# Comparative Genomic Hybridization Analysis ofYersinia enterocolitica and Yersinia pseudotuberculosis Identifies Genetic Traits to Elucidate Their Different Ecologies

Authors: Kaisa Jaakkola; Panu Somervuo; Hannu Korkeala
Journal: BioMed Research International
 (2015)

---

## Abstract

EnteropathogenicYersinia enterocolitica and Yersinia pseudotuberculosis are both etiological agents for intestinal infection known as yersiniosis, but their epidemiology and ecology bear many differences. Swine are the only known reservoir for Y. enterocolitica 4/O:3 strains, which are the most common cause of human disease, while Y. pseudotuberculosis has been isolated from a variety of sources, including vegetables and wild animals. Infections caused by Y. enterocolitica mainly originate from swine, but fresh produce has been the source for widespread Y. pseudotuberculosis outbreaks within recent decades. A comparative genomic hybridization analysis with a DNA microarray based on three Yersinia enterocolitica and four Yersinia pseudotuberculosis genomes was conducted to shed light on the genomic differences between enteropathogenic Yersinia. The hybridization results identified Y. pseudotuberculosis strains 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 and eukaryotic cells. These genetic traits are not found in Y. enterocolitica, and it appears that while Y. pseudotuberculosis has many tools beneficial for survival in varied environments, the Y. enterocolitica genome is more streamlined and adapted to their preferred animal reservoir.


---

## Body

## 1. Introduction

EnteropathogenicYersinia is the third most common cause of bacterial enteritis in European countries, even though a statistically significant decreasing 5-year trend in yersiniosis cases has been reported in the EU [1]. Infection is usually foodborne, with symptoms ranging from self-limiting diarrhea to reactive arthritis or erythema nodosum [2].Yersinia are Gram-negative rods belonging to Enterobacteriaceae. EnteropathogenicYersinia diverged around 41–185 million years ago, while the third human pathogenic species ofYersinia genus, the infamousYersinia pestis, is a relatively recent clone ofYersinia pseudotuberculosis[3]. The evolution of enteropathogenicYersinia is thought to have included multiple distinct ecological specializations that have separated the pathogenic strains from environmental, nonpathogenic lineages. This current hypothesis of parallel evolution [4] rejects the previous one suggesting that all pathogenicYersinia species share a common pathogenic ancestor [5].EnteropathogenicYersinia enterocolitica andY. pseudotuberculosis cause similar infections, but their epidemiology and ecology appear to differ in many aspects. BothY. enterocoliticaandY. pseudotuberculosis have been isolated from swine or pork, and yersiniosis has been associated with the consumption of uncooked pork [6–8]. Traditionally, most cases of yersiniosis are thought to occur sporadically, and cases caused byY. enterocolitica are mostly associated with pork products [7, 9–11]. In rare cases, the source of human infection has been traced back, for example, to milk, poultry meat, and ready-to-eat salad [12–14]. Within recent decades, several widespread outbreaks caused byY. pseudotuberculosis have been reported in Finland [15–18]. The sources of the infections have been traced back to fresh produce, such as iceberg lettuce [15] and grated carrots [18–20]. The epidemic strain involved in an outbreak caused by raw carrots was also recovered from the field and production line [19]. The genetic traits underlying the observed epidemiological differences remain poorly understood.Research has shown that the prevalence ofY. enterocolitica in swine is notably higher than that ofY. pseudotuberculosis, and swine are the only reservoir from whichY. enterocolitica 4/O:3 strains have regularly been isolated [2, 25–27]. The most common cause ofY. enterocolitica infection in humans in Africa, Europe, Japan, and Canada isY. enterocolitica4/O:3. Bioserotype 4/O:3 is considered as an emerging pathogen, while the prevalence of the second most common pathogenic bioserotype,Y. enterocolitica1B/O:8, is diminishing [2, 10, 28, 29]. Extensive research has been carried out to uncover the virulence factors ofY. enterocolitica and its different serotypes [30–35], and the virulence factors explaining the swine specificity ofY. enterocoliticaserotype O:3 were recently identified [22, 36].The differences in virulence gene expression patterns alter the surface adhesion properties and cytokine production profiles of O:3 strains and thus probably permit the asymptomatic infection and long-term colonization of the nasopharynx and intestine of swine [22].Pathogenic and nonpathogenicY. enterocoliticastrains are frequently found from wildlife samples such as water fowl and hares, but pathogenic strains have rarely been isolated from soil or water [2, 8, 37].Y. pseudotuberculosis strains have been isolated from a variety of sources, including fresh vegetables and wild animals, and contrary toY. enterocolitica, all strains are considered pathogenic [8, 15–17, 38]. Despite the frequent presence ofY. pseudotuberculosis in environmental samples, its reservoir is considered to be wildlife [38, 39].A comparative genomic hybridization (CGH) analysis with a DNA microarray based on threeY. enterocoliticaand fourY. pseudotuberculosis genomes was conducted to shed light on the genetic traits and ecological specializations explaining the epidemiological differences between enteropathogenicYersinia. Our hypothesis was that the genomes would contain operons elucidating the ways in whichY. enterocolitica has adapted to its mammal hosts and the ecology ofY. pseudotuberculosis. The strains hybridized on the microarray were isolated from human, animal, and environmental samples.The hybridization results revealed thatY. pseudotuberculosisstrainscarry many operons linked with the uptake of carbohydrates and use of aromatic substances that are absent fromY. enterocolitica. Phenolic compounds, polyamines, myoinositol, and aliphatic sulfonates are all substrates that are not commonly present in living animal tissue, but more abundant in plants and the soil environment.Y. pseudotuberculosisalso harbors an array of different types of type VI secretion systems (T6SSs), in contrast to just one found in theY. enterocoliticagenome. These T6SSs are likely to provide defense against other bacteria and single-celled organisms in the environment.


## 2. Materials and Methods

### 2.1. Bacterial Strains for Hybridization

A total of 60Y. enterocoliticaand 38Y. pseudotuberculosisstrains isolated from a variety of geographic locations and sources were used in this study (Table S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/760494). The strains were selected to represent the different biotypes and serotypes of enteropathogenicYersinia, and also included were three strains (Y. enterocolitica subsp.enterocolitica8081,Y. enterocolitica subsp.palearcticaY11 [DSM 13030], andY. pseudotuberculosis IP32953) used in the microarray design. These three strains were used as reference strains and as positive hybridization controls. The reference strains and one additional strain were hybridized in quadruplicate to assess the reproducibility of the hybridizations. The reference strains produced positive hybridization signals with 99.4–99.9% of the probes designed to hybridize with their sequences.In total, 41 strains represented the most common pathogenicY. enterocolitica bioserotype 4/O:3. The majority (79/98) of the strains had been isolated from swine or from swine slaughterhouses. The rest of the strains (n
=
19) were isolated from human patients, wild birds, and other animals.


### 2.2. DNA Microarrays

The DNA microarrays were designed based on seven genomes and 14 plasmid sequences (Table S2) obtained from the NCBI database. 29,786 sequences were clustered into 11,564 gene groups by Cd-hit-est [41]. The threshold value of identity was set to 95% with minimum alignment of at least 80% of the longer sequence. Stringent clustering parameters were chosen to avoid problems with uncomplimentary probes in the probe design. With these parameters, the number of unique sequences (gene groups containing 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 14 sequences were excluded from the probe design because of redundancy. The longest gene sequences were over 10,000 bases long (n
=
11), and for these a tiling method was used for the design of extra probes (10 per sequence). All probes were designed using Agilent Technologies Gene Expression Probe Design. Each of the eight subarrays of Agilent 8
∗
15 K custom arrays (Agilent, Santa Clara, CA, USA) contained an equal set of 11,661 probes.


### 2.3. Hybridization and Washes

Genomic DNA was isolated using a method described by Pitcher et al. [42]. A total of 500 ng of genomic DNA from eachYersiniastrain was fluorescently labeled with the BioPrime ArrayCGH labeling module (Invitrogen, Carlsbad, CA, USA) using either Cy3 or Cy5 (GE Healthcare, Buckinghamshire, UK). For each hybridization, one Cy3-labeled and one Cy5-labeled DNA sample were combined. The mixture was purified with a DNA purification kit (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The concentration of DNA and the incorporation of the dye were checked with the Nanodrop device (Nanodrop Technologies, Wilmington, MA, USA) before and after labeling. The differently labeled DNA sample pairs to be hybridized into one of the eight subarrays on each array slide were randomly selected.A volume of 2.2μL salmon sperm DNA (1 mg/mL) was added to 17.8 μL of labeled combined sample solution, and the mixture was heated at 95°C for 2 minutes for denaturation. A volume of 5 μL of 10x blocking agent (Agilent) and 25 μL of 2xGE (HI-RPI) hybridization buffer (Agilent) was added. A total of 45 μL of the solution was hybridized on each microarray at 65°C for 16 hours. The arrays were washed twice for 1 minute with Wash Buffer 1 (Agilent) and then for 1 minute with prewarmed Wash Buffer 2 (Agilent).


### 2.4. Scanning and Data Analysis

The CGH data analysis followed the routines set by Lindström et al. [43] and Lahti et al. [44]. The slides were scanned (Axon Genepix Autoloader 4200 AL, Molecular devices Inc., Sunnyvale, California, USA) with a resolution of 5 μm. Images were processed and manually checked using GenePix Pro 6.0/6.1 software. For data 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 formed two clear peaks in all hybridizations and a method conforming the positions of these density peaks was used to normalize the hybridization data. Standard normalization methods for microarrays are unsuited for CGH data since the distribution of intensities between different hybridizations cannot be assumed to be thesame. Conforming the positions of density peaks is based on an assumptionthat all hybridizations exhibit high densities of both positive andnegative hybridization signals but does not alter the distribution patternof intensities. By positive and negative hybridization signals, we here mean signals representing present and absent/divergent genes, that is, high and low intensity signals, respectively. Visualization and clustering of data were conducted using MEV [47].The distribution of logarithmic signal intensities was also 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 the corresponding genes as present, absent, or diverged in each strain. Intensity values of the threshold value ±0.3 were classified as diverged. The number of probes classified as “diverged” varied from 0 to 2 in all hybridizations, and these probes were considered as absent in further data analysis.The data discussed in this paper are compliant with the MIAME guidelines and were deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE67565 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE67565).


### 2.5. Phylogenetic Analysis of Type VI Secretion System Sequences

Phylogenetic analysis similar to analysis described by Schwarz et al. [48] was performed on VipA sequences stored in the NCBI database and annotated to TIGR category COG3516 (n
=
195). This TIGR sequence pool was supplemented by sequences annotated as VipA inY. pseudotuberculosis IP32953 (n
=
4),B. thailandensis (n
=
5),P. aeruginosa (n
=
1),Y. enterocoliticaY11 (n
=
1), andY. enterocolitica8081 (n
=
1). In total, 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 performed to identify the bidirectional best hits between the genomes used in microarray design. Any bidirectional best hit identified was assumed to represent an orthologous gene pair [51]. Information on orthologous gene pairs was used as an aid in the interpretation of the microarray data.


## 2.1. Bacterial Strains for Hybridization

A total of 60Y. enterocoliticaand 38Y. pseudotuberculosisstrains isolated from a variety of geographic locations and sources were used in this study (Table S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/760494). The strains were selected to represent the different biotypes and serotypes of enteropathogenicYersinia, and also included were three strains (Y. enterocolitica subsp.enterocolitica8081,Y. enterocolitica subsp.palearcticaY11 [DSM 13030], andY. pseudotuberculosis IP32953) used in the microarray design. These three strains were used as reference strains and as positive hybridization controls. The reference strains and one additional strain were hybridized in quadruplicate to assess the reproducibility of the hybridizations. The reference strains produced positive hybridization signals with 99.4–99.9% of the probes designed to hybridize with their sequences.In total, 41 strains represented the most common pathogenicY. enterocolitica bioserotype 4/O:3. The majority (79/98) of the strains had been isolated from swine or from swine slaughterhouses. The rest of the strains (n
=
19) were isolated from human patients, wild birds, and other animals.


## 2.2. DNA Microarrays

The DNA microarrays were designed based on seven genomes and 14 plasmid sequences (Table S2) obtained from the NCBI database. 29,786 sequences were clustered into 11,564 gene groups by Cd-hit-est [41]. The threshold value of identity was set to 95% with minimum alignment of at least 80% of the longer sequence. Stringent clustering parameters were chosen to avoid problems with uncomplimentary probes in the probe design. With these parameters, the number of unique sequences (gene groups containing 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 14 sequences were excluded from the probe design because of redundancy. The longest gene sequences were over 10,000 bases long (n
=
11), and for these a tiling method was used for the design of extra probes (10 per sequence). All probes were designed using Agilent Technologies Gene Expression Probe Design. Each of the eight subarrays of Agilent 8
∗
15 K custom arrays (Agilent, Santa Clara, CA, USA) contained an equal set of 11,661 probes.


## 2.3. Hybridization and Washes

Genomic DNA was isolated using a method described by Pitcher et al. [42]. A total of 500 ng of genomic DNA from eachYersiniastrain was fluorescently labeled with the BioPrime ArrayCGH labeling module (Invitrogen, Carlsbad, CA, USA) using either Cy3 or Cy5 (GE Healthcare, Buckinghamshire, UK). For each hybridization, one Cy3-labeled and one Cy5-labeled DNA sample were combined. The mixture was purified with a DNA purification kit (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The concentration of DNA and the incorporation of the dye were checked with the Nanodrop device (Nanodrop Technologies, Wilmington, MA, USA) before and after labeling. The differently labeled DNA sample pairs to be hybridized into one of the eight subarrays on each array slide were randomly selected.A volume of 2.2μL salmon sperm DNA (1 mg/mL) was added to 17.8 μL of labeled combined sample solution, and the mixture was heated at 95°C for 2 minutes for denaturation. A volume of 5 μL of 10x blocking agent (Agilent) and 25 μL of 2xGE (HI-RPI) hybridization buffer (Agilent) was added. A total of 45 μL of the solution was hybridized on each microarray at 65°C for 16 hours. The arrays were washed twice for 1 minute with Wash Buffer 1 (Agilent) and then for 1 minute with prewarmed Wash Buffer 2 (Agilent).


## 2.4. Scanning and Data Analysis

The CGH data analysis followed the routines set by Lindström et al. [43] and Lahti et al. [44]. The slides were scanned (Axon Genepix Autoloader 4200 AL, Molecular devices Inc., Sunnyvale, California, USA) with a resolution of 5 μm. Images were processed and manually checked using GenePix Pro 6.0/6.1 software. For data 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 formed two clear peaks in all hybridizations and a method conforming the positions of these density peaks was used to normalize the hybridization data. Standard normalization methods for microarrays are unsuited for CGH data since the distribution of intensities between different hybridizations cannot be assumed to be thesame. Conforming the positions of density peaks is based on an assumptionthat all hybridizations exhibit high densities of both positive andnegative hybridization signals but does not alter the distribution patternof intensities. By positive and negative hybridization signals, we here mean signals representing present and absent/divergent genes, that is, high and low intensity signals, respectively. Visualization and clustering of data were conducted using MEV [47].The distribution of logarithmic signal intensities was also 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 the corresponding genes as present, absent, or diverged in each strain. Intensity values of the threshold value ±0.3 were classified as diverged. The number of probes classified as “diverged” varied from 0 to 2 in all hybridizations, and these probes were considered as absent in further data analysis.The data discussed in this paper are compliant with the MIAME guidelines and were deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE67565 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE67565).


## 2.5. Phylogenetic Analysis of Type VI Secretion System Sequences

Phylogenetic analysis similar to analysis described by Schwarz et al. [48] was performed on VipA sequences stored in the NCBI database and annotated to TIGR category COG3516 (n
=
195). This TIGR sequence pool was supplemented by sequences annotated as VipA inY. pseudotuberculosis IP32953 (n
=
4),B. thailandensis (n
=
5),P. aeruginosa (n
=
1),Y. enterocoliticaY11 (n
=
1), andY. enterocolitica8081 (n
=
1). In total, 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 performed to identify the bidirectional best hits between the genomes used in microarray design. Any bidirectional best hit identified was assumed to represent an orthologous gene pair [51]. Information on orthologous gene pairs was used as an aid in the interpretation of the microarray data.


## 3. Results

CGH analysis of 60Y. enterocolitica and 38Y. pseudotuberculosis strains was conducted with a DNA microarray based on threeY. enterocolitica and fourY. pseudotuberculosis genomes to shed light on genomic differences between enteropathogenicYersinia.Y. enterocoliticastrains andY. pseudotuberculosisstrains grouped into two distinct clusters (Figure 1).Y. enterocoliticastrains belonging to four different biotypes formed distinct subclusters within theY. enterocoliticagroup. Strains belonging toY. enterocolitica biotype 2 or 3 (n
=
10) clustered together (Figure 1). The distance between strains, based on Pearson’s correlation on a scale from 0 to 2 (0 indicating identical samples), was 0.25 betweenY. enterocoliticabiotypes 2–4 and biotypes 1A and 1B. Within theY. pseudotuberculosis group, the distance was 0.15. The distance betweenY. enterocolitica andY. pseudotuberculosisgroup was 1.36.Figure 1
Yersinia enterocoliticaandYersinia pseudotuberculosis strains group into two distinct clusters andY. enterocoliticastrains belonging to four different biotypes form distinct subclusters within theY. enterocoliticagroup. The majority ofY. pseudotuberculosis strains obtained from swine samples cluster separately (“swine group”) from strains obtained from human and wildlife samples (“diverse group”). The diverse group ofY. pseudotuberculosis also includes some strains isolated from English swine. Hierarchical clustering was constructed using R [40].All hybridized strains produced positive signals on 459 probes, which is the equivalent of 320–360 genes depending on reference genome used. This means that around 8% of the genome is fully conserved across the two species. In the seven genomes used in array design, the core genome based on bidirectional best hits contained 2772 sequences, implying that 68–76% of genes in each sequenced genome have orthologous equivalents in the rest.Comparing the 320 shared probes in hybridization results and 2772 in orthologous gene pairs, it becomes clear just how sensitive microarray hybridization is as a research method. Out of the 3547 probes (gene clusters) deemed present in allY. enterocolitica strains, 1130 did not show a positive hybridization signal in anyY. pseudotuberculosis strain and were thus considered specific forY. enterocolitica(Table S3). When these 1130 gene clusters were further pruned using the information on orthologous gene pairs, only 448 gene clusters remained truly specific forY. enterocolitica. Similarly, in theY. pseudotuberculosis group, 906 gene clusters were deemed conserved and specific (Table S3).Y. enterocolitica bioserotype 4/O:3 strains (n
=
42) shared 51 gene clusters that were only extant in strains of this bioserotype. This represents around 1% of genes in the sequencedY. enterocolitica 4/O:3 genome Y11.Y. pseudotuberculosis strains shared three large operons coding type VI secretion systems that were missing fromY. enterocolitica strains.Y. pseudotuberculosis strains also shared a variety of gene clusters that based on their annotation are likely to be involved in the use and/or uptake of various substrates, including phenolic compounds, rhamnose, xylose, myoinositol, opines/polyamines, and aliphatic sulfonates (Table 1).Y. enterocoliticastrains share six ATP-binding cassette (ABC) transporters and seven phosphotransferase systems (PTS) that are all absent fromY. pseudotuberculosis strains (Table 1). In addition, theY. enterocoliticastrains, excluding the highly virulent 1B strains, carry an operon involved in the utilization of N-acetylgalactosamine.Y. pseudotuberculosis strains share 18 ABC transporters and 2 PTS transporters that are absent fromY. enterocolitica strains.Table 1
Main differences in gene clusters between enteropathogenicYersinia enterocolitica(YE) and Yersinia pseudotuberculosis (YP) strains.
Present in
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 a source of nitrogen inE. coli. Genes rutA and rutG are interrupted in Y. enterocolitica.
YE
YP
CBY28018.1-CBY28009.1
scsBCD
Resistance
Suppressor for copper sensitivity operon 2. Similar operon described inE. 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, phosphotransferase system (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, andβ-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 inY. 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 similar gene cluster of unknown function has been described inE. coli.
YP
YE
CAH20313.1-CAH20316.1
—
Transportation
ABC transporter (myoinositol), dehydrogenase.
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.
YE 4/O:3
YE BT 1–3, YP
CBY26503.1-CBY26517.1
Serotype O:3 antigen
dDTP-L-rhamnose biosynthesis [22, 23].
YE 4/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 BT 2–4
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 BT 2–4, 1
YE BT 1A, 1B; YP
CBY29000.1-CBY29007.1
sseDBCEBF
Virulence, type III secretion
Type III secreting effectors and chaperones.Salmonella type III secretion Sse operon is involved in interaction with macrophages.
YE BT 2–4, 1A
YE BT 1B
CBY28981.1-CBY29013.1
ysp
Virulence, type III secretion
Not fully conserved in biotype 1A strains [24].
YE BT 2–4, 1A
YE BT 1B
CBY26978.1-CBY26985.1
agaRZVWEFSY
N-Acetylgalactosamineutilization
Use of intestinal mucin as a carbon source [24].
YE =Y. enterocolitica, YP = Y. pseudotuberculosis, BT = biotype(s).Phylogenetic analysis of the different T6SSs shows that three distinct types of T6SS are present inY. pseudotuberculosis (Figure 2). One type present in two copies inY. pseudotuberculosis (CAH19881.1, CAH21904.1 inY. pseudotuberculosis IP32953) is present in one copy inY. enterocolitica genomes (CAL12724.1 in strain 8081) and bears strong similarity to several T6SSs found in other bacteria. These include H1-T6SS found inPseudomonas aeruginosa and four T6SSs found inBurkholderia thailandensis. Copies of the second type of T6SS (CAH20722.1 and CAH22490.1 inY. pseudotuberculosis IP32953) clustered together with uncharacterized T6SSs found inY. pestis (Figure 2). The function of T6SSs belonging to this type is unknown. The third distinct type of T6SS identified inY. pseudotuberculosis genomes (CAH22876.1 in IP32953) shared strong similarity with the T6SSs ofVibrio cholerae andB. thailandensis, which are both considered to have cytotoxic effects against unicellular organisms and macrophages.Figure 2
Phylogenetic relationships of type VI secretion systems (T6SSs) inYersinia pseudotuberculosis and T6SSs of other species were compared to evaluate the different types of T6SS. VipA sequences were used to represent T6SS and the alignment of 206 VipA proteins is shown here as an unrooted phylogenetic tree visualized by BioNJ. Type VI secretion systems ofY. pseudotuberculosisnamed YPTB 1–5 belong to three distinct branches of T6SSs. T6SSs ofVibrio cholerae,Pseudomonas aeruginosa, andBurkholderia thailandensis (BTHAI 1–6) are marked on the branches of the phylogenetic tree and the one VipA/T6SS present inY. enterocolitica is also shown.Relatively few gene cluster differences were observed between the hybridization results of differentY. enterocoliticastrains (Figure 3). These included an operon coding for type III secretion system shared by low-pathogenicY. enterocolitica, genes involved in drug resistance, and the operon coding from O:3 antigen. Many of the other differences are annotated as putative phages or flagellar components.Figure 3
A heatmap presentation ofYersinia enterocolitica hybridization results produced with R. Biotypes cluster separately and major gene clusters differing between different biotypes are shown. A green color signifies that the gene is present in the given strain.The majority ofY. pseudotuberculosis strains obtained from swine samples (“swine group” in Figure 1) in Finland, Sweden, Estonia, Russia, England, and Belgium (n
=
23) clustered separately from human and wildlife samples (“diverse group” in Figure 1). Five of the 11Y. pseudotuberculosisstrains isolated from English pigs clustered together with the human and wildlife samples.


## 4. Discussion

Y. enterocoliticaandY. pseudotuberculosis strains grouped into two distinct clusters, andY. enterocoliticastrains belonging to four different biotypes formed distinct subclusters within theY. enterocoliticagroup. On the gene level, the most interesting differences betweenY. enterocoliticaandY. pseudotuberculosisstrains included genes involved in T6SSs, the catabolism of phenolic compounds, and the transport of many carbohydrates (rhamnose, fructose, ribose, myoinositol, and xylose) and other compounds (aliphatic sulfonates, opines) (Table 1).T6SS forms a needle-like injectisome between the bacterial cell and the target cell [52]. First described under ten years ago, T6SS is now one of the most common large specialized secretion systems found in over 120 bacteria [48, 53]. T6SSs were first considered as virulence factors, but their abundance in nonpathogenic bacteria and further studies have suggested that most of these systems play a role in interbacterial interaction and defense against competitive bacteria and unicellular organisms [54]. The mechanism requires 15 conserved genes and direct contact with other cells and is thus thought to be especially useful in the stationary growth phase [52, 55, 56]. It is notable that diverse collections of T6SSs have been reported in many environmental bacteria with facultative pathogenic potential, such asPseudomonas aeruginosa,Burkholderia mallei, andBurkholderia pseudomallei, as well as in bacteria with multiple hosts and the ability to survive in diverse environmental conditions (Y. pestis,V. cholerae) [53, 57–60].Y. pseudotuberculosis is also considered a facultative pathogen with multiple host species and able to persist in the environment. The genome ofY. pseudotuberculosis has four conserved systems and one smaller, perhaps partial system [55]. Only one of these systems is shared withY. enterocolitica. The loci coding VipA protein is used to indicate the location of each T6SS. Two of theY. pseudotuberculosis T6SSs (CAH19881.1, CAH21904.1 in IP32953) and the solitary T6SS inY. enterocolitica(CAL12724.1 in strain 8081) group in phylogenetic analysis together withB. thailandensis T6SS (BTHAI-1) and H1-T6SS ofP. aeruginosa. The latter two have been reported to target other bacteria and give some competitive advantage to the bacterium itself  [48, 56]. Having this mechanism could enhance the growth ofY. pseudotuberculosis when other bacteria are present on a shared growth surface. Another T6SS (CAH22876.1 in IP32953) is similar to the T6SSs inV. cholerae andB. thailandensis, which are described as being cytotoxic against single-celled organisms and macrophages [48, 53, 61]. T6SSs like this are beneficial against protists living in the soil and water environment but are also possible pathogenicity factors. To better understand the function of each T6SS ofY. pseudotuberculosis,in vivo studies are required. Epidemiologically, T6SSs could probably helpY. pseudotuberculosis to survive and multiply in such ecological niches in the environment from which it could easily end up as a contaminant of the food chain. The lack of T6SSs inY. enterocoliticaimplies that the organism in its current ecological niche has no need for them. The lack of T6SSs might actually be beneficial for the organism, as a T6SS with cytotoxic effects against the macrophages of the mammal host might encumber the invasion and survival ofY. enterocolitica cells.Y. pseudotuberculosis strains also carry a variety of gene clusters involved in the uptake and/or utilization of various substrates (Table 1). The Hpa operon (CAH20875.1–CAH20884.1 in IP32953), also known as the 4-hydroxyphenylacetate degrading operon, is involved in the catabolism of phenolic and aromatic compounds [62, 63]. Based on database queries, Hpa sequences inY. pseudotuberculosisare homologous to those inE. coli andSalmonella. Phenols are products of plant secondary metabolism and often have bactericidal effects. Phenols are widely present in soil and the water environment, but their abundance in the intestines of animals has also been suggested [62, 63]. Interestingly, the hpa genes are expressed inSalmonella enterica serovar Typhimurium cells during the infection in swine, and it has been suggested that the operon is somehow beneficial for enteropathogenic bacteria [64]. Evidently, the lack of an Hpa operon does not seem to hinder the prevalence ofY. enterocoliticain swine.BothY. enterocoliticaandY. pseudotuberculosishave many transport and uptake systems that are not present in other species.Y. enterocoliticastrains share six ABC transporters and seven PTS transporters that are all absent fromY. pseudotuberculosis strains. Putative substrates for these systems include iron and metallic ions, glycosides, and lactose/cellobiose. Interestingly, one putative urea ABC transporter was noted as present in all otherY. enterocoliticastrains, but absent from reference strain 8081. This highlights the benefits of having multiple reference strains.Y. enterocoliticastrains shared notably fewer specific genes (n
=
448) between them thanY. pseudotuberculosisstrains (n
=
906). This is likely to reflect the greater heterogeneity withinY. enterocolitica biotypes and the ecological adaptation 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 and 2 PTS transporters absent fromY. enterocolitica. Putative substrates for these systems include rhamnose, fructose, xylose, myoinositol, iron, aromatic acids, polyamines, sorbitol, sulfonates, and 7 systems for unspecified sugars. TheY. pseudotuberculosisgenome appears to be equipped with many extra tools for moving substances in and out of its cell compared to theY. enterocoliticagenome, which appears more streamlined and likely to have adapted to another ecological niche, such as swine tonsils and gut, where variety in substrate transportation is not required. A recent hypothesis on the evolution of enteropathogenicYersinia assumes thatYersinia species have evolved to become more ecologically specific and metabolically more limited to their reservoirs by genome decay and gene loss [4]. An evolutionary path such as this appears plausible, asY. enterocolitica seems better adapted to living in a mammal host and to have lost many genes involved in survival in the environment.The theory of genome decay and gene loss, however, does not explain the differences betweenY. enterocolitica biotypes. Lipase activity, hydrolysis of B-glycosides such as salicin and esculin, use of xylose, and indole production are some of the biochemical tests belonging toY. enterocoliticabiotyping schema [2]. However, relatively few clusters of genes differentiate pathogenic and nonpathogenicY. enterocolitica strains from each other. Recent results have identified the changes in gene expression patterns for pathogenicity factors explaining the swine specificity ofY. enterocolitica 4/O:3 [22]. It seems likely that the adaptation and differences in pathogenicity ofY. enterocolitica biotypes are due to point mutations and changes in gene expression rather than gene loss.Y. pseudotuberculosis strains obtained from swine samples mostly clustered separately from human and wildlife samples (Figure 1). Notably, five of the 11Y. pseudotuberculosisstrains isolated from English swine clustered together with the human and wildlife samples. Niskanen et al. [39] have previously reported on the homogeneity ofY. pseudotuberculosis strains isolated from swine samples based on pulsed-field gel electrophoresis analysis. Our results further confirm this finding. Martínez et al. [25–27] noted that the prevalence and diversity ofY. pseudotuberculosisstrains appears to be higher in English swine than in swine of other European countries. This finding is also supported by the present results, as 5 of the 11 EnglishY. pseudotuberculosisstrains showed a marked genetic distance to other swine strains. In these results, no defining gene cluster setting the swine group and diverse group apart could be identified. In this type of study, the results are dependent on the reference strains used. It is important to note that because of the limitations of the method, many genes present in the studied strains might be absent from the reference genomes used and thus from the designed microarray and the further results. It would be interesting to have a wholly sequenced genome from the “swine group” ofY. pseudotuberculosisfor further research on the differences between these two groups.The high prevalence ofY. pseudotuberculosisin the English pork chain is probably explained by the more available access to outdoors of English swine compared to their continental counterparts. Swine and pork products are not considered to be a notable source of sporadicY. pseudotuberculosiscases, and animals having greater contact with the environment are more likely to have strains of soil and wildlife origins passing through their intestines. This would also explain why someY. pseudotuberculosisnot belonging to the “swine group” ofY. pseudotuberculosishave been isolated from English swine.


## 5. Conclusions

The hybridization results revealed thatY. pseudotuberculosisstrains carry many operons linked with the use of carbohydrates and other substrates that are absent fromY. enterocolitica. Phenolic compounds, polyamines, myoinositol, and aliphatic sulfonates are all substrates that are not commonly present in living animal tissue but are more abundant in soil and the environment.Y. pseudotuberculosisalso harbors an array of different type VI secretion systems, in contrast to just one found in theY. enterocoliticagenome. Type VI secretion systems target single-celled organisms and other bacteria but are also possible pathogenicity factors. These defense and interaction systems could helpY. pseudotuberculosis to survive and multiply in such ecological niches in the environment from which it could easily end up as a contaminant of the food chain.TheY. pseudotuberculosis genome holds many tools, such as type VI secretion systems and transporters for various substrates, which are likely to be beneficial for survival in varied growth environments and multiple host species. By comparison, the genome ofY. enterocolitica appears more streamlined and likely to have adapted to a different ecological niche where these survival systems are not needed or beneficial. ForY. enterocolitica bioserotype 4/O:3, this niche is with certainty swine.



---

*Source: 760494-2015-10-28.xml*