Introduction:

Rhodococcus:

The bacterial genus Rhodococcus is a member of the nocardia form actinomycetes and is
classified as a gram-positive, fully aerobic, highly in G+C content, somewhat acid-fast,
nonmotile, catalase-positive, or non-endospores pathogenic group of bacteria (Goodfellow and
Alderson, 1977). Rhodococcus belongs to the Corynebacterineae suborder, Actinobacteridae
family, Actinomycetales order, and Actinobacteria phylum (Barka et al., 2016). Among the other
well-known genera in the Corynebacterineae suborder are Mycobacteria and Nocardia. All
Corynebacterineae members, with the exception of Corynebacterium amycolatum, have a unique
cell wall made of mycolic acid that sets them apart from other Gram-positive bacteria.
Depending on the chemical composition of the cell wall, the genus Rhodococcus refers to the
Corynebacterium, Nocardia, and Mycobacterium (CMN) group, which may be recognised by its
chemotype IV peptidoglycan. The rhodococcal cell wall contains mycolic acids with lengths
ranging from 34 to 64 carbon atoms (Majidzadeh and Fatahi-Bafghi, 2018). In groundwater, soil,
marine, desert, and polar habitats, as well as in animals and plants, at least 57 species of
Rhodococcus have been identified (Majidzadeh and Fatahi-Bafghi, 2018);(Vázquez-Boland and
Meijer, 2019).

One of the biggest microbial groupings on Earth is the prototypical genus Rhodococcus. Due to
their diverse metabolic capabilities and biodegradative characteristics, many of the common
rhodococcal species are beneficial for biotechnology. The animal and human pathogen
Rhodococcus equi, a facultative parasite, has had its genome analysed (Letek et al., 2010). The
complex mixture of mycolic acids along with lipids that compose up this kind of gram-ve cell
wall creates a membrane with a repellent permeable barrier, providing the genus an inherent
resistance to various stresses and harsh surroundings, allowing rhodococci to survive in a wide
range of habitats (Majidzadeh and Fatahi-Bafghi, 2018);(Vázquez-Boland and Meijer, 2019).

Only R. fascians, which causes gall foliar infection in plants, and R. equi, which infects
numerous mammals, are hazardous rhodococci. The metabolic and catabolic diversity of
Rhodococcus species is quite diverse. As a result, many have come to be recognised as
promising candidates for biotechnological applications such biodegradation, biocatalysis,
biomedicine, and biosynthesis.Some of the most well-liked rhodococci biosynthetic products are
biosurfactants, triacylglycerols, carotenoids, and antibiotics (Cappelletti et al., 2020).
Rhodococci have long been used commercially to break down environmental pollutants. They
may degrade nitriles, aromatic and halogenated hydrocarbons, as well as other organic
compounds (Kim et al., 2018). The enormous diversity of catabolic and biosynthetic genes, as
well as the intricate regulatory systems that allow for adaptations to varied environments, all
contribute to the genus's high metabolic capacity and flexibility. After lateral gene transfer, most
of these genes emerged by recombination mechanisms, which are usually controlled by plasmids
(Letek et al., 2010b).
Rhodococcus equi

Rhodococcus equi, a high-G+C Gram-positive, facultative intracellular coccobacillus, parasitizes

macrophages and infects people and several animal species with pulmonary and extrapulmonary
pyogranulomatous diseases. Since being discovered by H. Magnusson in Sweden in 1923 as the
pathogen responsible for purulent bronchopneumonic illness in foals, R. equi has gained
widespread recognition in veterinary medicine as a significant horse pathogen (Vázquez-Boland
and Meijer, 2019). R. equi is widely distributed in soil, grows in herbivore faeces and the large
intestine, and likely spreads by faecal-oral cycling in the farm setting (Muscatello et al., 2007)
(Vazquez-Boland et al., 2013) (Vázquez-Boland and Meijer, 2019). It is likely that inhaling
airborne dust particles containing R. equi will result in lung infections (Muscatello et al., 2006;
Cohen et al., 2008); (Petry et al., 2017) (Vázquez-Boland and Meijer, 2019).
Rhodococcus equi is a disease that preys on immunocompromised individuals, such as AIDS
patients and transplant recipients. A important human pathogen, R. equi, has emerged as a result
of the HIV epidemic (Majidzadeh and Fatahi-Bafghi, 2018). Similar to other rhodococci that
accomplish fast ecological adaptation through transferable extrachromosomal replicons, the
pathogenicity of R. equi depends on a virulent plasmid (Cheng, 2022).

R. equi genome:

R. equi's strain 103S (= NCTC 13926 = DSM 104936), a representative horse clinical isolate, is
the only full and manually curated genome sequence that is currently accessible (Letek et
al., 2010) (Vázquez-Boland and Meijer, 2019). The reference 103S genome has 4,598 predicted
genes, a circular chromosome of 5.04 Mbp, and a G+C content of 68.8%. Its accession number is
FN563149.1. The virulence plasmid, which harbours the vap pathogenicity island (PAI), is a
second crucial element of the genome. It is an 80.6 kb circular plasmid in 103S with the name
pVAPA1037. The scarcity of DNA mobility genes or insertion sequences and lack of substantial
recombination suggest that the R. equi chromosome is genetically stable. It is likely under strong
selection since there are just a few pseudogenes (14 in 103S, the majority in areas that were
acquired horizontally) (Vázquez-Boland and Meijer, 2019).

R. equi is genetically homogenous and clonal, with a large core genome that accounts for around
80% of the genes in an isolate, according to comparative genomic investigations. With a 99.13%
Average Nucleotide Identity (ANI) and 100% 16S rDNA sequence identity, it is a clearly
characterised taxon. R. equi isolates radiate at a close genetic distance from one another in a
core-genome phylogenomic tree (0.001-0.002 substitutions per site), which is compatible with a
recent evolutionary origin and a quick clonal diversification from the common progenitor
(Vázquez-Boland and Meijer, 2019).

R. equi possesses an open pangenome, similar to many other bacteria. A major amount of the
accessory genome (60%) is only present in one or two isolates, accounting for the intraspecific
variability, even though non-core genes make up just around 20% of the gene content of each
strain. Gene gain/loss mechanisms and horizontal gene transfer (HGT) events contribute
significantly to the development of the R. equi genome (Letek et al., 2010) (Anastasi et al., 2016)
(Vázquez-Boland and Meijer, 2019). In R. equi, phages are widespread and probably have a
significant impact on HGT-driven genome plasticity (Vázquez-Boland and Meijer, 2019).

Core R. equi traits:

The majority of features thought to be crucial for R. equi biology and niche adaption are found in
the core genome, according to comparative genomic analyses (Anastasi et al., 2016). All
possible pathogenicity determinants discovered on the 103S chromosome are included in here,
especially several mycobacterial virulence gene homologs (Letek et al., 2010). The core genome
also contains genes that are tolerant to desiccation and oxidative stress, which are likely
necessary for life in dry soil and transmission by aerosolized dust. Additionally, there is a
conserved intrinsic resistome that contains several potential aminoglycoside
phosphotransferases, multidrug efflux pumps, and -lactamases. These most likely contribute to
R. equi's reported varied resistance to various antibiotics, as shown, for instance, with -lactams
and quinolones (Vázquez-Boland and Meijer, 2019).

The entire lack of phosphoenolpyruvate: carbohydrate transport system (PTS) components,

which is compatible with an essentially asaccharolytic metabolism, is a distinguishing feature of
R. equi. Only its closely related Rhodococcus defluvii, among the rhodococci, is deficient in the
PTS sugar transport system, indicating unique gene loss in the progenitor of the R. equi-R.
defluvii sublineage. The lack of PTS homologues is fairly unusual among Actinobacteria; two
notable instances are the obligate intracellular pathogen Tropheryma whipplei and
Mycobacterium TB, both of which are parasites of macrophages. This implies that in this
particular bacterial population, loss of PTS sugar transport may be linked to intracellular
parasitism adaption (Vázquez-Boland and Meijer, 2019).

In the R. equi core genome, potential non-PTS transporters for the sugars glucose (GlcP) and
ribose (RbsCB) have recently been discovered. Although utilisation of ribose and, in particular,
glucose by R. equi 103S is inefficient (and variable for the latter) in comparison to favoured
carbon sources like lactate or acetate, both permeases appear to be functioning (Letek et
al., 2010) (Anastasi et al., 2016). Since R. equi mostly uses short-chain organic acids and lipid
catabolism to ingest carbon, these two sugar transporters may occasionally improve "nutritional
fitness" in particular settings (Vázquez-Boland and Meijer, 2019).

The R. equi core genome also encodes a wide variety of -oxidation enzymes and lipases, in
addition to monocarboxylate and dicarboxylate transporters. Additionally, there are three full
mce (or "mammalian cell entry") systems that function as specialised lipid transport channel
mechanisms (Vázquez-Boland and Meijer, 2019). ICL facilitates the removal of acetylCoA
produced by fatty acid -oxidation or acetate oxidation from the TCA cycle intermediates needed
for gluconeogenesis and carbohydrate biosynthesis. This suggests that R. equi uses lipids as a
growth substrate in vivo, similar to the tubercle bacillus. Interestingly, R. equi has a putative
bifunctional D-xylulose 5-phosphate (X5P)/fructose 6-phosphate (F6P) phosphoketolase (Xfp),
despite not being a fermentative organism (Meile et al., 2001). By converting intermediates from
the glycolytic process and the pentose phosphate pathway (PPP) into acetyl phosphate (and
acetate/acetyl-CoA), this enzyme may offer flexibility in the metabolism of carbon and energy
(Vázquez-Boland and Meijer, 2019).

With a dedicated transporter (LldP) and factors for its conversion into acetate, either directly (L-
lactate monooxygenase) or via pyruvate and combined with pyruvate decarboxylation via
pyruvate dehydrogenase [cytochrome]), R. equi appears to be particularly well adapted for
growth on exogenous L-lactate. Along with having a NarK nitrate/nitrite transporter, a NarGHIJ
nitrate reductase, and a NirBD nitrite reductase, R. equi is also capable of denitrification. A
urease and an ATP-dependent urea carboxylase enable it to also grow with urea as the only
nitrogen source (Letek et al., 2010) ( Anastasi et al., 2016) (Vázquez-Boland and Meijer, 2019).

The disruption of the thiCD locus by an HGT island, which makes R. equi auxotrophic to
thiamin, is another essential characteristic. Apart from this, R. equi requires little in the way of
nutrients and can thrive with just the presence of inorganic N (such as ammonium chloride as a
source of carbon) and an organic acid. The nutritional and metabolic profile of R. equi, along
with its alkalophily (optimal growth between pH 8.5 and 10), may give it a competitive edge in
manure and the large intestine, its natural reservoirs, where there is easy access to thiamine and
lactate/short-chain fatty acid fermentation products derived from the microbiota. R. equi
possesses the capacity to use H2, generated by microbial metabolic activity, via a NiFe-type
hydrogenase, perhaps assisting in survival (Letek et al., 2010) ( Anastasi et al., 2016) (Vázquez-
Boland and Meijer, 2019).

Circular and linear genomes: a matter of size

Despite being monophyletic, it became clear from the identification of the entire 103S genome
sequence that Rhodococcus spp. varied in chromosomal structure. Covalently closed circular
chromosomes are found in R. equi 103S, R. erythropolis PR4, and R. opacus B4, whereas linear
chromosomes are found in R. jostii RHA1 and R. opacus B4. Surprisingly, not only do the four
species share a subgroup within the genus Rhodococcus, but R. erythropolis and R. jostii/R.
opacus also share a terminal clade and are members of sister sublineages. The only noticeable
variation between the four chromosomes is that R. jostii and R. opacus have somewhat bigger
sizes (around 7.25 Mb and 8 Mb, respectively), similar to Streptomyces spp., which likewise
have linear genomes. Therefore, rather than phylogenetic background, actinobacterial
chromosomal linearization seems to be a function of increasing size. This is similar to the
situation with rhodococcal plasmids, which tend to be linear over 100 kb regardless of the host
species (Letek et al., 2010) ( Anastasi et al., 2016) (Vázquez-Boland and Meijer, 2019).

Potential treatments as well as clinical manifestations

Equine infection

Pneumonia is estimated to be one of the most common causes of illness and mortality in foals
worldwide. R. equi is thought to be the most common culprit behind severe pneumonia in horses
(Giguère, 2017), despite the fact that a wide variety of germs can cause foal pneumonia. Between
1 and 6 months is the most typical age range for a foal to acquire R. equi pneumonia. For horses,
subacute to chronically purulent bronchopneumonia with abscessation is the most typical R. equi
infection (Vázquez-Boland and Meijer, 2019). Coughing, fever, sluggishness, and increased
respiratory stress are examples of clinical indications. Infected foals can occasionally develop
severe respiratory issues and pass away in a matter of hours or days (Jones et al., 2013).
According to Prescott (1991), up to 50% of the infected foals have disseminated the illness to
neighbouring areas. This is why enteric disorders like ulcerative intestine inflammation, which
induces lymphatic tissue inflammation, can coexist with R. equi pneumonia (Reuss et al., 2009).
Extrapulmonary conditions, however, can also arise even in the absence of pneumonia (Vázquez-
Boland and Meijer, 2019).

Human infection:

A male immunosuppressed patient with tumours in his lungs contracted the first R. equi infection
in humans in 1967 (GOLUB et al., 1967). R. equi infections in HIV patients have grown because
to the AIDS epidemic (Donisi et al., 1996). Immunosuppressive medications for other disorders
and organ transplants also raise the risk of R. equi diseases. Typically, Rhodococcus equi is
isolated and examined from the lungs of infected individuals. Most R. equi-infected AIDS
patients get necrotizing respiratory infections, that are similar to pulmonary TB, (Verville et al.,
1994) the most common clinical presentation of a human R. equi infection is its therapeutic
manifestation. R. equi infections in humans are still incredibly rare; they affect just 0.3% of
people with HIV. Infections brought on by more common bacteria like mycobacteria and
coryneform are different from this. R. equi infections are therefore frequently incorrectly
identified, which has a negative impact on clinical outcomes (Vázquez-Boland et al., 2013).

Non-equine animals infection:

Pigs and cattle are two more types of animals beyond horses that can get R. equi infection. R.
equi is frequently expelled by pigs whose submaxillary lymphatic system is affected by
granulomatous lymphoma. Disease in foals is different from this illness. Similar to how TB does,
infection with R. equi in cattle typically presents initially as an abscessation of the pulmonary
lymph nodes. Goats, cats, and dogs can occasionally contract R. equi. Clinical signs in these
animals can include, but are not limited to, vaginitis, hepatitis, joint infections, lymph node
abscesses, splenic abscesses, liver abscesses, and wound infections (Tkachuk-Saad et al., 1998)
( Takai et al., 2003).

Diagnostics Methods:

Ultrasonographic screening is the most common technique for early R. equi pneumonia
identification in many farms because it can reduce death rates and stop the development of
pathogenic R. equi (Muscatello, 2012b). The inclusion of virulence-associated genes and marker
genes in PCR analysis enhances both the accuracy and responsiveness of the clinical diagnosis
(Ocampo-Sosa et al., 2007). There is no reliable R. equi vaccine. The choice and effectiveness of
antibacterial medications are restricted by R. equi's intracellular reproduction. A macrolide plus a
rifampin such as azithromycin, erythromycin, or clarithromycin have been used in combination
for many years. Although the standard therapy is effective, R. equi resistance to macrolide or
rifampin has grown (Giguère et al., 2017). Most macrolide-resistant isolates have lately been
shown to be rifampin resistant. The increase of multi-drug resistant strains calls for the creation
of alternate therapeutic techniques. Growing understanding of the complex host-pathogen
interactions is likely to be beneficial for the development of efficient alternative therapies and/or
an R. equi vaccine (Álvarez-Narváez et al., 2021).

Toxin-Antitoxin (TA) Systems

The standard TA system has two parts: a toxin which can interfere with cellular function and an
antitoxin which serves as a countermeasure to the toxin. Escherichia coli, Mycobacterium, and
Salmonella exhibit virulence and chronic infections that are mediated by TA systems. Based on
the way that toxin and antitoxin interact, they are divided into six kinds. The antitoxin antisense
RNA interacts with the toxin mRNA in the type I TA system via an RNA-RNA pathway,
whereas the toxin and antitoxin interact via a protein-protein pathway in the type II TA system.
While toxin and antitoxin proteins compete for the same target in the type IV TA system, the
interaction between the toxic protein and antitoxin RNA inhibits the function of the toxic protein
in the type III TA system. The toxin mRNA is cut by the antitoxin protein in the type V TA
system. The antitoxin in the newly discovered type VI TA system is distinct from others in that it
acts as an adapter to promote toxin proteolysis. It has been hypothesised that the prevalence of
TA loci has arisen by horizontal gene transfer, a process by which TA systems effectively
integrate into the bacterial genome and play a role in both genetic stability and pathogenic
potential. The unstable antitoxin is broken down under unfavourable circumstances including
famine, bacteriophage assault, heat shock, and antibiotic stressors, allowing the toxin to target
crucial cellular functions like translation, transcription, and replication. The research of TA
systems in S. aureus is at a relatively early stage compared to that of TA systems in E. coli and
Mycobacterium (Habib et al., 2018).

Important physiological processes like DNA replication and translation are hampered by the
toxin. When the poison remains indefinitely active (without an antitoxin), the cells display a
severe growth restriction, which is a feature of TA toxins (Harms et al., 2018). It's possible that
TA toxins and other factors that control growth are what give bacteria antibiotic resistance
(Pontes and Groisman, 2020).

With 15 systems, the HigBA TA family has the most systems, followed by five RelBE TA
systems, six GCN5-related acetyltransferases (GNATs), and six GNATs. Other TA families
include transporter TA systems, abortive infection (Abi) TA systems, MazEF, VapBC, HicBA,
CcdBA, ParDE, HipBA, and those with a domain of uncertain function. We also ectopically
expressed a few toxin and antitoxin genes to evaluate the activities of their expected products
(Habib et al., 2018).

Escherichia coli research has been the main source of our knowledge on the subject, even though
other species have TA system homologs encoded in their genomes. For instance, the TA
Database reports that the E. coli K-12 strain possesses 31 potential TA toxins in its chromosomal
region, 26 of which have passed empirical testing. In contrast, only 4 of Salmonella enterica's
anticipated 18 TA systems have been tested in an experimental setting (Xie et al., 2018). Many
TA toxins, particularly those from the MazEFs and VapBCs families of TA systems, exhibit
ribonuclease (RNase) activity. For example, the MazeF toxins produced by Bacillus subtilis and
Staphylococcus aureus both cleave RNA at the location 5'-UACAU-3' (Park et al., 2011, Zhu et
al., 2009).

While their antitoxins did not exhibit any inhibitory effects, the majority of type II toxins did
decrease cell growth. We overexpressed the growth-inhibiting RelE, MazF, and HigB toxins.
Seven type I TA systems were found, including the previously identified type I toxins Fst and
TxpA. The bacterial cell wall was examined using transmission electron microscopy (TEM),
which showed that the TxpA and holin toxins promoted septation while the Fst toxin damaged
the cell wall. The toxin and antitoxin of a novel TA system that we discovered did not exhibit
any growth inhibition upon overexpression. The antitoxin may connect to the promoter and
autoregulate the operon in this novel TA system, allowing toxin and antitoxin to co-transcribe.
The antitoxin's ability to perform its job and the antitoxin's ability to attach to the promoter were
both blocked by the toxin protein (Habib et al., 2018).

Antitoxin is the corresponding antidote, and the toxin gene codes for a toxic protein. Families
and TA kinds are determined by the toxin and antitoxin sequence domains. The DNA-binding
domain of the transcriptional regulator, such as the helix-turn-helix (HTH) or ribbon-helix-helix
(RHH), is mostly present in the antitoxin. For instance, the most varied family of RelB antitoxins
in E. coli binds to the promoter region and autoregulates the TA operon. The most prevalent TA
family in Mycobacterium is VapBC, and the VapBC protein complex has the ability to
autoregulate the TA operon. A subfamily of the RelBE superfamily is the host inhibition of
growth (HigBA) TA family. The HigBA TA system is distinct from the other type II TA families
because it has a rare genomic annotation for type II TA systems—the toxin gene is located
upstream of the antitoxin. HigA antitoxin follows HigB as a ribonuclease in function, and the
two can combine to create a heterotetrameric complex. The toxin attacks the 50S ribosomal
subunit, whereas the antitoxin has an HTH-Xre domain and may attach to DNA (Habib et al.,
2018).

MazFEs-expressing cells' transcriptome study revealed no primary rRNA cleavage or elevated

ACA cleavage in mRNA leader regions (Culviner and Laub, 2018). But new study indicates that
MazFEs can cleave mRNAs and/or rRNA. The lone uncommon example in the genome of M.
tuberculosis is the tRNA-cleaving toxin MazF-mt9 (Schifano and Woychik, 2017, Schifano et
al., 2016).

The virulence-associated protein, or VapBC, has a higher incidence of tRNA breakage. The
conserved aspartic and glutamic acid residues that make up the enzyme's active are found in the
RNase-associated PilT N-terminal (PIN) domain of the VapBC family of toxins (Matelska et al.,
2017). Early biochemical characterizations of VapBCs family members were made using VapCs
in S enterica serovar Typhimurium LT2 chromosomes and a S. flexnerii plasmid (Winther and
Gerdes, 2011).
Methodology
Protein sequences representing toxins and antitoxins from diverse TA system families were
gathered from authoritative databases such as NCBI and UniProt. This dataset included
sequences from families like MazEF, HigBAs, ParDE, VapBC, Novel TA, and RelBE sourced
from E. coli and Mycobacterium as model organisms Additionally, sequences of proteins
containing PIN domains and previously documented toxin-antitoxin complexes were compiled
for comprehensive analysis.

BLASTP and tBLASTn Searches:

a. Database Preparation:

The genome sequence of R. equi was obtained and compiled into a local protein database.

b. BLASTP Searches:

Protein sequences from various TA system families were used as queries. BLASTP searches
were executed against the local R. equi protein database. Results were filtered based on stringent
criteria, including E-values and alignment scores, to identify significant hits. The identified hits
were validated using conserved domain analysis, ensuring their authenticity.

C. TBLASTN Searches

tBLASTn searches were conducted to explore genomic sequences, ensuring a thorough

investigation of both protein-coding and non-coding regions.

Genomic Island Analysis:

Using the Rhodococcus equi 103S genome, we conducted genomic island analysis to identify
potential TA systems. Genomic islands are regions in bacterial genomes often associated with
horizontal gene transfer and can harbor TA loci. We explored these islands for homologous
structures of toxin and antitoxin proteins.

Classification and Grouping:

Identified homologous sequences were manually curated, and TA systems were classified into
distinct families based on similarity scores, sequence alignments, and conserved domains.
Grouping was performed to categorize similar sequences, enhancing the accuracy of the
classification.

Distance Tree Analysis:

For the families ParDE, RelBE, and VapBC, multiple sequence alignments were conducted, and
evolutionary relationships were inferred using distance-based methods. Distance tree analysis
provided insights into the evolutionary history and relatedness of the identified TA systems.

Conserved Domain Analysis:

Conserved domain analysis was employed using databases such as Pfam and SMART to validate
the identified TA systems. This analysis ensured that the identified sequences contained
conserved domains characteristic of known TA systems, confirming their classification.

Genomic Structural Analysis:

In addition to sequence similarity, genomic structural analysis was performed independently. We

identified paired genes with comparable genomic structures, even if they lacked sequence
similarity to known TA systems. This approach enabled the discovery of novel TA loci.

Results
The standard toxin-antitoxin (TA) system, which is widely distributed in bacterial genomes,
consists of a stable toxin and an unstable antitoxin. Both the toxin and the antitoxin are
conjugative pairs of genes that co-transcribe, participate in a variety of cellular functions, and,
most critically, balance one another out. Each pathogen has a different percentage of the TA
system encoded in its DNA. Salmonella typhimurium carries 27, Mycobacterium carries 88, E.
coli carries 40, Pseudomonas aeruginosa has 26 TA systems (habib et al., 2018).

It was anticipated that a multi-method search for putative TA systems in the R. equi genome
would yield better results. To do this, we developed three strategies based on tested TA systems.
The NCBI BLASTP searches on the R. equi genome started with toxins and anti-toxins protein
sequences from the 5 key TA systems, families: MazEF, HigBAs, ParDE, VapBC, Novel TA
and RelBE. Firstly screened the Blastp, tBlastn for finding homologs, then genomic island
analysis for the prediction of TA system in Rhodococcus equi.

Both gram positive and gram negative species should be utilised to locate homologs. Second,
added to these list proteins containing PIN domains and toxin-antitoxin complexes identified in
past studies. In the R. equi genome, paired genes were discovered using a method that is
sequence independent. These genes are unrelated to well-known TA systems but have a
comparable genomic structure (Brown and Shaw, 2003).

Comphrensive Analysis:

In the present study, we searched the R. equi genome using a variety of approaches for possible
TA loci. While looking for TA genes, we discovered numerous potential TA systems with the
use of BLASTP. To improve the accuracy of the classification, TA systems of E. coli or
Mycobacterium were utilised as a modal or individually curated, grouping them into several
families.

We employed the TA systems of E. coli and Mycobacterium as modal and manually curated
each TA system, classifying them into several families to increase the classification's accuracy.
On the assumption of software prediction and conserved domain analysis, we discovered type I,
type II, type III, type IV, and type V TA systems in Rhodococcus equi.

Antitoxin protein Toxin protein

Helix-turn helix domain
DNA binding protein
Ribbon-helix-helix domain
Xre-transcriptional
Homologues of anti-toxin
interferase and nuclease domain
Inhibitor of transcription and translation
Pathogenesis related protein
Homologus of toxin

Databases
NCBI
Ensemble

Families Types
MazEF Type I
HigBA Type II
ParDE Type III
VapBC Type IV
RelBE Type V
Novel TA

Figure 1: Description of selected TA families screening procedures and TA systems are shown

Protein BLAST(Blastp):

In the present study, HigBA, Novel TA and MazEF shown no significant similarities with
Rhodococcus equi. While ParDE show 2 similarities, RelBE have 100 and VapBC have 8
similarities found with Rhodococcus equi

ParDE:
 There are two similarities found with Rhodococcus equi. Sequence producing significant
alignment.

The graphical summary shows 80-120 alignment scores. The graphical view of the similarities in
ParDE is:

Distance tree view of ParDE is:

RelBE:

There are 100 similarities found with Rhodococcus equi. Sequence producing significant
alignment.

The graphical summary shows 50-80 and 80-120 alignment score. The Graphical view of
similarities in RelBE is :
Distance tree view of RelBE is:

VapBC:

There are 8 sequence similarties found with Rhodococcus equi. Sequence producing significant
alignment.

The graphical summary show 80-299 alignment score. The graphical view of similarities in
VapBC is:
Distance tree view of VapBC is:

Tblastn:

In the current study, higBA, MazEF, Novel TA, ParDE and VapBC have no significant
simialrties are found with Rhodococcus equi while RelBE shows 3 similarities with
Rhodococcus equi

RelBE:

There are three similarities are found with Rhodococcus equi. Sequence producing significant
alignment.
The graphical summary shows 50-80 alignment scores. The graphical view is:

Genomic island Analysis:

Figure 2: Genomic analysis of Rhodococcus equi (103S)

By using Rhodococcus equi 103S, there is no identified TA system in R.equi tend to cluster in
genomic island. There is no homologues structure of any toxin and antitoxin protein. The
circular diagram of Rhodococcus equi 103S shows no sequence identity.