Pseudomonas genus is considered a major opportunistic pathogen, isolated from diverse ecological niches including, soil, water, and clinical specimens (Stover et al., 2000), and possess intrinsically advanced antibiotic resistance gene clusters (Lambert, 2002). Many strains have been associated with serious illness-causing nosocomial infections and various sepsis syndromes (Planquette et al., 2013; Philippart et al., 2015). The pathogenicity is attributed to the presence of virulence features like pili, flagella, exotoxin A, secretion system, and quorum-sensing proteins (Lyczak et al., 2000). Among various members, P. aeruginosa is a Gram-negative, aerobic, rod-shaped bacterium and environmental isolates of this bacterium easily adapt to a large variety of natural ecosystems (Selezska et al., 2012). As every P. aeruginosa has a certain pathogenic potential, they are all classified as risk group two and the degree of virulence varies substantially between strains (Hilker et al., 2015).

Besides, soil-dwelling Pseudomonas species form close relationships with plants, thereby positively affecting plant growth and nutrition (Cheng et al., 2017; Zamioudis et al., 2013), and also exhibits potent antagonistic activity toward pathogenic microorganisms (Biessy et al., 2019). However, the infection caused by various phytopathogens leads to a loss of ~25% crop production globally (Kim et al., 2017a, b). Additionally, the excessive use of chemical pesticides and fertilizers used to control phytopathogens imposes a serious effect on human and environmental health (Alvarez et al., 2012). Under these circumstances, microbial inoculants belonging to the genera Pseudomonas holds a promising substitute for conventional fertilizers and antibiotics (Stewart, 2001). Pseudomonas spp. influence plant health also by the production of diverse set of secondary metabolites (Nguyen et al., 2016; Stringlis et al., 2018), and through secreted proteins (Rangel et al., 2016). The production of phenazines and anthranilate by the P. aeruginosa contribute to the antagonism against plant pathogens (Anjaiah et al., 1998), and is thereby used as biocontrol agents and biofertilizers (Kwak and Weller, 2013). Moreover, Pseudomonas spp. also employ diverse mechanisms to colonize the plant rhizosphere (Little et al., 2019) and suppress a range of plant pathogens including bacteria (Arseneault et al., 2015), fungi (Michelsen et al., 2015), and insects (Flury et al., 2017), however, these attributes vary from strain to strain.

The genome of Pseudomonas spp. generally divided into a core genome containing sequences common to the species and an accessory genome, with the restriction of sequences present in some strains (Kung et al., 2010; Ozer et al., 2014). The virulence of Pseudomonas spp. is generally affected by the variation in both the core and accessory genome. However, many of the genomes investigation are still going, therefore, accessory genes associated with virulence in addition to mutation may be missed. This may limit the prediction of antibiotic resistance or virulence genes on mobile genetic elements (MGE) or standard chromosome regions as well as their prevalence in general (Beatson and Walker, 2014). It was suggested that the large genome size and complexity of several P. aeruginosa strains such as P. aeruginosa PAO1 (6.2 Mbp, 5683 genes), P. aeruginosa PA14 (6.5 Mbp, 5965 genes), and P. aeruginosa PA7 (6.5 Mbp, 6369 genes) reflects environmental adaptation with the highest proportion of regulatory genes, and a large number of genes involved in metabolism, transport and efflux systems allows the bacterium to survive in diverse environments (Stover et al., 2000; Winsor et al., 2011). The higher number of regulatory genes modulates the metabolic and genetic capacity of P. aeruginosa in varying environmental conditions. The increased availability of genes, genomics data, and further use of genomic tools suggests the potential applications of the strain for diverse biotechnological applications (Xia et al., 2018). Many of the Pseudomonas strains possess good industrial applications in-terms of production of highly stable enzymes (Grbavčić et al., 2011), biosurfactants (Rikalović, 2013), and polysaccharides (Dimitrijević et al., 2011). Additionally, many of the strains have been reported to tolerate high concentrations of heavy metals (Izrael-Zivkovic et al., 2018), which makes it industrial useful.

Due to the spectrum of ecological, biochemical and metabolic characteristics of the Pseudomonas genus, it is clearly evident that diversity among this bacterium extends to the genomic level. Klockgether et al. (2011) have suggested that sequencing of P. aeruginosa strains from environmental habitats provides an unbiased overview of the genetic repertoire. More than 200 P. aeruginosa genome sequences are available on the National Centre of Biotechnology Information (NCBI), however, less than 10% are of environmental strains. The increase in robust sequencing technologies has resulted in the economic cost of sequencing a bacterial genome. Next-generation sequencing provides valuable insight into the genome of organisms and allows the comprehensive analysis of genomic features (Roy et al., 2013). Furthermore, the functional annotation of genomic features can be utilized as a powerful tool for the development of genetically modified bacteria with improved functionality. Additionally, comparative genomics has emerged as a robust tool to identify and compare functionally important genomic elements (Wu et al., 2010). A comparison of genomes within the Pseudomonas group demonstrated evidence about the ecological and physiological diversity of these bacteria extends to the genomic level (Loper et al., 2012).

In the present study, we focused on the detailed genomic characterization of environmental isolate P. aeruginosa S-8 isolated from waste-dumping soil sample, which showed good antagonistic activity against tested bacterial and fungal pathogens. Therefore, the in-depth genome and comparative genome analysis will fill the gap in the genomic studies of environmental Pseudomonas strains. Additionally, the whole-genome analysis (WGS) of this strain will provide opportunities to identify genes involved in the biocontrol of pathogens, plant growth promotion (PGP), and genes involved in the production of secondary metabolites, etc. The available WGS data of many Pseudomonas strains in the public database (https://www.ncbi.nlm.nih.gov/datasets/genome/) have been used for the comparative genome analysis and pan-genome analysis with WGS data of P. aeruginosa S-8.

Utilizing the sequencing data of newly isolated strains, it is possible to make in silico-based predictions about the gene repository and virulence potential of a newly isolated strain P. aeruginosa S-8. This robust method decreases the need for a laborious trial, error-type wet lab experiments, and animal testing. In the present work, we investigated the in-depth genome, pan-genome, and comparative genome analysis of P. aeruginosa to assess the genomic differences and similarities between closely related strains. This type of study will provide insights into the adaption process of an environmental isolate to a particular environmental habitat. Moreover, the environmental isolates due to adaption to different habitats show more closeness among themselves than the clinical isolates (Sánchez et al., 2014). The genomes of several Pseudomonas-type strains have been deciphered, which has contributed to an improved understanding of the evolution and diversity of the Pseudomonas genus (Silby et al., 2011).

In particular, more P. aeruginosa isolates are being sequenced and showed the presence of various pathogenicity and virulence features (Klockgether et al., 2011; Marcelletti et al., 2011). Among the Pseudomonas genus, P. syringae comprise plant pathogens secreting effector’s proteins into plant cells by the type III secretion system (Lindeberg et al., 2012), whereas, several P. fluorescens are known for their biocontrol properties (Haas and Defago, 2005). In contrast, P. putida strains are mostly non-pathogenic and show robust metabolic capacities under stress conditions (Rojas et al., 2001). Flagella are used by bacteria for motility and many genes for flagella formation were annotated in the S-8 strain. The presence of flagella improves bacterial motility and consequently pathogenicity, and also provides the bacterium resistance to surfactant protein A (SP-A) (Zhang et al., 2007), a potent lung innate immune protein that kills microbial pathogens through opsonization (Tan et al., 2014a). Additionally, specific pili like type IV pili enhance the motility and antimicrobial resistance in P. aeruginosa (Tan et al., 2014b), and have been reported in severe cases of pneumonia, bacteremia, and increased mortality (Tang et al., 1995).

The KEGG analysis revealed that strain S-8 harbored the genes for the gluconeogenesis pathway, pentose phosphate pathway, purine and pyrimidine synthesis, fatty acid and peptidoglycan synthesis pathway. In terms of nitrogen metabolism, S-8 could take up ammonia using the ammonium family transporters. Besides sulfur metabolism, strain S-8 harbored the gene set for sulfate, phosphate, alaknesulfate and lipopolysaccharide, which enable S-8 to obtain and utilize nutrients such as nitrogen, carbon, phosphorus and sulfur from the environment to facilitate its survival in different environments. The genome analysis showed that S-8 could synthesize various amino acids including alanine, glycine, glutamate, serine, threonine, cysteine, valine, glutamine, proline, arginine, and phenylalanine. Moreover, genes for vitamin metabolism, peroxidase, superoxide dismutase, lipopolysaccharide biosynthesis, β-lactam resistance, and cationic antimicrobial peptide were also noted, which could enable the Pseudomonas strain S-8 to adapt to a complex and changeable environment.

Genome annotation identified the various metal resistance genes in S-8 genome. Genes coding for copper-translocating P-type ATPases (opA, copB, cusR, cusS, cueR) were identified which are a large family of transmembrane transporters and their role in ion homeostasis and tolerance of heavy-metal ions is well established. A previous study showed that P-type ATPases pumped out the metal ions from the cytoplasm to the periplasm. The cusRS is a two-component signal transduction system that activates the expression of cusCFBA operon and is involved in copper detoxification (Gilles-Gonzalez and Gonzalez, 2005). Copper-translocating P-type ATPases acts as an efflux pump in order to pump out excess Cu²⁺ from the cell (Ladomersky and Petris, 2015). Genes involved in lead resistance zntA and cadC, arsenic resistance arsC, arsR, genes involved in cobalt resistance czcD, and chromium resistance chrA were detected ( Supplementary Table 2 ). Nickel-responsive regulator protein nikR and copper chaperone protein copZ were noted which belong to drug/metabolite transporter superfamily, and are presumed to expel the toxic metabolites out of the cell (Gudhka et al., 2015). P. aeruginosa S-8 was motile and we identified chemotaxis regulator proteins encoded by CheA, CheB, CheB1, CheR, CheV, CheY, CheW, and CheZ gene. Additionally, chemotaxis-associated proteins encoded by McpC, McpH, McpP, McpQ, PctA, PctB and CtpH genes along with flagellar proteins (FlhABF, FliCDEFGOP, MotAB) were also identified. Genes involved in the regulation of Fe uptake e.g. Fur and PchhR were detected in the S-8 genome ( Supplementary Table 3 ).

S-8 genome also showed the presence of osmotic stress genes e.g. mdoB (Phosphoglycerol transferase I), mdoH (Glucans biosynthesis glucosyltransferase H), mdoG (Glucans biosynthesis protein G precursor), and mdoD (Glucans biosynthesis protein D precursor). Production of cold-shock and heat-shock proteins by microorganisms can help their survival in harsh environments and facilitate environmental adaption. The S-8 genome carries the various heat-shock and the cold-shock protein ( Supplementary Table 4 ). Moreover, the S-8 genome encodes numerous proteins to protect the cell from oxidative stress including five peroxidases, three catalases, four superoxide dismutases, two hydroperoxide reductases and 11 glutathione S-transferases (GST). GST is a detoxification enzyme involved in cell protection against stress-induced reactive oxygen species (Oakley, 2011). Furthermore, many dehydrogenases/oxidoreductases were found in the S-8 genomes which play an important role as an oxidative protection in bacteria in response to heavy metals (Kaur et al., 2006; Williams et al., 2007). S-8 genome also encoded ions scavenging systems, such as thioredoxin (1 gene), thioredoxin reductase (2 genes), ferredoxin (3 genes), and glutaredoxin (2 gene) which act as defense proteins to protect microorganisms from oxidative damage (Gorriti et al., 2014).

AntiSMASH informatics predicted that S-8 genome has many secondary metabolite synthesis gene clusters with gene cluster types such as NRPS, PKS, T3PKs, and was able to predict pyoverdine, lankacidin, pyochelin, and pseudopaline etc. These gene clusters may all be involved in the antimicrobial process. It has been shown in the literature that pyoverdine, as a broad-spectrum antibacterial substance present in many Pseudomonas spp., exhibits bactericidal activity mainly by destabilizing the phospholipid membrane of the target pathogen, leading to cell lysis (Liu et al., 2021). The biosynthetic gene clusters of pyochelin has demonstrated other biological activity recently other than being only a chelating compound. This compound can particularly inhibit bacterial pathogens in a study conducted by Adler et al. (2012) and Ong et al. (2016). In this study, we found that S-8 contains a cluster of biosynthetic genes for many of the above-mentioned inhibitory substances based on genome sequencing, indicating that the bacterium not only has broad inhibitory activity against bacteria but also has the potential to inhibit pathogenic fungi.

Secondary metabolites are organic molecules that have diverse and powerful biological functions which facilitate the bacterial strain to adapt to the environment (Abegaz and Kinfe, 2019). The respective genes involved in the synthesis of these secondary metabolites are often clustered into biosynthetic gene clusters (Medema et al., 2015). In S-8 genome, we identified several gene clusters for secondary metabolites production which act as defensive molecules for the organism producing them (James, 2017). Non-ribosomal peptides (NRPs) are synthesized through enzyme-mediated condensation of amino acid residues where > 300 different precursor molecules help in the assemblage of NRPs (Saxena et al., 2020), and fall into the class of secondary metabolites with diverse properties as toxins, siderophores, antibiotics, immune-suppressants, and anticancer agents (Wang et al., 2014; Martínez-Núñez and López, 2016). All these BGCs comprised approximately 14.6% of the genome. Various gene clusters associated to NRPSs toxic for prokaryotes and eukaryotes were identified in S-8 genome (Rojas Murcia et al., 2015). Some of the NRPSs clusters showed similarity to the pyoverdine cluster with less than 100% similarity. Pyoverdine is a common siderophore found within P. aeruginosa species and could represent a novel drug or vaccine target (Fothergill et al., 2012).

Region 1.5 represented the clusters of gene for the biosynthesis of bicarbonate transport ATP-binding protein CmpD, nitrate import ATP-binding protein NrtD, cysteine/O-acetylserine efflux protein, purine ribonucleoside efflux pump NepI, alpha-ketoglutarate-dependent taurine dioxygenase, gamma-glutamyl putrescine oxidoreductase, and FMNH2-dependent monooxygenase SfnC ( Supplementary Table 6 ). The nitrate import ATP-binding protein facilitates the nitrate uptake, nitrite transport, and responsible for energy coupling to the transport system (Watzer et al., 2019). The bicarbonate transport ATP-binding protein is involved in the specific and high affinity binding of nitrate and nitrite, and shows structural similarities to integral membrane subunits of ABC transporters (Poschenrieder et al., 2018).

The seventh cluster (Region 1.7) contained genes for the various transporters belonging to antibiotic efflux pump outer membrane protein (ArpC), multidrug ABC transporter permease (YbhR, YbhS, YbhF), gramicidin dehydrogenase (LgrE), vitamin B12 transporter (BtuB), L-lactate transporter, and helix-turn-helix (HTH)-type transcriptional regulator (MalT, YofA, DmlR) ( Supplementary Table 7 ). The antibiotic efflux pump outer membrane protein form trimeric channels that facilitate the export of a variety of substrates in gram-negative bacteria, whereas, ABC transporter complex YbhFSR could be involved in efflux of cefoperazone (Feng et al., 2020). Vitamin B₁₂ (or cobalamin) is an enzymatic cofactor essential for bacteria, however, it can be synthesized only by few microorganisms, so most bacteria need to take it up from the environment through the TonB-dependent transport system. Its import to bacterial cells occurs through the outer-membrane receptor called BtuB which forms a β-barrel with inner luminal domain and extracellular loops. Vitamin B₁₂ binds with high affinity to the extracellular side of the BtuB protein (Pieńko and Trylska, 2021). The HTH family of transcription regulators is involved in the development of antibiotic resistance (Eckstein et al., 2021). Another enzymes, methane and alkanesulfonate monooxygenase belongs to the family of oxidoreductases and catalyzes the chemical reaction with O₂ as oxidant and incorporation or reduction of oxygen (Liew et al., 2021).

The 8^th cluster (Region 1.8) contained various genes for phosphoethanolamine transferase (EptA), phosphate-import ATP-binding protein (PhnC), acyl carrier protein, inner membrane transport protein (YdhP), ABC transporter phosphite binding protein (PhnD1), and transcriptional regulator (SlyA) ( Supplementary Table 8 ). The phosphoethanolamine transferase (EptA) is an intramembrane enzyme that modifies the lipid-A portion of lipopolysaccharide (LPS) or lipooligosaccharide (LOS) by the addition of phosphoethanolamine, which resulted in reduction of overall net-negative charge of the outer membrane of some gram-negative bacteria, conferring resistance to polymyxin. This resistance mechanism has resulted in a global public health issue due to the increased use of polymyxin as last-resort antibiotic treatments against multi-drug-resistant pathogens (Samantha and Vrielink, 2020). The phosphate-import ATP-binding protein is involved in phosphonates, phosphate esters, phosphite and phosphate import, responsible for energy coupling to the transport system (Junhong et al., 2023). Another enzyme phosphate-import ATP-binding protein belongs to a larger family that includes phosphate, phosphite, and phosphonate transporters, which binds strongly to inorganic phosphate (Feingersch et al., 2012). The transcriptional regulators SlyA are often involved in the regulation of genes important for bacterial virulence and stress response. However, the slyA deletion mutant (ΔslyA) of Enterococcus faecalis showed more virulence in an insect infection model (Galleria mellonella), exhibited increased persistence in mouse kidneys and liver, and survives better inside peritoneal macrophages (Michaux et al., 2011).

The 12^th cluster (Region 1.12) genes included efflux pump periplasmic linker (BepF) and membrane transporter (BepE), toluene efflux pump outer membrane protein (Ttg), Iron import ATP-binding/permease protein (IrtB & IrtA), D-alanine–D-alanyl carrier protein ligase, and regulatory protein (PchR) ( Supplementary Table 9 ). Additionally, this region also possessed thioesterase (PikA5), isochorismate pyruvate lyase, salicylate biosynthesis isochorismate synthase, Inner membrane transport protein (YajR) and UvrABC system protein A. The UvrABC play critical role in DNA repair by nucleotide excision repair, replacing these aberrant nucleotides, involves the removal of twelve nucleotides where a genetic mutation has occurred followed by a DNA polymerase, and completing the DNA repair (Kraithong et al., 2021). Furthermore, this cluster possessed phenazine-1-carboxylate N-methyltransferase, which is involved in the biosynthesis of pyocyanin, a toxin produced and secreted by the P. aeruginosa, and plays a role in virulence (Mavrodi et al., 2001).

The 13^th cluster (Region 1.13) genes included pseudopaline exporter and synthase, metal-pseudopaline receptor CntO, biosynthetic arginine decarboxylase and putative Nudix hydrolase YfcD ( Supplementary Table 10 ). The pseudopaline genes are involved in biosynthesis of metallophores, their export in the extracellular medium, and the recovery of a metal-metallophore complex under metal scarce conditions (Lhospice et al., 2017). The Nudix hydrolase contribute to cellular ‘housekeeping’ through the breakdown of a wide range of nucleoside diphosphate derivatives (Tong et al., 2009).

A number of secretion systems were identified in S-8 genome such as type II secretion systems (T2SS) that secrete the folded proteins such as pseudolysin (lasB), phospholipase C (PlcH), or lipase (LipA) from the periplasm to extracellular milieu (Costa et al., 2015). The presence of type III secretion system (T3SS) allows the translocation of bacterial effectors proteins into the host cell (de Bentzmann and Plesiat, 2011; Girlich et al., 2004), and was associated with bacterial persistence in the lungs and increased mortality in patients suffering from acute respiratory infections (Anantharajah, 2017). Type VI (T6SS) plays crucial role in bacterial competition and pathogenesis (Wood et al., 2019). The presence of these diverse secretion systems also facilitates the survival of S-8 under different environment niche.

The VFDB and CARD based analysis revealed various genes associated with antibiotic resistance and virulence activity of strain S-8. Notably, the presence of gene encoded β-lactamases enzyme bla _PDC-142 and bla _PME-1 is consistent with carbapenem resistance. Additionally, the following cephalosporin-resistant genes, bla _PDC-2, bla _PDC-7, and bla _PDC-9 was observed and one each for aminoglycoside (aph(3′)-IIb) fosfomycin (fosA) and chloramphenicol (catB7) resistance genes was present. The gene arnA modifies the lipid A with 4-amino-4-deoxy-l-arabinose (Ara4N), which allows Gram-negative bacteria to resist antimicrobial peptides and antibiotics such as polymyxin. A previous study (Udaondo et al., 2016) reported that P. putida strains that share 85% of the coding regions with P. aeruginosa bear the various genes encoded for transporters, enzymes and regulators for amino acid metabolism and reveals their environmental applications by the capacity to degrade pollutants and ability to promote plant growth. This research represents an effort to find the mechanisms underlying the ecology, pathogenicity and evolution history of Pseudomonas spp. that able to develop biotechnological advances. Cho et al., 2015 reported P. fluorescens PCL1751 genome and comparative genome study that reveals the integration of prophages that play an important role in genome rearrangements and this strain achieves biological control of pathogens through effective competition for nutrients including niches.

Exposure to metal stressors significantly inhibits the respiratory enzymatic activities as well as corresponding transcript level (Samanta et al., 2020). In S-8 genome, a large inventory of CAZymes was noted including sugar metabolizing enzymes, which confers respiratory metabolism under metal stressors, facilitating the high energy metabolism activity. P. aeruginosa shows a large degree of genomic heterogeneity both through variation in sequences found throughout the species (core genome) and through the presence or absence of sequences in different isolates (accessory genome) (Pincus et al., 2020). P. aeruginosa isolates also differ markedly in their ability to cause disease. In this study, we investigated the genomic features of a newly isolated environmental strain P. aeruginosa S-8. We showed that core genome, alone or in combination with the accessory genome are also predictive of virulence. Another interesting genomic trait, the presence of several GIs was observed in S-8 genome. In bacteria, these GIs are decorated with additional genes acquired via horizontal gene transfer (HGT) mechanism. The presence of these genes may render additional metabolic functions including adaptive traits and genome plasticity which may facilitate evolutionary survival (Pérez-Pantoja et al., 2013). Recent genomic investigation has revealed GIs encoded functional traits which are classified into PAIs (pathogenicity islands) encoding virulence genes, MIs (metabolic islands) encoding biosynthesis of secondary metabolites, RIs (resistance islands) encoding resistance genes to antibiotics, and SIs (symbiotic islands) encoding genes for symbiotic association of the host to microorganism. The observed results suggest a strong possibility that in S-8 strain GIs were recruited via HGT to facilitate the survival in different environmental conditions.

The microbial genome variation is essential for understanding microbial functions, microbe-host interactions, and to understand the effects of genetic variation on function or phenotype. The small genomic differences may influence the phenotype provides evidence about the functional consequence of sequence variation. Over decades, microbiologists have identified the function of numerous genes across multiple species mainly through investigating the effect of gene loss. The diversity of functional genetic features may greatly exceed the taxonomic diversity due to horizontal gene transfer (HGT) and rapid evolutionary adaptation (Wiedenbeck and Cohan, 2011). The BGCs clusters of S-8 strains showed both strain-specific and unidentified characteristics, which support the idea that bacteria perform metabolic activity exclusively for survival in a particular ecological environment and potentially construct alternative routes for new bioactive metabolite production. The test isolate S-8 showed a higher number of virulence genes as compared to other tested genomes, therefore, genome analysis of S-8 reveals the number of potential features that might be considered candidates for future studies to explore the virulence mechanism deployed by this bacterium. P. aeruginosa enriched with conserved core genome of low sequence diversity and variable genome components that communicate with other Pseudomonas by horizontal gene transfer (Klockgether et al., 2011).

In summary, several genomic features of P. aeruginosa S-8 were identified based on the whole-genome analysis. Phylogenetic based analysis showed that strain S-8 has a high similarity to P. aeruginosa strains. Comparative genomic analysis revealed that S-8 possesses genomic islands, prophages, etc. We identified some putative virulence factors and future studies should expand the number of isolates, so as to increase the confidence of results generated in the present study. The ability of the genome to predict antibiotic resistance genes opens the door for sequencing of new strains and it will further supplement or replace the traditional antimicrobial susceptibility testing. However, future studies are needed to provide detailed understandings of the role that genetic variation plays the ability of P. aeruginosa to cause disease by using suitable model system. By being aware of the potentially high virulence of the organisms, personal safety measurements can be increased to avoid an accidental exposition of the organism. The presence of various CAZymes illustrate its importance in industry regarding complex polysaccharide degradation and further energy production. Using newly sequenced data and their investigation can help to substantially speed up research in the future and to draw wider, more general conclusions.