# Exploration of Nitrate Reductase Metabolic Pathway inCorynebacterium pseudotuberculosis

Authors: Sintia Almeida; Cassiana Sousa; Vinícius Abreu; Carlos Diniz; Elaine M. S. Dorneles; Andrey P. Lage; Debmalya Barh; Vasco Azevedo
Journal: International Journal of Genomics
 (2017)

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## Abstract

Based on the ability of nitrate reductase synthesis,Corynebacterium pseudotuberculosisis classified into two biovars: Ovis and Equi. Due to the presence of nitrate reductase, the Equi biovar can survive in absence of oxygen. On the other hand, Ovis biovar that does not have nitrate reductase is able to adapt to various ecological niches and can grow on certain carbon sources. Apart from these two biovars, some other strains are also able to carry out the reduction of nitrate. The enzymes that are involved in electron transport chain are also identified by in silico methods. Findings about pathogen metabolism can contribute to the identification of relationship between nitrate reductase and theC. pseudotuberculosis pathogenicity, virulence factors, and discovery of drug targets.


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## Body

## 1. Introduction

Corynebacterium pseudotuberculosis is a Gram-positive facultative intracellular pathogen [1–11].C. pseudotuberculosis can be classified into two biovars, based on their ability to convert nitrate to nitrite. The nitrate-positive biovar is Equi, which causes ulcerative lymphangitis in equines, while the nitrate-negative biovar is known as Ovis, which is the etiologic agent of caseous lymphadenitis in small ruminants [9]. Both diseases are globally distributed and cause large economic losses to goat, sheep, horse, and cattle farmers.The nitrate reduction is associated with the bacterium’s ability to breathe in the absence of oxygen and having two different metabolic pathways, (1) respiratory nitrate reductase and (2) dissimilatory nitrate reduction. In the first pathway, the denitrification process takes place where the nitrate is sequentially reduced to nitrite, nitric oxide, nitrous oxide, and finally to dinitrogen [12, 13]. In the second pathway nitrate is directly converted into ammonia, which is secreted from the cell; this process can be performed by organisms with thenrf gene. This is a less common method of nitrate reduction than denitrification in most ecosystems [12–14]. There are four classes of nitrate reductases, one in eukaryotes and three are in prokaryotes. Prokaryotic nitrate reductases include a class of assimilatory enzymes and two classes of respiratory enzymes; all contain a guanylate molybdenum cofactor but differ in their substructures, cellular location, and requirement for cofactor. Variability among enzyme is also found into the classes [12, 13].Among the Actinobacteria, respiratory nitrate reductase (Nar) is observed [15–18]. This class of enzymes is mostly membrane-bound and is having three different subunits: respiratory nitrate reductase fromPseudomonas stutzeri [19, 20], nitrate reductase A, and nitrate reductase Z fromEscherichia coli K-12 [21–23]. InCorynebacterium glutamicum andMycobacterium tuberculosis the nitrate reduction reaction is catalyzed by the operonsnarKGHJI andnarGHJI, respectively [15, 16]. In these organisms, the first step in nitrate assimilation is nitrate reduction (N
O
3
-) to nitrite (N
O
2
-) and the second step is nitrite reduced (N
3
-) to ammonia (N
H
4
+) [15, 16].Nitrate respiration is associated with virulence and adoption of pathogenic organisms. InBrucella suis it helps in intramacrophagic multiplication, inM. bovis it allows survival inside the host, and inM. tuberculosis it confers resistance to stress [17, 24, 25]. However, the role of nitrate respiration is not known inC. pseudotuberculosis. We presume that Nar may be associated with pathogenicity inC. pseudotuberculosis biovar Equi, providing a generate energy pathway when it grows under oxygen deprived condition allowing the survival ofC. pseudotuberculosis inside the host.With the increase in genome sequencing projects, understanding of the biological capabilities of the organisms has also increased. The availability of the annotated genomes allows the information of metabolic pathways to be interpreted and used for computational reconstruction at genomic scale. Thus the reconstruction of metabolic pathways through comparative analysis can help identify potential targets and find the mechanisms that cause a disease.In the present study, the genomes ofC. pseudotuberculosis strains deposited in GenBank were used to identify the genes involved in the nitrate reduction pathway; a comparative genomic study was performed on 15C. pseudotuberculosis strains of the biovars Ovis and Equi to identify the molecular bases of nitrate reduction in eachC. pseudotuberculosis biovar Equi. Thus, we hope that understanding of Nar’s role inC. pseudotuberculosis can help in identifying potent targets both for the development of more effective diagnostics and therapeutics and for the treatment and prevention of disease.


## 2. Material and Methods

### 2.1. Identification of Nitrate Reductase Genes, Metabolic Pathways, and Nitrate Reduction Enzymes

C. pseudotuberculosis genomes Table 1 were obtained from the NCBI GenBank. The sequence and arrangement analysis of nitrate reductase genes innarKGHJI operon was performed using the Artemis software (http://www.sanger.ac.uk/Software/Artemis) and manual curation based annotations are performed. Pathway Tools software developed by SRI International [45] and MetaCyc database [46] were used for computational prediction of metabolic pathways inC. pseudotuberculosis.Table 1
Strains used in phenotypic and genotypic analysis regarding the nitrate reductase activity.
Strain
Biovar
Genome size (MB)
Nitrate testa
NCBI access
Ref
1002
Ovis
2.33511
Yes
NC_017300.1
[26]
C231
Ovis
2.32821
Yes
NC_017301.1
[26]
FRC41
Ovis
2.33791
Yes
NC_014329.1
[27]
I19
Ovis
2.33773
Yes
NC_017303.1
[28]
PAT10
Ovis
2.33532
Yes
NC_017305.1
[29]
42/02-A
Ovis
2.33761
Yes
NC_017306.1
[30]
3/99-5
Ovis
2.33794
Yes
NC_016781.1
[30]
267
Ovis
2.33763
Yes
NC_017462.1
[31]
P54B96
Ovis
2.33794
Yes
NC_017031.1
[32]
CIP5297
Equi
2.32059
Yes
NC_017307.1
[33]
1/06 -A
Equi
2.27912
Yes
NC_017308.1
[34]
316
Equi
2.31041
Yes
NC_016932.1
[35, 36]
258
Equi
2.36982
Yes
NC_017945.1
[37]
162
Equi
2.29346
Yes
NC_018019.1
[38]
31
Equi
2.38969
Yes
NC_017730.1
[39]
262
Equi
2.32575
Yes
NZ_CP012022.1
—
MB20
Equi
2.36309
Yes
JPUV01
[40]
E19
Equi
2.36796
Unknown
NZ_CP012136.1
—
CCUG27541
Equi
2.37942
Unknown
JPJB01
[41]
aBiochemical test.


### 2.2.narKGHJIOperon Analysis

The families of proteins as well as the conserved protein domains present in the operonnarKGHJI and adjacent genes were analyzed using InterProScan database (https://www.ebi.ac.uk/interpro). Prediction of transmembrane helices in protein was performed using TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). The similarity analysis of genes involved in the nitrate reduction pathway was performed using the UniProtKB database in category BLASTp. Clustal Omega was used to multiple sequence alignment between operonnarKGHJIofC. pseudotuberculosis biovar Equi and homologous sequences of multiple organisms (http://www.ebi.ac.uk/Tools/msa/clustalo/), and Jalview was used to analyze similarity (http://www.jalview.org/) among these sequences.


## 2.1. Identification of Nitrate Reductase Genes, Metabolic Pathways, and Nitrate Reduction Enzymes

C. pseudotuberculosis genomes Table 1 were obtained from the NCBI GenBank. The sequence and arrangement analysis of nitrate reductase genes innarKGHJI operon was performed using the Artemis software (http://www.sanger.ac.uk/Software/Artemis) and manual curation based annotations are performed. Pathway Tools software developed by SRI International [45] and MetaCyc database [46] were used for computational prediction of metabolic pathways inC. pseudotuberculosis.Table 1
Strains used in phenotypic and genotypic analysis regarding the nitrate reductase activity.
Strain
Biovar
Genome size (MB)
Nitrate testa
NCBI access
Ref
1002
Ovis
2.33511
Yes
NC_017300.1
[26]
C231
Ovis
2.32821
Yes
NC_017301.1
[26]
FRC41
Ovis
2.33791
Yes
NC_014329.1
[27]
I19
Ovis
2.33773
Yes
NC_017303.1
[28]
PAT10
Ovis
2.33532
Yes
NC_017305.1
[29]
42/02-A
Ovis
2.33761
Yes
NC_017306.1
[30]
3/99-5
Ovis
2.33794
Yes
NC_016781.1
[30]
267
Ovis
2.33763
Yes
NC_017462.1
[31]
P54B96
Ovis
2.33794
Yes
NC_017031.1
[32]
CIP5297
Equi
2.32059
Yes
NC_017307.1
[33]
1/06 -A
Equi
2.27912
Yes
NC_017308.1
[34]
316
Equi
2.31041
Yes
NC_016932.1
[35, 36]
258
Equi
2.36982
Yes
NC_017945.1
[37]
162
Equi
2.29346
Yes
NC_018019.1
[38]
31
Equi
2.38969
Yes
NC_017730.1
[39]
262
Equi
2.32575
Yes
NZ_CP012022.1
—
MB20
Equi
2.36309
Yes
JPUV01
[40]
E19
Equi
2.36796
Unknown
NZ_CP012136.1
—
CCUG27541
Equi
2.37942
Unknown
JPJB01
[41]
aBiochemical test.


## 2.2.narKGHJIOperon Analysis

The families of proteins as well as the conserved protein domains present in the operonnarKGHJI and adjacent genes were analyzed using InterProScan database (https://www.ebi.ac.uk/interpro). Prediction of transmembrane helices in protein was performed using TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). The similarity analysis of genes involved in the nitrate reduction pathway was performed using the UniProtKB database in category BLASTp. Clustal Omega was used to multiple sequence alignment between operonnarKGHJIofC. pseudotuberculosis biovar Equi and homologous sequences of multiple organisms (http://www.ebi.ac.uk/Tools/msa/clustalo/), and Jalview was used to analyze similarity (http://www.jalview.org/) among these sequences.


## 3. Results and Discussion

### 3.1. Correlation between Nitrate Reduction Phenotype and Genotype

NineteenC. pseudotuberculosis strains that had genome information in GenBank are presented Table 1. To perform the phenotyping analysis, 18 strains that were sequenced in our lab were used. All these strains were biochemically tested in order to verify their ability of nitrate reduction; see Table 1. Among theC. pseudotuberculosisstrains, 31, 258, 262, and MB20 strains showed concordance between phenotypic and genotypic results. Strains 1/06-A, 316, 162, and CIP52.97 are found positive phenotype for reduction of nitrate; however they do not have genes involved in nitrate reduction in their genome.


### 3.2. Genes Involved in Respiratory Nitrate Reductase Metabolic Pathway

We first compared the autoassigned pathways by the Pathologic software with the pathways in databases such as KEGG (http://www.genome.jp/kegg/) and resolved possible discrepancies using manual curation. Comparative genome analysis revealed thatC. pseudotuberculosisbiovar Equi possessnarKGHJI gene clusters that participate via the respiratory anaerobic process of the nitrate reduction similarly toE. coli(Figure S8) [47]. TheC. pseudotuberculosis narKGHJI gene cluster showed great similarity with the protein sequences found in other Actinomycetes, such asC. diphtheriae,C. glutamicum, andM. tuberculosis (Table 2). All biovar Ovis strains do not present any gene of thenarKGHJI operon in their genomes (Figure 1).Table 2
Similarity sequence analysis ofnarKGHJI gene clusters of C. pseudotuberculosis.
C. pseudotuberculosis protein
Species
Length (aa)
ID (%)
Similarity (%)
Access number/UniProtKB
Nitrate reductase alpha subunit (NarG)—1240aa
Corynebacterium diphtheriae
1,240
97
99
Q6NJA9
Corynebacterium glutamicum
1,248
77
87
Q8NR68
Mycobacterium tuberculosis
1,232
56
70
P9WJQ3
Escherichia coli
1,247
46
64
P09152
Nitrate reductase beta subunit (NarH)—533aa
Corynebacterium diphtheriae
533
95
98
H2G670
Corynebacterium glutamicum
531
79
89
S5Y1U5
Mycobacterium tuberculosis
538
64
75
A0A049E025
Escherichia coli
512
54
72
P11349
Nitrate reductase gamma subunit (NarI)—259aa
Corynebacterium diphtheriae
259
96
98
H2HVN2
Corynebacterium glutamicum
259
75
89
S5YHX1
Mycobacterium tuberculosis
241
48
68
Q7D8Q6
Escherichia coli
225
28
51
P11350
Nitrate reductase chaperone (NarJ)—209aa
Corynebacterium diphtheriae
209
89
96
H2H309
Corynebacterium glutamicum
228
63
80
Q6M5Z0
Mycobacterium tuberculosis
206
37
49
Q7D8Q7
Escherichia coli
236
30
46
P0AF26
Nitrate/nitrite transporter (NarK)—443aa
C. diphtheriae
443
93
97
H2HGI1
C. glutamicum
445
66
78
I0LIN6
M. tuberculosis
395
22
35
P9WJY7
E. coli
463
33
51
P10903Figure 1
Graphical circular map showing BLAST between nineteenC. pseudotuberculosis strains. (a) From center to the outside: GC content in black and GC skew in pink and green; in red and yellow Equi strains and in blue Ovis strains. From the inner to outer circle on (a) and (b): the biovarEqui strains are the following: 258, 31, 262, MB20, E19, CCUG27541, CIP52.97, 1/06-A, 316, and 162, and the biovar Ovis strains are the following: 1002, C231, FRC41, I19, PAT10, 42/02-A, 3/99-5, 267, and P54B96. (b) Zoom to the nitratelocus, absent in the biovarEqui strains CIP52.97, 1/06 –A, 316, and 162 and all biovar Ovis strains.
(a)
(b)The nitrate locus that containsnarKGHJI operon inC. pseudotuberculosis is composed of gene cluster encoding the molybdopterinsmoeB, moaE, molB, molA, moeY, moaC, moeA, andmoa(Figure 2).Molybdopterin is a cofactor that is indispensable for activity of nitrate reductase [48]. The organization and conservation of thenarKGHJI gene cluster was assessed amongC. pseudotuberculosisandC. diphtheriae,C. glutamicum, M. tuberculosis and,E. coli.The analysis showed thatnarKGHJI gene cluster is conserved among these species; however gene orientation of the five genes is only conserved amongC. pseudotuberculosis, C. diphtheriae, andC. glutamicum.In addition, inC. pseudotuberculosis we foundnarT gene is located at the upstream of thenarKGHJI cluster. ThenarT gene encodes a nitrate transporter and is not present inE. coli andC. glutamicum.Figure 2
Nitratelocus fromC. pseudotuberculosis biovar Equi. (a) Thislocuscontains the following: genes encoding the nitrate reductasenarK,narG,narH,narJ, andnarI, and the genes encoding the molybdopterinsmoeB, moaE, molB, molA, moeY, moaC, moeA, andmoaA. Insertion betweenansA andrpsH: genes encoding the nitrate reductase are lacking in nitrate-negativeC. pseudotuberculosisbiovar Ovis strains. Arrows represent open reading frames and their orientations. (b) Bacterial nitrate respiration. The NarG and NarH bind to the membrane via the interaction between a hydrophobic patch of NarH and NarI which is buried within the membrane. The NarJis a chaperone that is involved in folding, maturation, and molybdenum cofactor insertion of nitrate reductase.
(a)
(b)


### 3.3. Enzymes Involved in Electron Transport Chain

We found conserved functional domains of the genes present in thenarGHIcomplex (involved in electron transport chain) inC. pseudotuberculosisbiovar Equi (Table 3). To infer the functionality ofnarGHI complex, we used tridimensional structure fromE. coli (PDB accession 1Q16) as a model fornar genes, as the structure of these enzymes is resolved in these bacteria.Table 3
Residues analyzed regarding the conservation between thenarGHI protein sequences of E. coli and C. pseudotuberculosis.
E. coli
C. pseudotuberculosis
Conservation (%)
Function
REF
Residues NarG
∗
His49(H)
His55
100
Mo ion coordination
[42]
Cys53(C)
Cys 59
100
Mo ion coordination
[42]
Cys57(C)
Cys 63
100
Mo ion coordination
[42]
Cys92(C)
Cys 98
100
Mo ion coordination
[42]
Asp222 (D)
Asp 228
100
Mo-bisMGD ligand
[42]
Val 578 (V)
Val 584
100
Left relatively hydrophobic environment to allow binding within the active site, with one face of the side chain carboxylate exposed of Asp222. Optimal interaction with the Mo of the cofactor
[43]
Tyr 220 (Y)
Tyr 226
100
Left relatively hydrophobic environment to allow binding to within the active site, with one face of the side chain carboxylate exposed of Asp222. Allowing optimal interaction with the Mo of the cofactor
[43]
His1092(H)
His 1099
100
Form a hydrogen bond network that links the solvent interface withA
s
n
A
52 and could be important for structural integrity and/or proton delivery to the active site
[42]
His1098(H)
His 1105
100
Form a hydrogen bond network that links the solvent interface withA
s
n
A
52 and could be important for structural integrity and/or proton delivery to the active site
[42]
His1163(H)
His 1170
100
Form a hydrogen bond network that links the solvent interface withA
s
n
A
52 and could be important for structural integrity and/or proton delivery to the active site
[42]
Arg 94(R)
Arg 100
100
Forms a hydrogen bond with the Cys92 ligand of FS0
[43]
Residues NarH
∗
Cys196 (C)
Cys 196
100
Binding of iron atoms of FS4 cluster[
3
F
e
-
4
S
]
[44]
Cys217(C)
Cys 217
100
Binding of iron atoms of FS4 cluster[
3
F
e
-
4
S
]
[44]
Cys223(C)
Cys 223
100
Binding of iron atoms of FS4 cluster[
3
F
e
-
4
S
]
[44]
Cys184(C)
Cys 184
100
Binding of iron atoms of FS3 cluster[
4
F
e
-
4
S
]
[44]
Cys187(C)
Cys 187
100
Binding of iron atoms of FS3 cluster[
4
F
e
-
4
S
]
[44]
Cys192(C)
Cys 192
100
Binding of iron atoms of FS3 cluster[
4
F
e
-
4
S
]
[44]
Cys 227(C)
Cys 227
100
Binding of iron atoms of FS3 cluster[
4
F
e
-
4
S
]
[44]
Cys26(C)
Cys 26
100
Binding of iron atoms of FS2 cluster[
4
F
e
-
4
S
]
[44]
Cys244(C)
Cys 244
100
Binding of iron atoms of FS2 cluster[
4
F
e
-
4
S
]
[44]
Cys247(C)
Cys 247
100
Binding of iron atoms of FS2 cluster[
4
F
e
-
4
S
]
[44]
Cys259(C)
Cys 259
100
Binding of iron atoms of FS2 cluster[
4
F
e
-
4
S
]
[44]
Cys16(C)
Cys 16
100
Binding of iron atoms of FS1 cluster[
4
F
e
-
4
S
]
[44]
Cys19(C)
Cys 19
100
Binding of iron atoms of FS1 cluster[
4
F
e
-
4
S
]
[44]
Cys22(C)
Cys 22
100
Binding of iron atoms of FS1 cluster[
4
F
e
-
4
S
]
[44]
Cys263 (C)
Cys 263
100
Binding of iron atoms of FS1 cluster[
4
F
e
-
4
S
]
[44]
Residues NarI
∗
His66 (H)
His66
100
Iron atoms coordination of hemeb
D
[43]
His187 (H)
His190
100
Iron atoms coordination of hemeb
D
[43]
His56 (H)
His56
100
Iron atoms coordination of hemeb
P
[43]
His205 (H)
His208
100
Iron atoms coordination of hemeb
P
[43]
Arg112
Arg113
100
Hydrogen bond network electron transfer between the redox centerb
P, FS4, and NarG
[43]
Arg202
Arg205
100
Hydrogen bond network electron transfer between the redox centerb
P, FS4, and NarG.
[43]
Ser39
Ser39
100
Hydrogen bond network electron transfer between the redox centerb
P, FS4, and NarG.
[43]
Ser40
Ser40
100
Hydrogen bond network electron transfer between the redox centerb
P, FS4, and NarG.
[43]
Tyr213(Y)
Tyr216
100
Involved in electrostatic interactions and hydrogen bond, are involved in the formation of NarGHI heterotrimer
[43]
Arg216(R)
Arg219
100
Involved in electrostatic interactions and hydrogen bond, are involved in the formation of NarGHI heterotrimer
[43]
Arg222(R)
Arg225
100
Involved in electrostatic interactions and hydrogen bond, are involved in the formation of NarGHI heterotrimer
[43]
Ser201(S)
Ser204
100
A strong link at Ser201 of NarI allows distinctly shorter distances between the hemes in NarI than the distances observed between the hemes in the cytochrome bc1 complex
[43]
∗Shown is the position of the residues at E. coli and C. pseudotuberculosis sequences and as well as the function of each residue in the E. colinarGHI structure.In thenarGHI complex in Figure 2, thenarG gene is a member of a superfamily of enzymes that use a Mo-bisMGD cofactor (bisMGD) for their catalytic activity. Residues present in the active site of the molybdenum atom (Mo) are highly conserved in NarG subunits of Gram-negative and Gram-positive bacterial species [21]. The NarH belongs to the superfamily of electron transfer subunit (ferredoxins) of bacterial oxidoreductases. NarH has three clusters [
4
F
e
-
4
S
] (FS1, FS2, and FS3) and a [
3
F
e
-
4
S
] cluster (FS4) [21, 42, 44]. The general structure of NarH inE. coli is composed of a central region of a linker region that is inserted between the two subdomains in coordination of the iron-sulfur clusters and forms an extended connection between the NarG and NarH subunits and an extended C-terminus. InC. pseudotuberculosis it was observed that the nine cysteine residues responsible for coordinating three clusters [
4
F
e
-
4
S
] and one cluster [
3
F
e
-
4
S
] are conserved in all analyzed sequences. NarI is the transmembrane subunit that anchors NarGH to the cytoplasmic side of the membrane and provides the connection between the quinone and the site of oxidation, referred to as “Site Q.” This subunit coordinates two hemes, one of which is located towards the side of cytoplasmic end of NarI and is referred to as the proximal heme (heme b
P), while the other is located towards the periplasmic side of the same and is referred to as the distal heme (heme b
D). Such hemes act as mediators in electron transfer from Site Q to the clusters [Fe-S] in NarH. NarI ofC. pseudotuberculosis presented five transmembrane helices S1 Figure, which is in agreement with the literature when compared to theE. coli crystal structure [49, 50]. This C-terminal segment presents highly conserved residues in the family NarI proteins and also is present inC. pseudotuberculosis.It is interesting to note that NarI is considerably preserved in relation to its primary sequence between orthologous proteins found in species of theCorynebacterium genus, particularly in relation toC. diphtheriae, which has 96% identity and 98% similarity. These results suggest that the enzymatic complexnarGHI is probably very similar with respect to its three dimensional structure among the species ofC. pseudotuberculosis biovar Equi andC. diphtheriae in view of the high identity found among NarG, NaH, and NarI subunits of these species. TheC. pseudotuberculosis biovar Equi presents 48% and 43% identity in relation to the orthologous proteins fromM. tuberculosis andB. subtilis, respectively.


### 3.4. NarJ (Molybdenum-Cofactor-Assembly Chaperone)

The NarJ is a chaperone that is involved in folding, maturation, and molybdenum cofactor insertion of nitrate reductase inE. coli [51, 52]. The similarity values found betweenC. pseudotuberculosisbiovar Equi and other sequences of orthologous proteins by BLAST, including the genusCorynebacterium, were slightly lower than those found in other proteins (NarG, NarH, and NarI) that form the three subunits of the enzyme nitrate reductase (Table 2). However,narJremained conserved betweenC. pseudotuberculosisbiovar Equi strains, and results of biochemical tests (Table 1) indicate that its function has remained preserved, since NarJ is important for nitrate reductase expression in others organisms [53].


### 3.5. Nitrate and Nitrite Transporters

The nitrate and nitrite export is mediated by nitrate and nitrite transporters that are members of the major facilitator superfamily (MFS) [54]. In bacteria such carriers are thenarK family represented by NarK and NarU proteins inE. colithat transport both nitrate and nitrite [47]. However, NarK is expressed at higher levels than NarU and it remains unclear till date if NarK is a nitrate/nitrite antiporter or symporter [55]. InC. pseudotuberculosisbiovar Equi, we found that the two genesnarK andnarT are responsible for coding two probable nitrate and nitrite carriers and inC. pseudotuberculosisbiovar Equi is present at the upstream of thenarGHJIoperon (Figure 2). Both proteins are found to be composed of 12 transmembrane helices similar to other members of the superfamily of bacterial transporters; see S2 and S3 Figures. InC. glutamicum thenarK is in close proximity tonarG, narH, narJ,andnarI, which together form thenarKGHJIoperon [56], but inE. coli,narGHJI operon is transcribed individually andnarT is upstream of the operonnarKGHJI[57]. Since we did only the in silico analysis, we cannot state the precise transcription and regulation of these genes in vivo. We presume that thisnarKGHJI cluster inC. pseudotuberculosisbiovar Equi may be regulated together as found inC. glutamicum. However, experimental confirmation is required.


### 3.6. Nitrite Reductase inC. pseudotuberculosis

Genes inC. pseudotuberculosis are found to encode two different types of nitrite reductase enzymes. One is the nitric oxide-forming enzyme that is encoded bynirK gene and shown to be a copper dependent enzyme. The second groups of enzymes are ammonium nitrite reductase that are encoded bynrfA gene that encodes catalytic subunit andnrfH that encodes for a membrane associated electron transfer subunit. In allC. pseudotuberculosisstrains we found NrfA and NrfH subunits of nitrite reductase NrfAH complex that catalyze the reduction of nitrite to ammonium. BesideC. pseudotuberculosis, such genes are found only inC. ulcerans and are absent in all other species from this genus. InC. pseudotuberculosis, the two genesnrfA andnrfH are located in a “cluster” of genes, S4 Figure, responsible for coding biogenesis system I proteins cytochrome c that appears to be a common feature among bacterial genomes.C. pseudotuberculosis also has a nitrite reductase enzyme likely dependent on copper (CuNIR). The gene for CuNIR is nirK that encodes a protein composed of 882 residues and is conserved in all strains ofC. pseudotuberculosis; see Figure S5 of the Supplementary Material available online at https://doi.org/10.1155/2017/9481756. The sequence similarity analysis ofnirK was performed using the BLAST and it was observed that such protein has an N-terminal region of about 500 amino acids, S6 Figure, compared to the most homologous proteins that have been experimentally well characterized [58–61]. Within the genusCorynebacterium, the CuNIR proteins are found to have similar sizes to that ofC. pseudotuberculosis, such asCorynebacterium durum (WP_006063127.1) andCorynebacterium vitaeruminis (WP_051483598.1), with 40% identity between the sequences; see Table S1. Furthermore, it was found that the N-terminal region of about 500 residues is probably composed of 13 transmembrane helices inC. pseudotuberculosis, S7 Figure, which indicates that it can be a membrane associated protein. To verify the residues conservation, we usedG. kaustophilus CuNIR proteins as a reference sequence because its crystal structure has recently been determined (pdb∣3WIA∣A). All residues responsible for binding to the copper atoms and the catalytic site were analyzed. It is observed that the C-terminal region is 100% conserved inC. pseudotuberculosis; see S6 Figure.


### 3.7. Reconstruction ofC. pseudotuberculosis Biovar Equi Metabolic Network

A metabolic network reconstruction is assembled piece-by-piece by compiling data on known enzymes, and annotated genome together with data from the literature is used to assemble a network reconstruction [62]. InC. pseudotuberculosis after the similarity analysis we observed that genomic sequences participating in the nitrate reduction pathway are conserved, and the residues were analyzed, indicating a possible conserved function. Thus we can infer thatC. pseudotuberculosis biovar Equi is different from that occurring inC. glutamicum, where nitrate is excreted out of the cells as the major end product under the conditions of anaerobic respiration, and nitrate and nitrite are not used as sources of nitrogen for aerobic or anaerobic growth [18] soC. pseudotuberculosis biovar Equi has a likely route of denitrification composed of the nitrate reductase Nar, nitrite reductase dependent copper, and nitric oxide reductase quinol-dependent, and this is a way of incomplete denitrification, in which nitrous oxide is the final product, and, therefore, there is no presence of nitrous oxide reductase enzyme and subsequent reduction to nitrogen gas; see Figure 3. FurthermoreC. pseudotuberculosis biovar Equi also shows the pathway of ammonification in dissimilatory nitrate reduction to ammonium, what has been proposed as catalyzed by the enzyme nitrate reductase Nar and nitrite reductase complex NrfAH, a membrane-associated cytochrome; see Figure 3.E. coli not only reduce nitrite to ammonia but also catalyze the reduction of nitrite to NO, which can be used as an electron acceptor for anaerobic respiration [63]. The presence of enzymes and pathways of nitrate reduction proposals was also compared between biovar Ovis and biovar Equi strains and with respect to enzymes, the main difference was the presence of the nitrate locus that contains the genes encoding the molybdopterin and the genes encoding the nitrate reductase anda region situated upstream of theansA gene and this locus has a 19,606 pb length.Figure 3
Model building of nitratereductase fromC. pseudotuberculosis biovar Equi. Showed here is respiratory nitrate reduction to nitrite; incomplete denitrification of nitrite in which nitrous oxide is the final product; and Nrf-dependent ammonification.


## 3.1. Correlation between Nitrate Reduction Phenotype and Genotype

NineteenC. pseudotuberculosis strains that had genome information in GenBank are presented Table 1. To perform the phenotyping analysis, 18 strains that were sequenced in our lab were used. All these strains were biochemically tested in order to verify their ability of nitrate reduction; see Table 1. Among theC. pseudotuberculosisstrains, 31, 258, 262, and MB20 strains showed concordance between phenotypic and genotypic results. Strains 1/06-A, 316, 162, and CIP52.97 are found positive phenotype for reduction of nitrate; however they do not have genes involved in nitrate reduction in their genome.


## 3.2. Genes Involved in Respiratory Nitrate Reductase Metabolic Pathway

We first compared the autoassigned pathways by the Pathologic software with the pathways in databases such as KEGG (http://www.genome.jp/kegg/) and resolved possible discrepancies using manual curation. Comparative genome analysis revealed thatC. pseudotuberculosisbiovar Equi possessnarKGHJI gene clusters that participate via the respiratory anaerobic process of the nitrate reduction similarly toE. coli(Figure S8) [47]. TheC. pseudotuberculosis narKGHJI gene cluster showed great similarity with the protein sequences found in other Actinomycetes, such asC. diphtheriae,C. glutamicum, andM. tuberculosis (Table 2). All biovar Ovis strains do not present any gene of thenarKGHJI operon in their genomes (Figure 1).Table 2
Similarity sequence analysis ofnarKGHJI gene clusters of C. pseudotuberculosis.
C. pseudotuberculosis protein
Species
Length (aa)
ID (%)
Similarity (%)
Access number/UniProtKB
Nitrate reductase alpha subunit (NarG)—1240aa
Corynebacterium diphtheriae
1,240
97
99
Q6NJA9
Corynebacterium glutamicum
1,248
77
87
Q8NR68
Mycobacterium tuberculosis
1,232
56
70
P9WJQ3
Escherichia coli
1,247
46
64
P09152
Nitrate reductase beta subunit (NarH)—533aa
Corynebacterium diphtheriae
533
95
98
H2G670
Corynebacterium glutamicum
531
79
89
S5Y1U5
Mycobacterium tuberculosis
538
64
75
A0A049E025
Escherichia coli
512
54
72
P11349
Nitrate reductase gamma subunit (NarI)—259aa
Corynebacterium diphtheriae
259
96
98
H2HVN2
Corynebacterium glutamicum
259
75
89
S5YHX1
Mycobacterium tuberculosis
241
48
68
Q7D8Q6
Escherichia coli
225
28
51
P11350
Nitrate reductase chaperone (NarJ)—209aa
Corynebacterium diphtheriae
209
89
96
H2H309
Corynebacterium glutamicum
228
63
80
Q6M5Z0
Mycobacterium tuberculosis
206
37
49
Q7D8Q7
Escherichia coli
236
30
46
P0AF26
Nitrate/nitrite transporter (NarK)—443aa
C. diphtheriae
443
93
97
H2HGI1
C. glutamicum
445
66
78
I0LIN6
M. tuberculosis
395
22
35
P9WJY7
E. coli
463
33
51
P10903Figure 1
Graphical circular map showing BLAST between nineteenC. pseudotuberculosis strains. (a) From center to the outside: GC content in black and GC skew in pink and green; in red and yellow Equi strains and in blue Ovis strains. From the inner to outer circle on (a) and (b): the biovarEqui strains are the following: 258, 31, 262, MB20, E19, CCUG27541, CIP52.97, 1/06-A, 316, and 162, and the biovar Ovis strains are the following: 1002, C231, FRC41, I19, PAT10, 42/02-A, 3/99-5, 267, and P54B96. (b) Zoom to the nitratelocus, absent in the biovarEqui strains CIP52.97, 1/06 –A, 316, and 162 and all biovar Ovis strains.
(a)
(b)The nitrate locus that containsnarKGHJI operon inC. pseudotuberculosis is composed of gene cluster encoding the molybdopterinsmoeB, moaE, molB, molA, moeY, moaC, moeA, andmoa(Figure 2).Molybdopterin is a cofactor that is indispensable for activity of nitrate reductase [48]. The organization and conservation of thenarKGHJI gene cluster was assessed amongC. pseudotuberculosisandC. diphtheriae,C. glutamicum, M. tuberculosis and,E. coli.The analysis showed thatnarKGHJI gene cluster is conserved among these species; however gene orientation of the five genes is only conserved amongC. pseudotuberculosis, C. diphtheriae, andC. glutamicum.In addition, inC. pseudotuberculosis we foundnarT gene is located at the upstream of thenarKGHJI cluster. ThenarT gene encodes a nitrate transporter and is not present inE. coli andC. glutamicum.Figure 2
Nitratelocus fromC. pseudotuberculosis biovar Equi. (a) Thislocuscontains the following: genes encoding the nitrate reductasenarK,narG,narH,narJ, andnarI, and the genes encoding the molybdopterinsmoeB, moaE, molB, molA, moeY, moaC, moeA, andmoaA. Insertion betweenansA andrpsH: genes encoding the nitrate reductase are lacking in nitrate-negativeC. pseudotuberculosisbiovar Ovis strains. Arrows represent open reading frames and their orientations. (b) Bacterial nitrate respiration. The NarG and NarH bind to the membrane via the interaction between a hydrophobic patch of NarH and NarI which is buried within the membrane. The NarJis a chaperone that is involved in folding, maturation, and molybdenum cofactor insertion of nitrate reductase.
(a)
(b)


## 3.3. Enzymes Involved in Electron Transport Chain

We found conserved functional domains of the genes present in thenarGHIcomplex (involved in electron transport chain) inC. pseudotuberculosisbiovar Equi (Table 3). To infer the functionality ofnarGHI complex, we used tridimensional structure fromE. coli (PDB accession 1Q16) as a model fornar genes, as the structure of these enzymes is resolved in these bacteria.Table 3
Residues analyzed regarding the conservation between thenarGHI protein sequences of E. coli and C. pseudotuberculosis.
E. coli
C. pseudotuberculosis
Conservation (%)
Function
REF
Residues NarG
∗
His49(H)
His55
100
Mo ion coordination
[42]
Cys53(C)
Cys 59
100
Mo ion coordination
[42]
Cys57(C)
Cys 63
100
Mo ion coordination
[42]
Cys92(C)
Cys 98
100
Mo ion coordination
[42]
Asp222 (D)
Asp 228
100
Mo-bisMGD ligand
[42]
Val 578 (V)
Val 584
100
Left relatively hydrophobic environment to allow binding within the active site, with one face of the side chain carboxylate exposed of Asp222. Optimal interaction with the Mo of the cofactor
[43]
Tyr 220 (Y)
Tyr 226
100
Left relatively hydrophobic environment to allow binding to within the active site, with one face of the side chain carboxylate exposed of Asp222. Allowing optimal interaction with the Mo of the cofactor
[43]
His1092(H)
His 1099
100
Form a hydrogen bond network that links the solvent interface withA
s
n
A
52 and could be important for structural integrity and/or proton delivery to the active site
[42]
His1098(H)
His 1105
100
Form a hydrogen bond network that links the solvent interface withA
s
n
A
52 and could be important for structural integrity and/or proton delivery to the active site
[42]
His1163(H)
His 1170
100
Form a hydrogen bond network that links the solvent interface withA
s
n
A
52 and could be important for structural integrity and/or proton delivery to the active site
[42]
Arg 94(R)
Arg 100
100
Forms a hydrogen bond with the Cys92 ligand of FS0
[43]
Residues NarH
∗
Cys196 (C)
Cys 196
100
Binding of iron atoms of FS4 cluster[
3
F
e
-
4
S
]
[44]
Cys217(C)
Cys 217
100
Binding of iron atoms of FS4 cluster[
3
F
e
-
4
S
]
[44]
Cys223(C)
Cys 223
100
Binding of iron atoms of FS4 cluster[
3
F
e
-
4
S
]
[44]
Cys184(C)
Cys 184
100
Binding of iron atoms of FS3 cluster[
4
F
e
-
4
S
]
[44]
Cys187(C)
Cys 187
100
Binding of iron atoms of FS3 cluster[
4
F
e
-
4
S
]
[44]
Cys192(C)
Cys 192
100
Binding of iron atoms of FS3 cluster[
4
F
e
-
4
S
]
[44]
Cys 227(C)
Cys 227
100
Binding of iron atoms of FS3 cluster[
4
F
e
-
4
S
]
[44]
Cys26(C)
Cys 26
100
Binding of iron atoms of FS2 cluster[
4
F
e
-
4
S
]
[44]
Cys244(C)
Cys 244
100
Binding of iron atoms of FS2 cluster[
4
F
e
-
4
S
]
[44]
Cys247(C)
Cys 247
100
Binding of iron atoms of FS2 cluster[
4
F
e
-
4
S
]
[44]
Cys259(C)
Cys 259
100
Binding of iron atoms of FS2 cluster[
4
F
e
-
4
S
]
[44]
Cys16(C)
Cys 16
100
Binding of iron atoms of FS1 cluster[
4
F
e
-
4
S
]
[44]
Cys19(C)
Cys 19
100
Binding of iron atoms of FS1 cluster[
4
F
e
-
4
S
]
[44]
Cys22(C)
Cys 22
100
Binding of iron atoms of FS1 cluster[
4
F
e
-
4
S
]
[44]
Cys263 (C)
Cys 263
100
Binding of iron atoms of FS1 cluster[
4
F
e
-
4
S
]
[44]
Residues NarI
∗
His66 (H)
His66
100
Iron atoms coordination of hemeb
D
[43]
His187 (H)
His190
100
Iron atoms coordination of hemeb
D
[43]
His56 (H)
His56
100
Iron atoms coordination of hemeb
P
[43]
His205 (H)
His208
100
Iron atoms coordination of hemeb
P
[43]
Arg112
Arg113
100
Hydrogen bond network electron transfer between the redox centerb
P, FS4, and NarG
[43]
Arg202
Arg205
100
Hydrogen bond network electron transfer between the redox centerb
P, FS4, and NarG.
[43]
Ser39
Ser39
100
Hydrogen bond network electron transfer between the redox centerb
P, FS4, and NarG.
[43]
Ser40
Ser40
100
Hydrogen bond network electron transfer between the redox centerb
P, FS4, and NarG.
[43]
Tyr213(Y)
Tyr216
100
Involved in electrostatic interactions and hydrogen bond, are involved in the formation of NarGHI heterotrimer
[43]
Arg216(R)
Arg219
100
Involved in electrostatic interactions and hydrogen bond, are involved in the formation of NarGHI heterotrimer
[43]
Arg222(R)
Arg225
100
Involved in electrostatic interactions and hydrogen bond, are involved in the formation of NarGHI heterotrimer
[43]
Ser201(S)
Ser204
100
A strong link at Ser201 of NarI allows distinctly shorter distances between the hemes in NarI than the distances observed between the hemes in the cytochrome bc1 complex
[43]
∗Shown is the position of the residues at E. coli and C. pseudotuberculosis sequences and as well as the function of each residue in the E. colinarGHI structure.In thenarGHI complex in Figure 2, thenarG gene is a member of a superfamily of enzymes that use a Mo-bisMGD cofactor (bisMGD) for their catalytic activity. Residues present in the active site of the molybdenum atom (Mo) are highly conserved in NarG subunits of Gram-negative and Gram-positive bacterial species [21]. The NarH belongs to the superfamily of electron transfer subunit (ferredoxins) of bacterial oxidoreductases. NarH has three clusters [
4
F
e
-
4
S
] (FS1, FS2, and FS3) and a [
3
F
e
-
4
S
] cluster (FS4) [21, 42, 44]. The general structure of NarH inE. coli is composed of a central region of a linker region that is inserted between the two subdomains in coordination of the iron-sulfur clusters and forms an extended connection between the NarG and NarH subunits and an extended C-terminus. InC. pseudotuberculosis it was observed that the nine cysteine residues responsible for coordinating three clusters [
4
F
e
-
4
S
] and one cluster [
3
F
e
-
4
S
] are conserved in all analyzed sequences. NarI is the transmembrane subunit that anchors NarGH to the cytoplasmic side of the membrane and provides the connection between the quinone and the site of oxidation, referred to as “Site Q.” This subunit coordinates two hemes, one of which is located towards the side of cytoplasmic end of NarI and is referred to as the proximal heme (heme b
P), while the other is located towards the periplasmic side of the same and is referred to as the distal heme (heme b
D). Such hemes act as mediators in electron transfer from Site Q to the clusters [Fe-S] in NarH. NarI ofC. pseudotuberculosis presented five transmembrane helices S1 Figure, which is in agreement with the literature when compared to theE. coli crystal structure [49, 50]. This C-terminal segment presents highly conserved residues in the family NarI proteins and also is present inC. pseudotuberculosis.It is interesting to note that NarI is considerably preserved in relation to its primary sequence between orthologous proteins found in species of theCorynebacterium genus, particularly in relation toC. diphtheriae, which has 96% identity and 98% similarity. These results suggest that the enzymatic complexnarGHI is probably very similar with respect to its three dimensional structure among the species ofC. pseudotuberculosis biovar Equi andC. diphtheriae in view of the high identity found among NarG, NaH, and NarI subunits of these species. TheC. pseudotuberculosis biovar Equi presents 48% and 43% identity in relation to the orthologous proteins fromM. tuberculosis andB. subtilis, respectively.


## 3.4. NarJ (Molybdenum-Cofactor-Assembly Chaperone)

The NarJ is a chaperone that is involved in folding, maturation, and molybdenum cofactor insertion of nitrate reductase inE. coli [51, 52]. The similarity values found betweenC. pseudotuberculosisbiovar Equi and other sequences of orthologous proteins by BLAST, including the genusCorynebacterium, were slightly lower than those found in other proteins (NarG, NarH, and NarI) that form the three subunits of the enzyme nitrate reductase (Table 2). However,narJremained conserved betweenC. pseudotuberculosisbiovar Equi strains, and results of biochemical tests (Table 1) indicate that its function has remained preserved, since NarJ is important for nitrate reductase expression in others organisms [53].


## 3.5. Nitrate and Nitrite Transporters

The nitrate and nitrite export is mediated by nitrate and nitrite transporters that are members of the major facilitator superfamily (MFS) [54]. In bacteria such carriers are thenarK family represented by NarK and NarU proteins inE. colithat transport both nitrate and nitrite [47]. However, NarK is expressed at higher levels than NarU and it remains unclear till date if NarK is a nitrate/nitrite antiporter or symporter [55]. InC. pseudotuberculosisbiovar Equi, we found that the two genesnarK andnarT are responsible for coding two probable nitrate and nitrite carriers and inC. pseudotuberculosisbiovar Equi is present at the upstream of thenarGHJIoperon (Figure 2). Both proteins are found to be composed of 12 transmembrane helices similar to other members of the superfamily of bacterial transporters; see S2 and S3 Figures. InC. glutamicum thenarK is in close proximity tonarG, narH, narJ,andnarI, which together form thenarKGHJIoperon [56], but inE. coli,narGHJI operon is transcribed individually andnarT is upstream of the operonnarKGHJI[57]. Since we did only the in silico analysis, we cannot state the precise transcription and regulation of these genes in vivo. We presume that thisnarKGHJI cluster inC. pseudotuberculosisbiovar Equi may be regulated together as found inC. glutamicum. However, experimental confirmation is required.


## 3.6. Nitrite Reductase inC. pseudotuberculosis

Genes inC. pseudotuberculosis are found to encode two different types of nitrite reductase enzymes. One is the nitric oxide-forming enzyme that is encoded bynirK gene and shown to be a copper dependent enzyme. The second groups of enzymes are ammonium nitrite reductase that are encoded bynrfA gene that encodes catalytic subunit andnrfH that encodes for a membrane associated electron transfer subunit. In allC. pseudotuberculosisstrains we found NrfA and NrfH subunits of nitrite reductase NrfAH complex that catalyze the reduction of nitrite to ammonium. BesideC. pseudotuberculosis, such genes are found only inC. ulcerans and are absent in all other species from this genus. InC. pseudotuberculosis, the two genesnrfA andnrfH are located in a “cluster” of genes, S4 Figure, responsible for coding biogenesis system I proteins cytochrome c that appears to be a common feature among bacterial genomes.C. pseudotuberculosis also has a nitrite reductase enzyme likely dependent on copper (CuNIR). The gene for CuNIR is nirK that encodes a protein composed of 882 residues and is conserved in all strains ofC. pseudotuberculosis; see Figure S5 of the Supplementary Material available online at https://doi.org/10.1155/2017/9481756. The sequence similarity analysis ofnirK was performed using the BLAST and it was observed that such protein has an N-terminal region of about 500 amino acids, S6 Figure, compared to the most homologous proteins that have been experimentally well characterized [58–61]. Within the genusCorynebacterium, the CuNIR proteins are found to have similar sizes to that ofC. pseudotuberculosis, such asCorynebacterium durum (WP_006063127.1) andCorynebacterium vitaeruminis (WP_051483598.1), with 40% identity between the sequences; see Table S1. Furthermore, it was found that the N-terminal region of about 500 residues is probably composed of 13 transmembrane helices inC. pseudotuberculosis, S7 Figure, which indicates that it can be a membrane associated protein. To verify the residues conservation, we usedG. kaustophilus CuNIR proteins as a reference sequence because its crystal structure has recently been determined (pdb∣3WIA∣A). All residues responsible for binding to the copper atoms and the catalytic site were analyzed. It is observed that the C-terminal region is 100% conserved inC. pseudotuberculosis; see S6 Figure.


## 3.7. Reconstruction ofC. pseudotuberculosis Biovar Equi Metabolic Network

A metabolic network reconstruction is assembled piece-by-piece by compiling data on known enzymes, and annotated genome together with data from the literature is used to assemble a network reconstruction [62]. InC. pseudotuberculosis after the similarity analysis we observed that genomic sequences participating in the nitrate reduction pathway are conserved, and the residues were analyzed, indicating a possible conserved function. Thus we can infer thatC. pseudotuberculosis biovar Equi is different from that occurring inC. glutamicum, where nitrate is excreted out of the cells as the major end product under the conditions of anaerobic respiration, and nitrate and nitrite are not used as sources of nitrogen for aerobic or anaerobic growth [18] soC. pseudotuberculosis biovar Equi has a likely route of denitrification composed of the nitrate reductase Nar, nitrite reductase dependent copper, and nitric oxide reductase quinol-dependent, and this is a way of incomplete denitrification, in which nitrous oxide is the final product, and, therefore, there is no presence of nitrous oxide reductase enzyme and subsequent reduction to nitrogen gas; see Figure 3. FurthermoreC. pseudotuberculosis biovar Equi also shows the pathway of ammonification in dissimilatory nitrate reduction to ammonium, what has been proposed as catalyzed by the enzyme nitrate reductase Nar and nitrite reductase complex NrfAH, a membrane-associated cytochrome; see Figure 3.E. coli not only reduce nitrite to ammonia but also catalyze the reduction of nitrite to NO, which can be used as an electron acceptor for anaerobic respiration [63]. The presence of enzymes and pathways of nitrate reduction proposals was also compared between biovar Ovis and biovar Equi strains and with respect to enzymes, the main difference was the presence of the nitrate locus that contains the genes encoding the molybdopterin and the genes encoding the nitrate reductase anda region situated upstream of theansA gene and this locus has a 19,606 pb length.Figure 3
Model building of nitratereductase fromC. pseudotuberculosis biovar Equi. Showed here is respiratory nitrate reduction to nitrite; incomplete denitrification of nitrite in which nitrous oxide is the final product; and Nrf-dependent ammonification.


## 4. Conclusions

In this study, we found thatC. pseudotuberculosis strains 1/06-A, 316, 162, and CIP52.97 are able to do nitrate reduction but in their genomes they do not have genes associated with nitrate reduction, warning us about resequencing of these genomes. TheC. pseudotuberculosis nitrate locus is composed ofnarKGHJIgene cluster and by genes encoding the molybdopterinsmoeB,moaE,molB,molA,moeY,moaC,moeA, andmoa genes are similar to the other bacteria under Actinomycetes and all the biovar Ovis strains that lack the nitrate locus in their genomes. ThenarGHIcomplex inC. pseudotuberculosis biovar Equi shows conserved functional domains.narK andnarT genes inC. pseudotuberculosis biovar Equi encode two putative nitrate and nitrite carriers, respectively. Finally, thenirK, nrfA,andnrfH encoding nitrite reductase enzymes are conserved inC. pseudotuberculosis. Therefore, the study of pathogen metabolism can contribute to the identification of pathogen virulence, discovery of drug targets, host response, and generating hypotheses for experimental investigation.



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*Source: 9481756-2017-02-20.xml*