yersinia yersinia pseudotuberculosis...

Research ArticleComparative Genomic Hybridization Analysis of Yersiniaenterocolitica and Yersinia pseudotuberculosis IdentifiesGenetic Traits to Elucidate Their Different Ecologies

Kaisa Jaakkola, Panu Somervuo, and Hannu Korkeala

Department of Food Hygiene and Environmental Health, University of Helsinki, P.O. Box 66, 00014 Helsinki, Finland

Correspondence should be addressed to Kaisa Jaakkola; [email protected]

Received 2 April 2015; Accepted 28 September 2015

Academic Editor: Miguel Prieto

Copyright © 2015 Kaisa Jaakkola et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

EnteropathogenicYersinia enterocolitica andYersinia pseudotuberculosis are both etiological agents for intestinal infection known asyersiniosis, but their epidemiology and ecology bearmany differences. Swine are the only known reservoir forY. enterocolitica 4/O:3strains, which are themost common cause of human disease, whileY. pseudotuberculosis has been isolated from a variety of sources,including vegetables andwild animals. Infections caused byY. enterocoliticamainly originate from swine, but fresh produce has beenthe source for widespread Y. pseudotuberculosis outbreaks within recent decades. A comparative genomic hybridization analysiswith a DNA microarray based on three Yersinia enterocolitica and four Yersinia pseudotuberculosis genomes was conducted toshed light on the genomic differences between enteropathogenic Yersinia. The hybridization results identified Y. pseudotuberculosisstrains to carry operons linked with the uptake and utilization of substances not found in living animal tissues but present in soil,plants, and rotting flesh. Y. pseudotuberculosis also harbors a selection of type VI secretion systems targeting other bacteria andeukaryotic cells. These genetic traits are not found in Y. enterocolitica, and it appears that while Y. pseudotuberculosis has manytools beneficial for survival in varied environments, the Y. enterocolitica genome is more streamlined and adapted to their preferredanimal reservoir.

1. Introduction

Enteropathogenic Yersinia is the third most common causeof bacterial enteritis in European countries, even though astatistically significant decreasing 5-year trend in yersiniosiscases has been reported in the EU [1]. Infection is usu-ally foodborne, with symptoms ranging from self-limitingdiarrhea to reactive arthritis or erythema nodosum [2].Yersinia are Gram-negative rods belonging to Enterobac-teriaceae. Enteropathogenic Yersinia diverged around 41–185 million years ago, while the third human pathogenicspecies of Yersinia genus, the infamous Yersinia pestis, isa relatively recent clone of Yersinia pseudotuberculosis [3].The evolution of enteropathogenic Yersinia is thought tohave includedmultiple distinct ecological specializations thathave separated the pathogenic strains from environmental,nonpathogenic lineages. This current hypothesis of parallelevolution [4] rejects the previous one suggesting that all

pathogenic Yersinia species share a common pathogenicancestor [5].

Enteropathogenic Yersinia enterocolitica and Y. pseudo-tuberculosis cause similar infections, but their epidemiologyand ecology appear to differ in many aspects. Both Y.enterocolitica and Y. pseudotuberculosis have been isolatedfrom swine or pork, and yersiniosis has been associated withthe consumption of uncooked pork [6–8]. Traditionally,mostcases of yersiniosis are thought to occur sporadically, andcases caused by Y. enterocolitica are mostly associated withpork products [7, 9–11]. In rare cases, the source of humaninfection has been traced back, for example, to milk, poultrymeat, and ready-to-eat salad [12–14]. Within recent decades,several widespread outbreaks caused by Y. pseudotuberculosishave been reported in Finland [15–18]. The sources of theinfections have been traced back to fresh produce, such asiceberg lettuce [15] and grated carrots [18–20]. The epidemic

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015, Article ID 760494, 12 pageshttp://dx.doi.org/10.1155/2015/760494

2 BioMed Research International

strain involved in an outbreak caused by raw carrots was alsorecovered from the field and production line [19].The genetictraits underlying the observed epidemiological differencesremain poorly understood.

Research has shown that the prevalence ofY. enterocoliticain swine is notably higher than that of Y. pseudotuberculosis,and swine are the only reservoir from which Y. enterocolitica4/O:3 strains have regularly been isolated [2, 25–27]. Themost common cause of Y. enterocolitica infection in humansinAfrica, Europe, Japan, andCanada isY. enterocolitica 4/O:3.Bioserotype 4/O:3 is considered as an emerging pathogen,while the prevalence of the secondmost common pathogenicbioserotype, Y. enterocolitica 1B/O:8, is diminishing [2, 10, 28,29]. Extensive research has been carried out to uncover thevirulence factors ofY. enterocolitica and its different serotypes[30–35], and the virulence factors explaining the swinespecificity ofY. enterocolitica serotypeO:3 were recently iden-tified [22, 36]. The differences in virulence gene expressionpatterns alter the surface adhesion properties and cytokineproduction profiles of O:3 strains and thus probably permitthe asymptomatic infection and long-term colonization of thenasopharynx and intestine of swine [22].

Pathogenic and nonpathogenic Y. enterocolitica strainsare frequently found fromwildlife samples such as water fowland hares, but pathogenic strains have rarely been isolatedfrom soil or water [2, 8, 37]. Y. pseudotuberculosis strains havebeen isolated from a variety of sources, including fresh veg-etables and wild animals, and contrary to Y. enterocolitica, allstrains are considered pathogenic [8, 15–17, 38]. Despite thefrequent presence of Y. pseudotuberculosis in environmentalsamples, its reservoir is considered to be wildlife [38, 39].

A comparative genomic hybridization (CGH) analysiswith a DNA microarray based on three Y. enterocoliticaand four Y. pseudotuberculosis genomes was conducted toshed light on the genetic traits and ecological specializa-tions explaining the epidemiological differences betweenenteropathogenic Yersinia. Our hypothesis was that thegenomes would contain operons elucidating the ways inwhich Y. enterocolitica has adapted to its mammal hosts andthe ecology of Y. pseudotuberculosis. The strains hybridizedon the microarray were isolated from human, animal, andenvironmental samples.

The hybridization results revealed that Y. pseudotuber-culosis strainscarry many operons linked with the uptake ofcarbohydrates and use of aromatic substances that are absentfrom Y. enterocolitica. Phenolic compounds, polyamines,myoinositol, and aliphatic sulfonates are all substrates thatare not commonly present in living animal tissue, but moreabundant in plants and the soil environment. Y. pseudotu-berculosis also harbors an array of different types of type VIsecretion systems (T6SSs), in contrast to just one found intheY. enterocolitica genome.These T6SSs are likely to providedefense against other bacteria and single-celled organisms inthe environment.

2. Materials and Methods

2.1. Bacterial Strains for Hybridization. A total of 60 Y.enterocolitica and 38 Y. pseudotuberculosis strains isolated

from a variety of geographic locations and sources were usedin this study (Table S1 in Supplementary Material availableonline at http://dx.doi.org/10.1155/2015/760494). The strainswere selected to represent the different biotypes and serotypesof enteropathogenic Yersinia, and also included were threestrains (Y. enterocolitica subsp. enterocolitica 8081, Y. entero-colitica subsp. palearctica Y11 [DSM 13030], and Y. pseudo-tuberculosis IP32953) used in the microarray design. Thesethree strains were used as reference strains and as positivehybridization controls. The reference strains and one addi-tional strain were hybridized in quadruplicate to assess thereproducibility of the hybridizations. The reference strainsproduced positive hybridization signals with 99.4–99.9% ofthe probes designed to hybridize with their sequences.

In total, 41 strains represented the most commonpathogenic Y. enterocolitica bioserotype 4/O:3. The majority(79/98) of the strains had been isolated from swine or fromswine slaughterhouses. The rest of the strains (𝑛 = 19) wereisolated from human patients, wild birds, and other animals.

2.2. DNAMicroarrays. TheDNAmicroarrays were designedbased on seven genomes and 14 plasmid sequences (TableS2) obtained from the NCBI database. 29,786 sequences wereclustered into 11,564 gene groups by Cd-hit-est [41]. Thethreshold value of identity was set to 95% with minimumalignment of at least 80% of the longer sequence. Stringentclustering parameters were chosen to avoid problems withuncomplimentary probes in the probe design. With theseparameters, the number of unique sequences (gene groupscontaining a sole sequence) amounted to 3747.

One 45–60-mer probe was designed for each gene group(𝑛 = 11,564). Thirteen gene groups containing a total of 14sequences were excluded from the probe design because ofredundancy. The longest gene sequences were over 10,000bases long (𝑛 = 11), and for these a tiling method was usedfor the design of extra probes (10 per sequence). All probeswere designed using Agilent Technologies Gene ExpressionProbe Design. Each of the eight subarrays of Agilent 8 ∗ 15Kcustom arrays (Agilent, Santa Clara, CA, USA) contained anequal set of 11,661 probes.

2.3. Hybridization and Washes. Genomic DNA was isolatedusing a method described by Pitcher et al. [42]. A totalof 500 ng of genomic DNA from each Yersinia strain wasfluorescently labeled with the BioPrime ArrayCGH labelingmodule (Invitrogen, Carlsbad, CA, USA) using either Cy3or Cy5 (GE Healthcare, Buckinghamshire, UK). For eachhybridization, one Cy3-labeled and one Cy5-labeled DNAsample were combined. The mixture was purified witha DNA purification kit (QIAquick PCR Purification Kit,Qiagen, Hilden, Germany) according to the manufacturer’sinstructions. The concentration of DNA and the incorpo-ration of the dye were checked with the Nanodrop device(NanodropTechnologies,Wilmington,MA,USA) before andafter labeling.The differently labeled DNA sample pairs to behybridized into one of the eight subarrays on each array slidewere randomly selected.

A volume of 2.2 𝜇L salmon sperm DNA (1mg/mL) wasadded to 17.8 𝜇L of labeled combined sample solution, and the

BioMed Research International 3

mixture was heated at 95∘C for 2 minutes for denaturation.A volume of 5 𝜇L of 10x blocking agent (Agilent) and 25 𝜇Lof 2xGE (HI-RPI) hybridization buffer (Agilent) was added.A total of 45𝜇L of the solution was hybridized on eachmicroarray at 65∘C for 16 hours. The arrays were washedtwice for 1 minute with Wash Buffer 1 (Agilent) and then for1 minute with prewarmed Wash Buffer 2 (Agilent).

2.4. Scanning and Data Analysis. The CGH data analysisfollowed the routines set by Lindstrom et al. [43] and Lahti etal. [44]. The slides were scanned (Axon Genepix Autoloader4200 AL, Molecular devices Inc., Sunnyvale, California,USA) with a resolution of 5𝜇m. Images were processed andmanually checked using GenePix Pro 6.0/6.1 software. Fordata analysis, R software and the LIMMA package were used[40, 45]. For background correction, the normexp algorithm(offset 50) was applied [46].

The distribution of logarithmic signal intensities formedtwo clear peaks in all hybridizations and a method conform-ing the positions of these density peaks was used to normalizethe hybridization data. Standard normalization methods formicroarrays are unsuited for CGH data since the distributionof intensities between different hybridizations cannot beassumed to be thesame. Conforming the positions of densitypeaks is based on an assumptionthat all hybridizations exhibithigh densities of both positive andnegative hybridization sig-nals but does not alter the distribution patternof intensities.By positive and negative hybridization signals, we here meansignals representing present and absent/divergent genes, thatis, high and low intensity signals, respectively. Visualizationand clustering of data were conducted using MEV [47].

The distribution of logarithmic signal intensities wasalso used to set a threshold between the intensity peaks(lowest point of density) separately for each hybridization.This threshold was used to classify the probes and thecorresponding genes as present, absent, or diverged in eachstrain. Intensity values of the threshold value ±0.3 wereclassified as diverged. The number of probes classified as“diverged” varied from 0 to 2 in all hybridizations, and theseprobes were considered as absent in further data analysis.

The data discussed in this paper are compliant withthe MIAME guidelines and were deposited in NCBI’s GeneExpression Omnibus and are accessible through GEO Seriesaccession number GSE67565 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE67565).

2.5. PhylogeneticAnalysis of TypeVI Secretion SystemSequences.Phylogenetic analysis similar toanalysis described by Schwarzet al. [48] was performed on VipA sequences stored in theNCBI database and annotated to TIGR category COG3516(𝑛 = 195). This TIGR sequence pool was supplemented bysequences annotated asVipA inY. pseudotuberculosis IP32953(𝑛 = 4), B. thailandensis (𝑛 = 5), P. aeruginosa (𝑛 = 1), Y.enterocoliticaY11 (𝑛 = 1), andY. enterocolitica 8081 (𝑛 = 1). Intotal, 206 VipA sequences were aligned using MUSCLE [49]and the resulting alignment was visualized with BioNJ [50].

2.6. Orthologous Genes. Reciprocal Blast searches were per-formed to identify the bidirectional best hits between the

genomes used in microarray design. Any bidirectional besthit identified was assumed to represent an orthologous genepair [51]. Information on orthologous gene pairs was used asan aid in the interpretation of the microarray data.

3. Results

CGH analysis of 60 Y. enterocolitica and 38 Y. pseudotu-berculosis strains was conducted with a DNA microarraybased on three Y. enterocolitica and four Y. pseudotuberculosisgenomes to shed light on genomic differences betweenenteropathogenic Yersinia. Y. enterocolitica strains and Y.pseudotuberculosis strains grouped into two distinct clusters(Figure 1). Y. enterocolitica strains belonging to four differentbiotypes formed distinct subclusters within the Y. enteroco-litica group. Strains belonging to Y. enterocolitica biotype 2or 3 (𝑛 = 10) clustered together (Figure 1). The distancebetween strains, based on Pearson’s correlation on a scalefrom 0 to 2 (0 indicating identical samples), was 0.25 betweenY. enterocolitica biotypes 2–4 and biotypes 1A and 1B. Withinthe Y. pseudotuberculosis group, the distance was 0.15. Thedistance between Y. enterocolitica and Y. pseudotuberculosisgroup was 1.36.

All hybridized strains produced positive signals on 459probes, which is the equivalent of 320–360 genes dependingon reference genome used. This means that around 8% ofthe genome is fully conserved across the two species. Inthe seven genomes used in array design, the core genomebased on bidirectional best hits contained 2772 sequences,implying that 68–76% of genes in each sequenced genomehave orthologous equivalents in the rest.

Comparing the 320 shared probes in hybridization resultsand 2772 in orthologous gene pairs, it becomes clear just howsensitive microarray hybridization is as a research method.Out of the 3547 probes (gene clusters) deemed present inall Y. enterocolitica strains, 1130 did not show a positivehybridization signal in any Y. pseudotuberculosis strain andwere thus considered specific for Y. enterocolitica (Table S3).When these 1130 gene clusters were further pruned usingthe information on orthologous gene pairs, only 448 geneclusters remained truly specific for Y. enterocolitica. Similarly,in the Y. pseudotuberculosis group, 906 gene clusters weredeemed conserved and specific (Table S3). Y. enterocoliticabioserotype 4/O:3 strains (𝑛 = 42) shared 51 gene clusters thatwere only extant in strains of this bioserotype.This representsaround 1% of genes in the sequenced Y. enterocolitica 4/O:3genome Y11.

Y. pseudotuberculosis strains shared three large operonscoding type VI secretion systems that were missing fromY. enterocolitica strains. Y. pseudotuberculosis strains alsoshared a variety of gene clusters that based on their anno-tation are likely to be involved in the use and/or uptakeof various substrates, including phenolic compounds, rham-nose, xylose, myoinositol, opines/polyamines, and aliphaticsulfonates (Table 1). Y. enterocolitica strains share six ATP-binding cassette (ABC) transporters and seven phospho-transferase systems (PTS) that are all absent from Y. pseudo-tuberculosis strains (Table 1). In addition, the Y. enterocoliticastrains, excluding the highly virulent 1B strains, carry an


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K2Figure 1: Yersinia enterocolitica and Yersinia pseudotuberculosis strains group into two distinct clusters and Y. enterocolitica strains belongingto four different biotypes form distinct subclusters within the Y. enterocolitica group. The majority of Y. pseudotuberculosis strains obtainedfrom swine samples cluster separately (“swine group”) from strains obtained from human and wildlife samples (“diverse group”).The diversegroup of Y. pseudotuberculosis also includes some strains isolated from English swine. Hierarchical clustering was constructed using R [40].

operon involved in the utilization of N-acetylgalactosamine.Y. pseudotuberculosis strains share 18 ABC transporters and 2PTS transporters that are absent fromY. enterocolitica strains.

Phylogenetic analysis of the different T6SSs shows thatthree distinct types of T6SS are present in Y. pseudotuberculo-sis (Figure 2). One type present in two copies in Y. pseudotu-berculosis (CAH19881.1, CAH21904.1 in Y. pseudotuberculosisIP32953) is present in one copy in Y. enterocolitica genomes(CAL12724.1 in strain 8081) and bears strong similarity toseveral T6SSs found in other bacteria. These include H1-T6SS found inPseudomonas aeruginosa and fourT6SSs foundin Burkholderia thailandensis. Copies of the second type ofT6SS (CAH20722.1 and CAH22490.1 in Y. pseudotuberculo-sis IP32953) clustered together with uncharacterized T6SSsfound in Y. pestis (Figure 2).The function of T6SSs belongingto this type is unknown. The third distinct type of T6SSidentified in Y. pseudotuberculosis genomes (CAH22876.1 inIP32953) shared strong similarity with the T6SSs of Vibriocholerae and B. thailandensis, which are both consideredto have cytotoxic effects against unicellular organisms andmacrophages.

Relatively few gene cluster differences were observedbetween the hybridization results of different Y. enterocoliticastrains (Figure 3). These included an operon coding for typeIII secretion system shared by low-pathogenic Y. enterocolit-ica, genes involved in drug resistance, and the operon coding

fromO:3 antigen.Many of the other differences are annotatedas putative phages or flagellar components.

The majority of Y. pseudotuberculosis strains obtainedfrom swine samples (“swine group” in Figure 1) in Finland,Sweden, Estonia, Russia, England, and Belgium (𝑛 = 23) clus-tered separately from human and wildlife samples (“diversegroup” in Figure 1). Five of the 11 Y. pseudotuberculosis strainsisolated from English pigs clustered together with the humanand wildlife samples.

4. Discussion

Y. enterocolitica and Y. pseudotuberculosis strains groupedinto two distinct clusters, and Y. enterocolitica strains belong-ing to four different biotypes formed distinct subclusterswithin the Y. enterocolitica group. On the gene level, themost interesting differences between Y. enterocolitica and Y.pseudotuberculosis strains included genes involved in T6SSs,the catabolism of phenolic compounds, and the transport ofmany carbohydrates (rhamnose, fructose, ribose, myoinosi-tol, and xylose) and other compounds (aliphatic sulfonates,opines) (Table 1).

T6SS forms a needle-like injectisome between the bacte-rial cell and the target cell [52]. First described under ten yearsago, T6SS is now one of the most common large specializedsecretion systems found in over 120 bacteria [48, 53]. T6SSs


Table 1: Main differences in gene clusters between enteropathogenic Yersinia enterocolitica (YE) and Yersinia pseudotuberculosis (YP) strains.

Presentin Absent from Locus Gene names Role Description and comments

YE YP CBY25444.1-CBY25441.1 aapJQMP Transportation ABC transporter (L-amino acids).

YE YP CBY25938.1-CBY25944.1 sorEMABF Transportation Phosphotransferase system (sorbose) [21].

YE YP, YE str.8081

CBY25947.1-CBY25953.1 urtEDCBA Transportation ABC transporter (urea). This copy of operon is

absent from strain 8081.

YE YP CBY26058.1-CBY26056.1 aglBA Transportation Phosphotransferase system (alpha-glycosides).

YE YP CBY26159.1-CBY26152.1 — Transportation ABC transporter (metallic ion).

YE YP CBY26547.1-CBY26568.1 pduVUTONMLKJBA Propanediol

utilization 1,2-Propanediol utilization [21].

YE YP CBY26570.1-CBY26589.1 cbiGKNQO Propanediol

utilization Cobalamin synthesis [21].

YE YP CBY26648.1-CBY26640.1 citXFEDCAB Metabolism Citrate lyase, ability to ferment citrate in

anaerobic conditions.

YE YP CBY26805.1-CBY26815.1 rutGEFDCGR Nitrogen

metabolism

Pyrimidine utilization. Use of pyrimidines as asource of nitrogen in E. coli. Genes rutA and rutGare interrupted in Y. enterocolitica.

YE YP CBY28018.1-CBY28009.1 scsBCD Resistance Suppressor for copper sensitivity operon 2.

Similar operon described in E. coli.

YE YP CBY28023.1-CBY28018.1 — Transportation Phosphotransferase system (lactose/cellobiose).

YE YP CBY28059.1-CBY28057.1 ascGFB Transportation Phosphotransferase system (𝛽-glycosides).

YE YP CBY28068.1-CBY28065.1 yrbFE Transportation ABC transporter (YrbF/E).

YE YP CBY28213.1-CBY28205.1 gutQRMDBEA Transportation Phosphotransferase system (glucitol/sorbitol).

YE YP CBY28759.1-CBY28735.1

gldA, dhaKR,scrRBAYK

Metabolism,transportation

Glycerol metabolism operon, phosphotransferasesystem (sucrose).

YE YP CBY29405.1-CBY29415.1 bcsCBAFEG Gut colonization Cellulose biosynthesis [21].

YE YP CBY29454.1-CBY29443.1 manA, bglA, gmuD Transportation Phosphotransferase system (lactose/cellobiose),

maltoporin, and 𝛽-glucosidase

YP YE CAH19316.1-CAH19320.1 — Transportation ABC transporter (molybdate-malate).

YP YE CAH19585.1-CAH19591.1 — Resistance Methyltetrahydrofolate reduction, conserved with

ter operon

YP YE CAH19592.1-CAH19597.1 terZABCDE Resistance Tellurite/tellurium resistance. Similar to the

operon in Y. pestis plasmid.

YP YE CAH19782.1-CAH19785.1 frwDBC, pstA Transportation Phosphotransferase system (fructose).

YP YE CAH19879.1-CAH19896.1

impACG, hcp, vasG,icmF Type VI secretion YPTB T6SS-1, interbacterial interaction.

YP YE CAH20037.1-CAH20044.1 sgbK, araD Transportation ABC transporter (L-xylose), epimerase.

YP YE CAH20283.1-CAH20290.1 — Unknown

CDP-diacylglycerol synthesis operon. A similargene cluster of unknown function has beendescribed in E. coli.

YP YE CAH20313.1-CAH20316.1 — Transportation ABC transporter (myoinositol), dehydrogenase.


Table 1: Continued.


YP YE CAH20560.1-CAH20567.1 rpiA Transportation ABC transporter (sugar), dehydrogenase.

YP YE CAH20608.1-CAH20613.1 — Transportation ABC transporter (iron).

YP YE CAH20708.1-CAH20725.1 — Type VI secretion Conserved area before type VI secretion system.

YP YE CAH20725.1-CAH20742.1 impKL, hcp, vasGD Type VI secretion YPTB T6SS-2.

YP YE CAH20812.1-CAH20822.1 lidD Transportation Symport.

YP YE CAH20875.1-CAH20884.1 hpaRGEDFHIXBC Use of aromatic

substances Hpa operon.

YP YE CAH20923.1-CAH20928.1 — Transportation ABC transporter (sugar).

YP YE CAH21145.1-CAH21154.1 — Transportation ABC transporter (sorbitol).

YP YE CAH21162.1-CAH21172.1 — Transportation MFS transporter (aromatic acids).

YP YE CAH21251.1-CAH21255.1 potDCBA Transportation ABC transporter (polyamines).

YP YE CAH21445.1-CAH21448.1 tauB Transportation Transporter (taurine/sulfonate).

YP YE CAH21739.1-CAH21760.1 manB, mtlK Transportation MFS transporter (sugar), ABC transporter

(sugar), and CRISPR repeats.

YP YE CAH21766.1-CAH21777.1 gutB Transportation Carnitine transporter, tartrate dehydrogenase,

and ABC transporter (sorbitol).

YP YE CAH22045.1-CAH22050.1 goaG Transportation ABC transporter (opines/polyamines).

YP YE CAH22292.1-CAH22299.1 gspLKJHI Type II secretion General secretion pathway.

YP YE CAH22307.1-CAH22312.1 — Growth on

chondroitin sulfate Secreted chondroitin ABC lyase.

YP YE CAH22317.1-CAH22328.1 kduD2 Transportation Phosphotransferase system

(N-acetylgalactosamine), chondro-6-sulfatase.

YP YE CAH22333.1-CAH22353.1 lamb, bgaB Transportation ABC transporter (maltodextrin/maltose/ribose).

YP YE CAH22467.1-CAH22469.1 mglA Transportation ABC transporter (sugar).

YP YE CAH22662.1-CAH22687.1 yapF Transportation Na+/H+-antiport, ABC transporter (sugar).

YP YE CAH23038.1-CAH23046.1 — Transportation ABC transporter (ribose), two-component

system.YE4/O:3

YE BT 1–3,YP

CBY26503.1-CBY26517.1 Serotype O:3 antigen dDTP-L-rhamnose biosynthesis [22, 23].

YE4/O:3

YE BT 1–3,YP

CBY26512.1-CBY26517.1 Serotype O:3 antigen Conserved area posterior to the O:3 antigen.

Hypothetical proteins, transposon.YE BT2–4

YE BT 1A,1B; YP

CBY25728.1-CBY25740.1 aatBCAP, araC Resistance Multidrug efflux system. Cluster includes 6 genes

and 7 hypothetical insertion sequences.

YE BT2–4, 1

YE BT 1A,1B; YP

CBY29000.1-CBY29007.1 sseDBCEBF Virulence, type III

secretion

Type III secreting effectors andchaperones. Salmonella type III secretion Sseoperon is involved in interaction withmacrophages.


Table 1: Continued.


YE BT2–4, 1A YE BT 1B CBY28981.1-

CBY29013.1 ysp Virulence, type IIIsecretion Not fully conserved in biotype 1A strains [24].

YE BT2–4, 1A YE BT 1B CBY26978.1-

CBY26985.1 agaRZVWEFSYN-

Acetylgalactosamineutilization

Use of intestinal mucin as a carbon source [24].

YE = Y. enterocolitica, YP = Y. pseudotuberculosis, BT = biotype(s).

YPTB 3

V. cholerae

BTHAI 5

YPTB 4YPTB 2

BTHAI 1

YPTB 1

BTHAI 4 BTHAI 2

YPTB 5Y. enterocoliticaP. aeruginosa HI-T6SS

BTHAI 6

Figure 2: Phylogenetic relationships of type VI secretion systems (T6SSs) in Yersinia pseudotuberculosis and T6SSs of other species werecompared to evaluate the different types of T6SS. VipA sequences were used to represent T6SS and the alignment of 206 VipA proteinsis shown here as an unrooted phylogenetic tree visualized by BioNJ. Type VI secretion systems of Y. pseudotuberculosis named YPTB 1–5belong to three distinct branches of T6SSs. T6SSs of Vibrio cholerae, Pseudomonas aeruginosa, and Burkholderia thailandensis (BTHAI 1–6)are marked on the branches of the phylogenetic tree and the one VipA/T6SS present in Y. enterocolitica is also shown.

were first considered as virulence factors, but their abundancein nonpathogenic bacteria and further studies have suggestedthatmost of these systems play a role in interbacterial interac-tion and defense against competitive bacteria and unicellularorganisms [54]. The mechanism requires 15 conserved genes

and direct contact with other cells and is thus thought to beespecially useful in the stationary growth phase [52, 55, 56]. Itis notable that diverse collections of T6SSs have been reportedin many environmental bacteria with facultative pathogenicpotential, such as Pseudomonas aeruginosa, Burkholderia


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1A1B

2 and 34/O:3

Y. enterocolitica

Figure 3: A heatmap presentation of Yersinia enterocolitica hybridization results produced with R. Biotypes cluster separately andmajor geneclusters differing between different biotypes are shown. A green color signifies that the gene is present in the given strain.

mallei, and Burkholderia pseudomallei, as well as in bacteriawith multiple hosts and the ability to survive in diverseenvironmental conditions (Y. pestis, V. cholerae) [53, 57–60]. Y. pseudotuberculosis is also considered a facultativepathogen with multiple host species and able to persist inthe environment. The genome of Y. pseudotuberculosis hasfour conserved systems and one smaller, perhaps partialsystem [55]. Only one of these systems is shared with Y.enterocolitica.The loci codingVipA protein is used to indicatethe location of each T6SS. Two of the Y. pseudotuberculosisT6SSs (CAH19881.1, CAH21904.1 in IP32953) and the solitaryT6SS in Y. enterocolitica (CAL12724.1 in strain 8081) group

in phylogenetic analysis together with B. thailandensis T6SS(BTHAI-1) and H1-T6SS of P. aeruginosa.The latter two havebeen reported to target other bacteria and give some com-petitive advantage to the bacterium itself [48, 56]. Havingthis mechanism could enhance the growth of Y. pseudotuber-culosis when other bacteria are present on a shared growthsurface. Another T6SS (CAH22876.1 in IP32953) is similarto the T6SSs in V. cholerae and B. thailandensis, which aredescribed as being cytotoxic against single-celled organismsand macrophages [48, 53, 61]. T6SSs like this are beneficialagainst protists living in the soil and water environment butare also possible pathogenicity factors. To better understand


the function of each T6SS of Y. pseudotuberculosis, in vivostudies are required. Epidemiologically, T6SSs could probablyhelp Y. pseudotuberculosis to survive and multiply in suchecological niches in the environment from which it couldeasily end up as a contaminant of the food chain. The lackof T6SSs in Y. enterocolitica implies that the organism in itscurrent ecological niche has no need for them. The lack ofT6SSs might actually be beneficial for the organism, as aT6SS with cytotoxic effects against the macrophages of themammal host might encumber the invasion and survival ofY. enterocolitica cells.

Y. pseudotuberculosis strains also carry a variety of geneclusters involved in the uptake and/or utilization of var-ious substrates (Table 1). The Hpa operon (CAH20875.1–CAH20884.1 in IP32953), also known as the 4-hydroxyphen-ylacetate degrading operon, is involved in the catabolismof phenolic and aromatic compounds [62, 63]. Based ondatabase queries, Hpa sequences in Y. pseudotuberculosisare homologous to those in E. coli and Salmonella. Phenolsare products of plant secondary metabolism and often havebactericidal effects. Phenols are widely present in soil andthe water environment, but their abundance in the intestinesof animals has also been suggested [62, 63]. Interestingly,the hpa genes are expressed in Salmonella enterica serovarTyphimurium cells during the infection in swine, and ithas been suggested that the operon is somehow beneficialfor enteropathogenic bacteria [64]. Evidently, the lack of anHpa operon does not seem to hinder the prevalence of Y.enterocolitica in swine.

BothY. enterocolitica andY. pseudotuberculosishavemanytransport and uptake systems that are not present in otherspecies. Y. enterocolitica strains share six ABC transportersand seven PTS transporters that are all absent from Y.pseudotuberculosis strains. Putative substrates for these sys-tems include iron and metallic ions, glycosides, and lactose/cellobiose. Interestingly, one putative urea ABC transporterwas noted as present in all other Y. enterocolitica strains, butabsent from reference strain 8081.This highlights the benefitsof having multiple reference strains.

Y. enterocolitica strains shared notably fewer specificgenes (𝑛 = 448) between them than Y. pseudotuberculosisstrains (𝑛 = 906). This is likely to reflect the greater het-erogeneity within Y. enterocolitica biotypes and the ecologicaladaptation of biotypes 2–5 by gene loss and genome decay [4].Many of these specific genes were involved in transportation.Y. pseudotuberculosis strains shared 18 ABC transporters and2 PTS transporters absent from Y. enterocolitica. Putativesubstrates for these systems include rhamnose, fructose,xylose, myoinositol, iron, aromatic acids, polyamines, sor-bitol, sulfonates, and 7 systems for unspecified sugars. The Y.pseudotuberculosis genome appears to be equippedwithmanyextra tools for moving substances in and out of its cell com-pared to the Y. enterocolitica genome, which appears morestreamlined and likely to have adapted to another ecologicalniche, such as swine tonsils and gut, where variety in substratetransportation is not required. A recent hypothesis on theevolution of enteropathogenic Yersinia assumes that Yersiniaspecies have evolved to become more ecologically specificandmetabolically more limited to their reservoirs by genome

decay and gene loss [4]. An evolutionary path such as thisappears plausible, as Y. enterocolitica seems better adapted toliving in amammal host and to have lostmany genes involvedin survival in the environment.

The theory of genome decay and gene loss, however, doesnot explain the differences between Y. enterocolitica biotypes.Lipase activity, hydrolysis of B-glycosides such as salicin andesculin, use of xylose, and indole production are some ofthe biochemical tests belonging to Y. enterocolitica biotypingschema [2]. However, relatively few clusters of genes differen-tiate pathogenic and nonpathogenic Y. enterocolitica strainsfrom each other. Recent results have identified the changes ingene expression patterns for pathogenicity factors explainingthe swine specificity of Y. enterocolitica 4/O:3 [22]. It seemslikely that the adaptation and differences in pathogenicityof Y. enterocolitica biotypes are due to point mutations andchanges in gene expression rather than gene loss.

Y. pseudotuberculosis strains obtained from swine samplesmostly clustered separately from human and wildlife samples(Figure 1). Notably, five of the 11 Y. pseudotuberculosis strainsisolated from English swine clustered together with thehuman and wildlife samples. Niskanen et al. [39] have previ-ously reported on the homogeneity of Y. pseudotuberculosisstrains isolated from swine samples based on pulsed-fieldgel electrophoresis analysis. Our results further confirm thisfinding. Martınez et al. [25–27] noted that the prevalence anddiversity of Y. pseudotuberculosis strains appears to be higherin English swine than in swine of other European countries.This finding is also supported by the present results, as 5 ofthe 11 English Y. pseudotuberculosis strains showed a markedgenetic distance to other swine strains. In these results, nodefining gene cluster setting the swine group and diversegroup apart could be identified. In this type of study, theresults are dependent on the reference strains used. It isimportant to note that because of the limitations of themethod, many genes present in the studied strains mightbe absent from the reference genomes used and thus fromthe designed microarray and the further results. It wouldbe interesting to have a wholly sequenced genome from the“swine group” ofY. pseudotuberculosis for further research onthe differences between these two groups.

The high prevalence of Y. pseudotuberculosis in theEnglish pork chain is probably explained by the moreavailable access to outdoors of English swine compared totheir continental counterparts. Swine and pork productsare not considered to be a notable source of sporadic Y.pseudotuberculosis cases, and animals having greater contactwith the environment are more likely to have strains ofsoil and wildlife origins passing through their intestines.This would also explain why some Y. pseudotuberculosis notbelonging to the “swine group” of Y. pseudotuberculosis havebeen isolated from English swine.

5. Conclusions

The hybridization results revealed that Y. pseudotuberculosisstrains carry many operons linked with the use of carbohy-drates and other substrates that are absent from Y. entero-colitica. Phenolic compounds, polyamines, myoinositol, and


aliphatic sulfonates are all substrates that are not commonlypresent in living animal tissue but are more abundant insoil and the environment. Y. pseudotuberculosis also harborsan array of different type VI secretion systems, in contrastto just one found in the Y. enterocolitica genome. Type VIsecretion systems target single-celled organisms and otherbacteria but are also possible pathogenicity factors. Thesedefense and interaction systems could help Y. pseudotuber-culosis to survive and multiply in such ecological niches inthe environment from which it could easily end up as acontaminant of the food chain.

The Y. pseudotuberculosis genome holds many tools, suchas type VI secretion systems and transporters for varioussubstrates, which are likely to be beneficial for survival invaried growth environments and multiple host species. Bycomparison, the genome of Y. enterocolitica appears morestreamlined and likely to have adapted to a different ecolog-ical niche where these survival systems are not needed orbeneficial. For Y. enterocolitica bioserotype 4/O:3, this nicheis with certainty swine.

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.

Acknowledgments

The authors thank Paivi Lahti for advice and helpful discus-sions and Erika Pitkanen and Erja Merivirta for technicalassistance. The work was performed in the Centre of Excel-lence in Microbial Food Safety Research and funded by theAcademy of Finland (Grant nos. 118602 and 141140).

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