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First genome sequencing and comparative analyses of Corynebacterium pseudotuberculosis strains from Mexico

  • Doglas Parise
  • Mariana T D Parise
  • Marcus V C Viana
  • Adrian V Muñoz-Bucio
  • Yazmin A Cortés-Pérez
  • Beatriz Arellano-Reynoso
  • Efrén Díaz-Aparicio
  • Fernanda A Dorella
  • Felipe L Pereira
  • Alex F Carvalho
  • Henrique C P Figueiredo
  • Preetam Ghosh
  • Debmalya Barh
  • Anne C P Gomide
  • Vasco A C Azevedo
Open Access
Extended genome report

Abstract

Corynebacterium pseudotuberculosis is a pathogenic bacterium which has been rapidly spreading all over the world, causing economic losses in the agricultural sector and sporadically infecting humans. Six C. pseudotuberculosis strains were isolated from goats, sheep, and horses with distinct abscess locations. For the first time, Mexican genomes of this bacterium were sequenced and studied in silico. All strains were sequenced using Ion Personal Genome Machine sequencer, assembled using Newbler and SPAdes software. The automatic genome annotation was done using the software RAST and in-house scripts for transference, followed by manual curation using Artemis software and BLAST against NCBI and UniProt databases. The six genomes are publicly available in NCBI database. The analysis of nucleotide sequence similarity and the generated phylogenetic tree led to the observation that the Mexican strains are more similar between strains from the same host, but the genetic structure is probably more influenced by transportation of animals between farms than host preference. Also, a putative drug target was predicted and in silico analysis of 46 strains showed two gene clusters capable of differentiating the biovars equi and ovis: Restriction Modification system and CRISPR-Cas cluster.

Keywords

Phylogenetics Genomic sequencing Drug target CRISPR-Cas Restriction-modification systems 

Abbreviations

AQUACEN

National Reference Laboratory for Aquatic Animal Diseases

BHI

Brain heart infusion

CRISPR

Clustered regularly interspaced short palindromic repeats

LGMC

Laboratory of Cellular and Molecular Genetics

PATRIC

Pathosystems Resource Integration Center

PEPR

Phylogenomic Estimation with Progressive Refinement

RM systems

Restriction-modification

UFMG

Federal University of Minas Gerais

UNAM

National Autonomous University of Mexico

Introduction

Corynebacterium pseudotuberculosis is a Gram-positive bacterium that infects several different species of mammals. Strains of the biovar ovis infect sheep and goats, and strains of the biovar equi infect larger mammals such as horses, camels, and buffaloes. The manifestation of the infection depends on the host [1, 2, 3, 4]. This bacterium causes significant economic loss to animal production all over the world due to reduced production of wool, milk and meat, carcass condemnation, as well as the death of infected animals [4, 5, 6]. C. pseudotuberculosis can also affect humans, causing distinct kinds of lymphadenitis. Contamination occurs through contact with infected animals and consumption of infected food [4, 5, 7].

This organism affects several countries such as Australia, Brazil, Canada, Egypt, Israel, New Zealand, South Africa, United Kingdom and United States [4, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17]. Cases in other countries such as Portugal [18], Mexico [19] and Equatorial Guinea [20] have been reported in the recent years. In the United States, C. pseudotuberculosis infections are reemerging and considered endemic [19], and the state with the highest number of cases of this bacterium was Texas, which borders Mexico [21]. The spread of C. pseudotuberculosis to other countries brings out the importance of improving the understanding of this bacterium. In the present study, six Mexican C. pseudotuberculosis strains were investigated, two from the biovar equi and four from the biovar ovis. This is the first time that strains of this bacterium, isolated in Mexico, have been completely sequenced. Among those strains, these are the first isolates of the biovar equi coming from this country [19]. The characterization of these strains is important for achieving a better understanding of this species, considering they can present relevant features not yet identified in other strains.

Organism information

C. pseudotuberculosis is a pathogenic bacterium that belongs to the CMNR ( Corynebacterium , Mycobacterium , Nocardia , and Rhodococcus ) group. This group is characterized by high GC content (46–74%) and by the structure of the cell wall which is mainly composed of peptidoglycan, arabinogalactan and mycolic acids [4, 22]. C. pseudotuberculosis is placed in the phylum Actinobacteria , class Actinobacteria , order Actinomycetales , suborder Corynebacterineae and genus Corynebacterium [23, 24, 25, 26, 27, 28, 29, 30]. The species is considered a facultative intracellular pathogen [4, 31] which is Gram-positive, pleomorphic, non-motile, non-sporulating, mesophilic and can survive both in the host and in the soil [25, 31, 32, 33, 34, 35]. Its strains are classified into two biovars, ovis and equi, according to its host preference and nitrate reduction capacity, which is identified through the presence or absence of the narG gene in a PCR Multiplex test [36]. The biovar equi can reduce nitrate and affects mostly large ruminants. The biovar ovis is not able to reduce nitrate and affects mostly small ruminants [4]. More information about classification, general features of this species and some details about the target strains are shown in Table 1 (Additional file 1).
Table 1

Classification and general features of Corynebacterium strains MEX1, MEX9, MEX25, MEX29, MEX30, and MEX31 according to the MIGS recommendations [41]

MIGS ID

Property

Term

Evidence codea

 

Classification

Domain Bacteria

TAS [23]

Phylum Actinobacteria

TAS [24]

Class Actinobacteria

TAS [25]

Order Actinomycetales Suborder Corynebacterineae

TAS [25, 26, 27, 28]

Family Corynebacteriaceae

TAS [25, 28]

Genus Corynebacterium

TAS [29, 30]

Species Corynebacterium pseudotuberculosis

TAS [26, 29]

strain: MEX1 (Accession NZ_CP017711.1)

MEX9 (Accession NZ_CP014543.1),

MEX25 (Accession NZ_CP013697.1),

MEX29 (Accession NZ_CP016826.1),

MEX30 (Accession NZ_CP017291.1),

MEX31 (Accession NZ_CP017292.1)

 

Gram stain

Positive

TAS [31]

Cell shape

Pleomorphic

TAS [31]

Motility

Non-motile

TAS [31, 35]

Sporulation

non-sporulating

TAS [31]

Temperature range

Mesophilic

TAS [32, 35]

Optimum temperature

37 °C

TAS [32, 73]

pH range; Optimum

7.0–7.2

TAS [4, 35]

Carbon source

Glucose, fructose, maltose, mannose, and sucrose

TAS [11, 15]

MIGS-6

Habitat

Host and soil

TAS [25, 33, 34]

MIGS-6.3

Salinity

Up to 2 M NaCl

TAS [32]

MIGS-22

Oxygen requirement

Aerobic and facultative anaerobic

TAS [4, 35, 73]

MIGS-15

Biotic relationship

Facultative intracellular pathogen

TAS [4, 31]

MIGS-14

Pathogenicity

Sheep, goats, horses, cattle, camel, buffalo, rarely humans

TAS [4, 37, 74]

MIGS-4

Geographic location

MEX1 – Ixtenco, Tlaxcala, Mexico

MEX9 – Salamanca, Guanajuato, Mexico

MEX25 – Celaya, Guanajuato, Mexico

MEX29 - Río Frio, Estado de Mexico, Mexico

MEX30 and MEX31 – Valparaiso, Zacatecas, Mexico

TAS [19]

MIGS-5

Sample collection

MEX1–2014, MEX9 and MEX25 –2012,

MEX29 , MEX30 and MEX31 –2013

TAS [19]

MIGS-4.1

Latitude

MEX1 – 19 o 15’11” MEX9–20 o 34’26”

MEX25 –20 o 55’1” MEX29 –19 o 21’8”

MEX30 and MEX31 –22 o 46’16”

IDA

MIGS-4.2

Longitude

MEX1 – 97 o 53’45” MEX9 - 101 o 11’45”

MEX25 - 101 o 9’42” MEX29 - 98 o 40’17”

MEX30 and MEX31 –103 o 34’11”

IDA

MIGS-4.4

Altitude

MEX1–8236 ft MEX9–5623 ft

MEX25 –6502 ft MEX29 –9770 ft

MEX30 and MEX31 –6221 ft

IDA

aEvidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [75]

Six C. pseudotuberculosis strains were isolated in Mexico from different hosts and biovars. The strain MEX1 was isolated from a retropharyngeal abscess in a goat. The strain MEX9 was isolated from a prescapular abscess in a goat. The strain MEX25 was isolated from a parotidean abscess in a sheep. The strain MEX29 was isolated from a retropharyngeal abscess in a sheep. These four strains presented negative result for the presence of the narG gene in the PCR multiplex test and were classified as belonging to the biovar ovis. All ovis strains were obtained from outbreaks occurred relatively close to Mexico City. MEX30 and MEX31 were isolated from abscesses in the pectoral muscles of two horses [19]. These two strains were positive for the presence of the narG gene in PCR Multiplex. Consequently, they were classified as belonging to the biovar equi. Although both equi strains were obtained in the same city, they could be considered as isolated cases.

To verify the phylogenetic relationship of these strains to other strains of C. pseudotuberculosis , we generated a phylogenetic tree (Fig. 1) based on the core proteome and progressive refinement, using a bootstrap value of 100. The tree was generated using the PEPR software (https://github.com/enordber/pepr.git) with the Maximum-Likelihood method. The Mexican strains were clustered according to the respective biovars and host preferences, as shown in previous works) [1, 37].
Fig. 1

Phylogenetic tree of new Corynebacterium pseudotuberculosis strains of this work inside the rectangles, with other strains of the group CMNR. The blue rectangles highlight the biovar ovis strains and the red rectangle highlights the biovar equi strains of this work. The numbers near the nodes indicate bootstrap values

MEX30 and MEX31 were isolated in Valparaiso, in the first reported case of infection of horses in Mexico [19]. They clustered together probably because they came from the same source, that could be transported infected animals. Affected horses were identified in all regions of the US and the state of Texas, which borders Mexico, has the highest number of cases) [9, 21].

Ovis strains were isolated in Tlaxcala (MEX1) and Rio Frio de Juárez (MEX29), with a 50 Km distance from each other, and Guanajuato (MEX9 and MEX25), within a 400–450 Km distance from the two other isolation localities. However, the strains cluster by host rather than locality of isolation. MEX1 and MEX9 were isolated from goat and MEX25 and MEX29 were isolated from sheep. However, MEX25 and MEX29 (goat) clustered with isolates from lhama (USA) and cow (Israel), while MEX1 and MEX9 (sheep) clustered with isolates from goat and sheep (Brazil), all with a 100% bootstrap. Strains of Ovis biovar are more clonal but does not show the same degree of clustering by the host as Equi [1, 37]. Considering a maximum distance of 450 Km between localities of isolation, this genetic structure could better be explained by farming history than host preference. The goat and sheep farms could have different sources of Ovis strains. Transportation of infected animals and further contact and transmission of the disease probably occurred between farms of the same host species [38, 39, 40].

Genome sequencing information

Genome project history

The present project is a collaboration between the National Autonomous University of Mexico (UNAM), Mexico City, Mexico, and the Federal University of Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil. The six C. pseudotuberculosis strains were isolated by UNAM researchers. Sequencing was performed at the National Reference Laboratory for Aquatic Animal Diseases (AQUACEN), and the two processes of assembly and annotation were performed at the Laboratory of Cellular and Molecular Genetics (LGCM), both laboratories located at UFMG. All genomes are complete and available at the National Center for Biotechnology Information (NCBI). This information is shown in Table 2 and conforms with MIGS recommendations [41]. As mentioned above, the present study presents the first sequencing of C. pseudotuberculosis , and the first isolation of the biovar equi, from Mexico. This data can provide new insights into the diagnosis and treatment of diseases caused by this organism.
Table 2

Project information

MIGS ID

Property

Term

MIGS 31

Finishing quality

Finished

MIGS-28

Libraries used

Fragments

MIGS 29

Sequencing platforms

Ion Torrent PGM

MIGS 31.2

Fold coverage

115× (MEX1); 129× (MEX9); 99× (MEX25); 135× (MEX29); 81× (MEX30); 123× (MEX31).

MIGS 30

Assemblers

Newbler, SPAdes.

MIGS 32

Gene calling method

RAST

Locus Tag

CpMEX1_ (MEX1); CpMEX9_ (MEX9); AN397_ (MEX25); CpMEX29_ (MEX29); CpMEX30_ (MEX30); CpMEX31_ (MEX31);

Genbank ID

CP017711 (MEX1); CP014543(MEX9); CP013697 (MEX25); CP016826 (MEX29); CP017291 (MEX30); CP017292 (MEX31);

GenBank Date of Release

2017/01/30 (MEX1); 2016/05/27 (MEX9); 2015/12/23 (MEX25); 2016/11/03 (MEX29); 2016/12/27 (MEX30); 2016/12/27 (MEX1);

GOLD ID

- (MEX1); Go0366057 (MEX9); Go0139540 (MEX25); Go0364114 (MEX29); Go0364489 (MEX30); Go0364678 (MEX31);

BIOPROJECT

PRJNA348354 (MEX1); PRJNA312392 (MEX9); PRJNA294672 (MEX25); PRJNA335634 (MEX29); PRJNA343017 (MEX30); PRJNA341961 (MEX31);

MIGS 13

Source Material Identifier

BHI broth

Project relevance

Animal Pathogen, Medical

Growth conditions and genomic DNA preparation

The samples used in the present study are in the sample collection of LGCM. All six strains were grown in a brain-heart-infusion media (BHI-HiMedia Laboratories Pvt. Ltd., India) with 1.5% of bacteriological agar and supplemented with 0.5% of Tween 80, at 37 °C for 72 h under rotation. Genomic DNA was extracted following the protocol of Pacheco et al. [36].

Genome sequencing and assembly

The first step in sequencing each genome was the library construction, following manufacturer’s recommendations (IonXpress™ Plus gDNA Fragment Library Preparation). This was performed in three steps: (i) DNA fragmentation using the Ion Shear™ Plus Reagents Kit, (ii) addition of adapters using Ion Xpress™ Barcode Adapters and (iii) library amplification using the Ion PGM™ Template OT2 200 kit (all kits from Thermo Fisher Scientific, USA). The resulting library was put on the semiconductor chip Ion 318 Chip Kit v2 (Thermo Fisher Scientific) and then into the sequencer Ion Personal Genome Machine™ (Thermo Fisher Scientific). The number of reads and the mean read length of MEX1, MEX9, MEX25, MEX29, MEX30 and MEX31 strains are respectively: 1,100,551 and 244; 1,496,261 and 201; 1,117,243 and 206; 1,371,907 and 230; 1,127,325 and 186; and, 1,262,316 and 230.

The assembly process was managed using SIMBA software [42]. The quality assessment of the reads was performed using FastQC software [43]. The assemblies were performed using SPAdes version 3.6 [44] on MEX1 and MEX31; and, Newbler version 2.9 (Roche, USA) on MEX9, MEX25, MEX29, and MEX30. This produced the following contigs: 6 on MEX1, 7 on MEX9, 7 on MEX25, 9 on MEX29, 33 on MEX30 and 13 on MEX31. The N50 s were: 543,202 on MEX1, 372,309 on MEX9, 543,326 on MEX25, 367,275 on MEX29, 103,276 on MEX30 and 535,978 on MEX31. The QUAST software [45] was used to evaluate the quality of the assemblies for all strains. The scaffolds were constructed using CONTIGuator software version 2.0 [46] with C. pseudotuberculosis strain 29,156 (CP010795.1) as a reference to MEX9, MEX25 and MEX29, C. pseudotuberculosis strain MEX9 as a reference to MEX1, C. pseudotuberculosis strain 316 (CP003077.1) as a reference to MEX30 and C. pseudotuberculosis strain E19 (CP012136.1) as a reference to MEX31. Gap closure was performed using CLC Genomics Workbench 7 (Qiagen, USA). This process resulted in six complete genome sequences.

Genome annotation

Genome annotation was performed in two steps: automatic annotation and manual curation. The RAST [47] and tRNAscan-SE [48] software were used in the automated annotation. An in-house script was also employed to transfer the annotation from a reference genome. The Artemis software version 16.0.0 [49], the UniProt [50] and the National Center for Biotechnology Information (NCBI) databases [51] were used in the manual curation. Putative frameshifts were analyzed using CLC Genomics Workbench 7 (Qiagen, USA) and fixed whenever possible.

Genome properties

Genome sizes of the respective strains are: 2,337,090 bp (base pairs) on MEX1, 2,337,578 bp on MEX9, 2,337,529 bp on MEX25, 2,337,866 bp on MEX29, 2,368,140 bp on MEX30 and 2,367,880 bp on MEX31. The respective percentages of the predicted coding regions are: 86.16% on MEX1, 86.33% on MEX9, 85.94% on MEX25, 86.66% on MEX29, 83.06% on MEX30 and 86.64% on MEX31. These genome sizes and the G + C content (~ 52%) are consistent with other C. pseudotuberculosis studies [2, 6, 52]. There are 64 predicted RNA genes in strains of the biovar ovis (MEX1, MEX9, MEX25 and MEX29) and 66 from the biovar equi (MEX30 and MEX31). The numbers (and percentages) of predicted protein coding genes and pseudogenes of MEX1, MEX9, MEX25, MEX29, MEX30 and MEX31 strains are, respectively: 2021 (94.22%) and 60 (2.80%); 2025 (94.36%) and 57 (2.66%); 2016 (94.07%) and 63 (2.94%); 2032 (94.73%) and 49 (2.28%); 2008 (91.77%) and 114 (5.21%); and 2058 (94.32%) and 61 (2.80%). Table 3 shows detailed information about properties and statistics of these genomes. The number of genes associated with general COG functional categories [53, 54] was generated with the in-house script Blast Cog (https://github.com/aquacen/blast_cog) and are summarized in Table 4. The circular maps of C. pseudotuberculosis MEX1 and MEX30 strains in comparison with the other strains of the present study are shown in Figs. 2 and 3, respectively.
Table 3

Genome statistics

Attribute

MEX1

MEX9

MEX25

MEX29

MEX30

MEX31

Value

%

Value

%

Value

%

Value

%

Value

%

Value

%

Genome size (bp)

2,337,090

100.0

2,337,578

100.0

2,337,529

100.0

2,337,866

100.0

2,368,140

100.0

2,367,880

100.0

DNA coding (bp)

2,012,758

86.12

2,017,915

86.33

2008,915

85.94

2025,972

86.66

1,966,942

83.06

2,051,473

86.64

DNA G + C (bp)

1,219,520

52.18

1,219,842

52.18

1,219,763

52.18

1,219,957

52.18

1,234,064

52.11

1,233,547

52.10

DNA scaffolds

1

100.0

1

100.0

1

100.0

1

100.0

1

100.0

1

100.0

Total genes

2145

100.0

2146

100.0

2143

100.0

2145

100.0

2188

100.0

2182

100.0

Protein coding genes

2021

94.22

2025

94.36

2016

94.07

2032

94.73

2008

91.77

2058

94.32

RNA genes

64

2.98

64

2.98

64

2.99

64

2.98

66

3.02

63

2.89

Pseudo genes

60

2.80

57

2.66

63

2.94

49

2.28

114

5.21

61

2.80

Genes in internal clusters

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

Genes with function prediction

1579

73.61

1576

73.44

1583

73.87

1578

73.57

1605

73.36

1610

73.79

Genes assigned to COGs

2007

93.57

2013

93.80

2009

93.75

2020

94.17

1998

91.32

2046

93.77

Genes with Pfam domains

1679

78.28

1675

78.05

1670

77.93

1690

78.79

1664

76.05

1731

79.33

Genes with signal peptides

157

7.32

151

7.04

159

7.42

155

7.23

144

6.58

153

7.01

Genes with transmembrane helices

595

27.74

591

27.54

585

27.30

601

28.02

585

26.74

596

27.31

CRISPR repeats

0

00 0

1

0.05

1

0.05

1

0.05

4

0.18

4

0.18

Table 4

Number of genes associated with general COG functional categories

Code

MEX1

MEX9

MEX25

MEX29

MEX30

MEX31

Description

Value

%age

Value

%age

Value

%age

Value

%age

Value

%age

Value

%age

 

J

182

9.01

182

8.99

181

8.98

186

9.15

183

9.11

189

9.18

Translation, ribosomal structure and biogenesis

A

2

0.10

2

0.10

2

0.10

2

0.10

2

0.10

2

0.10

RNA processing and modification

K

138

6.83

139

6.87

137

6.80

138

6.79

136

6.77

134

6.51

Transcription

L

105

5.20

105

5.19

96

4.76

102

5.02

102

5.08

101

4.91

Replication, recombination and repair

B

0

0

0

0

0

0

0

0

1

0.05

0

0

Chromatin structure and dynamics

D

44

2.18

43

2.12

45

2.23

45

2.22

43

2.14

47

2.28

Cell cycle control, Cell division, chromosome partitioning

V

68

3.37

67

3.31

69

3.42

71

3.49

75

3.74

70

3.40

Defense mechanisms

T

99

4.90

101

4.99

99

4.91

103

5.07

98

4.88

98

4.76

Signal transduction mechanisms

M

124

6.14

122

6.03

119

5.90

120

5.91

119

5.93

117

5.69

Cell wall/membrane biogenesis

N

21

1.04

22

1.09

20

0.99

20

0.98

13

0.65

17

0.83

Cell motility

U

32

1.58

31

1.53

30

1.49

31

1.53

29

1.44

30

1.46

Intracellular trafficking and secretion

O

128

6.33

122

6.03

121

6.00

126

6.20

122

6.08

122

5.93

Posttranslational modification, protein turnover, chaperones

C

125

6.19

124

6.12

116

5.75

124

6.10

123

6.13

121

5.88

Energy production and conversion

G

158

7.82

154

7.61

151

7.49

156

7.68

151

7.52

161

7.82

Carbohydrate transport and metabolism

E

212

10.49

213

10.52

204

10.12

213

10.48

219

10.91

223

10.84

Amino acid transport and metabolism

F

82

4.06

82

4.05

79

3.92

82

4.04

78

3.88

81

3.94

Nucleotide transport and metabolism

H

143

7.08

141

6.96

135

6.70

141

6.94

151

7.52

152

7.39

Coenzyme transport and metabolism

I

92

4.55

91

4.49

90

4.46

93

4.58

86

4.28

87

4.23

Lipid transport and metabolism

P

162

8.02

157

7.75

162

8.04

162

7.97

166

8.27

168

8.16

Inorganic ion transport and metabolism

Q

49

2.43

46

2.27

46

2.28

48

2.36

52

2.59

50

2.43

Secondary metabolites biosynthesis, transport, and catabolism

R

170

8.41

164

8.10

157

7.79

167

8.22

167

8.32

169

8.21

General function prediction only

S

138

6.83

142

7.01

127

6.30

141

6.94

138

6.87

140

6.80

Function unknown

14

0.69

12

0.59

7

0.35

12

0.59

10

0.50

12

0.58

Not in COGs

The total is based on the total number of protein coding genes in the genome

Fig. 2

Circular map of C. pseudotuberculosis strain MEX1 (biovar ovis) in comparison with the other strains of this study. The cluster of methylation type III, which is only present in biovar ovis strains, is highlighted in blue

Fig. 3

Circular map of C. pseudotuberculosis strain MEX30 (biovar equi) in comparison with the other strains of this study. The cluster of CRISPR-Cas, which is only present in biovar equi strains, is highlighted in blue. The nitrate reductase gene cluster is highlighted by a black rectangle

Insights from the genome sequence

The nucleotide sequences, analyzed using the Gegenees software version 2.1 [55], show high similarity (> 92%) between the strains. Higher similarity (> = 99.7%) within strains belonging to the same biovar was found (Fig. 4). This is consistent with a previous study [1], using 15 strains of C. pseudotuberculosis , that shows similarity greater than 99% within the biovar ovis strains and at least 95% of sequencing similarity within the biovar equi strains. Moreover, the sequencing similarity among strains isolated from the same host is higher than the similarity among strains isolated from different hosts (Figs. 1 and 4).
Fig. 4

Alignment generated using Gegenees software showing the percentage similarity among the strains, based on the accessory genome. The blue rectangle highlights the grouping of the biovar ovis. The red rectangle highlights the grouping of the biovar equi

Traditionally, the two biovars are differentiated using a nitrate reduction test, in which equi is positive, and ovis is negative [56]. Figure 3 highlights the cluster of genes related to nitrate reduction in Mexican equi strains with the black rectangle. The Protein Family Sorter tool [57] was used to search for genes or clusters of genes that may be used to differentiate the biovars. Within the six genomes of the present study, we found the cluster of genes that is related to proteins of type III restriction-modification (RM) systems [58, 59] exclusively in the biovar ovis (highlighted in blue in Fig. 2). A cluster of genes related to the proteins of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas) systems, probably belonging to type I-E [60], was found exclusively in the biovar equi (highlighted in blue in Fig. 3). Both systems work as protection systems, defending the bacteria against exogenous DNA. We analyzed 40 other sequenced strains of C. pseudotuberculosis to confirm these results in other strains. The same pattern was observed.

RM systems have two main components, a DNA methyltransferase, and a restriction endonuclease. The first one methylates the DNA in possible cleavage sites; the second one is responsible for the cleavage of DNA from external sources [61]. A good review of RM systems can be found in [62]. CRISPR-Cas systems are adaptive immune systems in bacteria and archaea. They use a complex of proteins known as Cas that are responsible for acquiring new, short sequences of external sources (exogenous genetic elements). These short sequences are incorporated into the bacterial chromosome and are called CRISPRs. The CRISPRs are transcribed into small RNAs that guide the Cas proteins to recognize and cleave foreign DNA, protecting the bacterial genome [63]. Reviews of CRISPR-Cas systems can be found in [63, 64, 65].

Possible new drug targets were predicted using the Specialty Genes Search from the Pathosystems Resource Integration Center (PATRIC) bioinformatics resource center [66]. The result shows a new putative target, the gene nrdF2, for five of the six strains used in the present study. In the C. pseudotuberculosis MEX30 strain, this gene is annotated as a pseudogene, which can explain why it was not considered a putative target. The product of this gene is the small subunit of ribonucleotide reductase (RNR) which is involved in dNTP (deoxynucleotide triphosphate) synthesis that reduces ribonucleotides to nucleotides. The RNRs can be classified into three classes (I, II and III). Class I is oxygen dependent and has two subclasses (Ia and Ib). Class Ia is coded by nrdA and nrdB genes; class Ib is coded by nrdE and nrdF. Therefore, the RNR found in the biovar ovis strains belongs to class Ib [67]. Previous studies [68, 69, 70] show the importance of this gene for growth under normal conditions (in vitro) in Mycobacterium tuberculosis , Corynebacterium ammoniagenes and Corynebacterium glutamicum . Additionally, other studies have pointed to this gene as a potential target of M. tuberculosis vaccine [70, 71, 72].

Conclusions

In the present study, we investigated six strains of C. pseudotuberculosis from different hosts and their sequenced genomes, the first whole-genome investigation of this organism from Mexico. The phylogenomic analysis suggested that the genetic structure of Ovis is more influenced by animal transportation than host preference. An in silico analysis of protein families showed two important clusters that may differentiate the biovars equi and ovis. Also, the present work identified a new putative drug target against C. pseudotuberculosis , the gene nrdF2, which has been previously described as a potential vaccine target [70, 71, 72]. Further in silico and in vitro analyses are required to validate these findings. Those results could provide a better understanding of this organism and its mechanisms of virulence and pathogenesis, as well as develop new diagnoses, vaccines, and treatments.

Notes

Acknowledgements

The authors thank the Brazilian funding agencies, CNPq, CAPES, and FAPEMIG, for providing financial support to the National Institute of Science and Technology in Theranostics and Nanobiotechnology – INCT-TeraNano (CNPq/CAPES/FAPEMIG, Grant numbers CNPq-465669/2014-0 and FAPEMIG-CBB-APQ-03613-17). Debmalya Barh acknowledges the “TWAS-CNPq Postdoctoral Fellowship Programme” for granting a fellowship for postdoctoral studies.

Authors’ contributions

DP and MTDP wrote the paper. DP, MTDP, and MVCV performed assembly, annotation and in silico analyses. AVMB, ACP, BAR, and EDA isolated the samples. FAD, FLP, AFC and HCPF sequenced the samples. PG, DB, ACPG, and VACA worked on conception, design, coordination of this study and helped to write the paper. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary material

40793_2018_325_MOESM1_ESM.docx (17 kb)
Additional file 1: Contains tables Annotation Summary, GenBank Accession Summary, Strain ID Summary, Plant Name Summary, Scientific Name Summary and Reference Search Summary. (DOCX 17 kb)

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© The Author(s). 2018

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  • Doglas Parise
    • 1
  • Mariana T D Parise
    • 1
  • Marcus V C Viana
    • 1
  • Adrian V Muñoz-Bucio
    • 2
  • Yazmin A Cortés-Pérez
    • 2
  • Beatriz Arellano-Reynoso
    • 2
  • Efrén Díaz-Aparicio
    • 2
  • Fernanda A Dorella
    • 3
  • Felipe L Pereira
    • 3
  • Alex F Carvalho
    • 3
  • Henrique C P Figueiredo
    • 3
  • Preetam Ghosh
    • 4
  • Debmalya Barh
    • 1
    • 5
    • 6
  • Anne C P Gomide
    • 1
  • Vasco A C Azevedo
    • 1
  1. 1.Laboratory of Cellular and Molecular Genetics, Institute of Biologic SciencesFederal University of Minas GeraisBelo HorizonteBrazil
  2. 2.Department of Microbiology and Immunology, Faculty of Veterinary Medicine and ZootechnicsNational Autonomous University of MexicoMexico CityMexico
  3. 3.Aquacen - National Reference Laboratory for Aquatic Animal DiseasesFederal University of Minas GeraisBelo HorizonteBrazil
  4. 4.Department of Computer ScienceVirginia Commonwealth UniversityRichmondUSA
  5. 5.Centre for Genomics and Applied Gene TechnologyInstitute of Integrative Omics and Applied Biotechnology (IIOAB)Purba MedinipurIndia
  6. 6.Division of Bioinformatics and Computational GenomicsNITTE University Center for Science Education and Research (NUCSER), NITTE (Deemed to be University)MangaluruIndia

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