-sessed in Cherry Valley ducklings. Twenty-two mutants with dif-ferent COG function classifications were selected for virulenceevaluation. Deaths were observed from day 1 to day 7 postinfec-tion. The LD50s are summarized in Table 2. Of these 22 mutants, 3were attenuated 100-fold, 10 were attenuated 1,000-fold, 3 wereattenuated 10,000-fold, and 5 were attenuated 100,000-fold com-pared with wild-type Yb2. The results indicate that the genes iden-tified by Tn4351 mutagenesis screening are required for full viru-lence in Yb2.

DISCUSSION

Only in recent years have complete genomes of R. anatipestiferstrains been sequenced. The first published R. anatipestifer ge-nome was that of DSM15868, and several R. anatipestifer genomesequences are available in GenBank. In this study, the completegenome sequence of R. anatipestifer serotype 2 virulent strain Yb2was determined and compared to the reference genomes of R.anatipestifer strains DSM15868, RA-GD, RA-CH-1, and RA-CH-2. The Yb2 genome was found to be highly colinear with thefour reference genomes, except for a predominant rearrangementinvolving four inversions (Fig. 3). Pairwise and reciprocal com-parisons identified 4,548 SNPs between Yb2 and RA-GD, which isYb2’s closest phylogenetically related neighbor. The two genomesequences are 99.30% identical.

Strong variations in virulence, as assessed by mortality and

morbidity rates in outbreaks, have been reported for different R.anatipestifer strains. Despite the devastating losses it has caused inthe poultry industry, only a few virulence factors have been estab-lished for R. anatipestifer (10, 12). To better understand the po-tential pathogenicity of R. anatipestifer, genes involved in the fullvirulence of R. anatipestifer strain Yb2 were identified by genome-wide random transposon mutagenesis. A total of 49 genes wereconfirmed to be virulence associated, with protein products in-volved in metabolism, cellular processes and signaling, and infor-mation storage and processing. Transposon disruption of eithergene attenuated Yb2 virulence by �100-fold. Of these, mutantstrain RA2640, which showed significantly attenuated virulencedue to inactivation of the AS87_04050 gene, has been previouslyinvestigated (41).

Four genes that encode outer membrane proteins were identi-fied as virulence factors. In particular, genes AS87_09335 andAS87_08340, identified in mutants RA1072 and RA3107, respec-tively, are involved in transport processes. Both of them encodeouter membrane receptor proteins, also named TonB-dependentreceptors. The TonB-dependent receptors combine with an extra-cellular or intracellular messenger and transmit it into or out ofthe cytoplasm, leading to the transcriptional activation of targetgenes (42). The TonB protein of E. coli carries out high-affinitybinding and energy-dependent uptake of specific substrates intothe periplasmic space (43). The proteins that are currently knownto act as TonB-dependent receptors include HpuAB, ShuA, lacto-ferrin-binding protein (Lbp), and transferrin-binding protein(Tbp) (44–47). Furthermore, TonB-dependent receptor TbdR1has been identified as a cross-immunogenic antigen among R.anatipestifer serotypes 1, 2, and 10 (48). The TonB-dependentreceptor TdrA of Pseudomonas fluorescens, which is a protective

TABLE 2 LD50s of the virulence attenuation mutants in this study

Disrupted genea Description of gene LD50 (CFU)Virulenceattenuation (fold)b

AS87_08795 Gliding-motility protein GldM 1.97 � 107 184AS87_01220 Amidophosphoribosyltransferase 2.88 � 107 269AS87_01210 Phosphoribosylformylglycinamidine (FGAM) synthase 5.75 � 107 537AS87_00250 PorT protein 1.24 � 108 1,160AS87_01015 Glycosyltransferase family 2 1.27 � 108 1,190AS87_00910 Phosphoribosylaminoimidazole (AIR) synthetase 1.48 � 108 1,380AS87_04980 Antitermination protein NusB 2.43 � 108 2,270AS87_00505 Gliding-motility protein GldG 3.16 � 108 2,950AS87_05585 Beta-lactamase 3.16 � 108 2,950AS87_07625 Fructose-bisphosphate aldolase 4.16 � 108 3,890AS87_04765 Beta-carotene 15,15=-monooxygenase 4.27 � 108 3,990AS87_08785 Gliding-motility protein Gldk 7.5 � 108 7,000AS87_03285 Phosphoribosylaminoimidazole carboxylase (NCAIR synthetase) 1 � 109 9,300AS87_06385 Transcriptional regulator, LuxR family 3.83 � 109 35,800AS87_04750 Inosine-5-monophosphate dehydrogenase 8.13 � 109 76,000AS87_09295 Pyruvate dehydrogenase 1 � 1010 93,400AS87_10020 Hypothetical protein 1.10 � 1010 103,000AS87_09440 ATPase 1.76 � 1010 165,000AS87_04050 Vi polysaccharide biosynthesis protein VipB/TviC 1.91 � 1010 179,000AS87_01735 PncA 5.23 � 1010 488,000AS87_09530 8-Amino-7-oxononanoate synthase 8.22 � 1010 768,000AS87_05545 Biotin synthase 2.18 � 1011 2,040,000a Based on the R. anatipestifer Yb2 genome (accession no. CP007204).b The measured LD50 of wild-type strain Yb2 was 1.07 � 105 CFU. Fold virulence attenuation was calculated by dividing the LD50 of the mutant strain by the LD50 of wild-typestrain Yb2.

Genome Sequence and Virulence Genes of RA Strain Yb2

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immunogen, is likely to be required for iron acquisition and op-timal bacterial virulence (49). We speculate that the products ofAS87_09335 and AS87_08340 may play a similar role in the patho-genicity of R. anatipestifer.

Cell invasion or disruption may be considered the first step ofsystemic disease development. It has been proposed that R. anati-pestifer may gain entry into the systemic circulation through bloodand tissue. Gene AS87_01015, identified in mutant RA2634, en-codes a family 2 glycosyltransferase that is responsible for thetransfer of nucleotide-diphosphate sugars to substrates such aslipopolysaccharide (LPS) and lipids. LPS, which functions as anendotoxin, an adhesin, and a modulator of the immune response,is an important virulence factor for numerous bacteria (50–52).Pseudomonas aeruginosa LPS was specifically bound by the cysticfibrosis transmembrane conductance regulator and endocytosedby epithelial cells, leading to rapid nuclear translocation of thetranscription factor nuclear factor kappa-light-chain-enhancer ofactivated B cells (NF-�B) (53). Therefore, it is attempting to spec-ulate that AS87_01015 may affect invasion by inhibiting bacterialLPS assembly and capsule formation.

Five of our transposon-disrupted genes, namely, AS87_03285,AS87_04750, AS87_09910, AS87_01210, and AS87_01220, are in-volved in the de novo IMP biosynthetic process. The product ofAS87_01210 performs the fourth step in IMP biosynthesis, whilethe protein encoded by AS87_09910 is the fifth enzyme in thispathway. The reaction catalyzed by the product of AS87_04750 isthe first committed and rate-limiting step in the de novo synthesisof guanine nucleotides, and therefore, it plays an important rolein cell growth regulation (54, 55). The protein encoded byAS87_01220 is the rate-limiting enzyme in the de novo pathway ofpurine ribonucleotide synthesis, and it is regulated by feedbackinhibition by AMP and GMP (56). As a result of the disruptedgenes, virulence was �200- to 80,000-fold lower in the mutantsthan in wild-type strain Yb2, suggesting that de novo IMP biosyn-thesis is important for the full virulence of R. anatipestifer. A pre-vious study also showed that the purine biosynthetic enzymePurH, which catalyzes the final steps in purine IMP biosynthesis,is required for the pathogenesis and virulence of Bacillus anthracisin guinea pigs (57).

Among the virulence genes we found, AS87_09530 andAS87_05545 were involved in the biotin synthetic process. The8-amino-7-oxononanoate synthase encoded by AS87_09530 cat-alyzes the decarboxylative condensation of L-alanine withpimeloyl coenzyme A (pimeloyl-CoA) to form 8(S)-amino-7-ox-ononanoate, which is the first committed step in biotin biosyn-thesis (58). The predicted product of AS87_05545 is biotin syn-thase, which catalyzes the last step of the biotin biosyntheticpathway (59). Biotin is the essential cofactor of biotin-dependentcarboxylases such as pyruvate carboxylase and acetyl-CoA carbox-ylase (60); therefore, it is necessary for cell growth, the produc-tion of fatty acids, and the metabolism of amino acids. Addi-tionally, recent reports have revealed that biotin synthesis isessential for bacterial growth, infection, and survival duringthe latent phase of Mycobacterium marinum (61, 62). In thisstudy, the mutants inactivated in biotin synthesis-related geneswere attenuated �700,000- to 2,000,000-fold compared withwild-type strain Yb2, demonstrating that biotin synthesis plays animportant role in the pathogenicity of R. anatipestifer.

Various antibiotics are currently used to prevent and con-trol R. anatipestifer infection in ducks, but this accelerates the

emergence of drug-resistant strains. We predicted a GI containingthree antibiotic resistance genes (AS87_09605, AS87_09610, andAS87_09615) in this study. The products of these genes conferresistance to lincomycin, chloramphenicol, and tetracycline,respectively. In addition, three metal transporter genes (AS87_09680, AS87_09690, and AS87_09710) that are involved in trans-membrane transport are also located on this GI. Furthermore, theproduct of AS87_09680 is a mercury transporter that is exposed tothe cytoplasm and involved in the transport of Hg across the cy-toplasmic membrane (63). This mercury transporter enables bac-teria to respond to changes in the concentration of mercury in theenvironment (64). Our data may provide a genetic basis for drugresistance and persistence during R. anatipestifer infections. Insummary, we completed a whole-genome sequence analysis of R.anatipestifer strain Yb2 and identified 49 virulence genes by ge-nome-wide random transposon mutagenesis and in vivo screen-ing. Our data provide the foundation for future studies of R. anati-pestifer pathogenesis.

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation ofChina (31272591).

We thank the staff of Shanghai Personal Biotechnology Co., Ltd.(Shanghai, China), for sequencing the genome of R. anatipestifer strainYb2.

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