*3.2. BOX-Fingerprinting*

BOX-fingerprinting was performed on the isolates that scored positive on the indole/indigo test and thus possess dioxygenases that can have activity against naphthalene and toluene. Based on the differences in the fingerprint patterns and partial 16S rRNA gene identification, eight groups could be discriminated. Subsequently, those strains which gave a positive amplification band and blast hit to an aromatic compound dioxygenase were down-selected (in squares). A multiple sequence alignment of the Rieske subunit of the naphthalene dioxygenases showed a high similarity with the naphthalene dioxygenase large subunit (*nahAc*) of *Pseudomonas putida*, encoded on plasmid pAK5, and verified experimentally to use naphthalene (Figure S2). Based on this information, we chose to sequence two representative strains, one of the *Pseudomonas* sp. group (VI4.1) and *Pseudomonas veronii* group (VI4T1), Figure 3.

#### *3.3. General Features of the Draft Genomes of Strain VI4T1 and VI4.1*

The 7,150,343-bp genome of strain VI4T1 (GC 60.54%) contains 7962 genes, of which 7809 are Coding Sequences (CDSs), 9 are rRNAs (7, 1, 1 for 5S, 16S, 23S), and 60 are tRNAs comprising 20 different tRNA types. The 7,346,306-bp genome of *Pseudomonas* sp. VI4.1 (GC 60.02%) contains 8082 genes, of which 7922 are CDS, 9 are rRNAs (7, 1, 1 for 5S, 16S, and 23S, respectively), and 58 are tRNAs composed of 21 different tRNA types. The MaGe server classified 73.65% and 74.38% of the CDSs in at least one Cluster of Orthologous Group (COG) for VI4T1 and VI4.1, respectively. In addition, 83.90% and 82.87% of the CDSs are classified in at least one Evolutionary genealogy of genes: Non-supervised Orthologous Groups (EGGNOG) for VI4T1 and VI4.1 (Figure S3). The most abundant EGGNOG categories for VI4T1 are, amino acid and transport, transcription, inorganic ion transport and metabolism, energy production, while for VI4.1, the most dominant categories were secondary metabolite biosynthesis, inorganic ion transport, lipid transport and metabolism, and coenzyme transport and metabolism (Figure S3). In strain VI4.1, 21 CRISPR sequences and nine transposases were found, while in VI4T1, seven CRISPRs, two Cas (CRISPR-associated), and 10 transposases were present. CRISPR, clustered regularly interspaced short palindromic repeats, along with Cas are a bacterial defence system against bacteriophage predation. This all indicates that both strains have experienced extensive and complex genetic alterations and acquired new genetic elements by bacteriophages and exchanges by lateral gene transfer.

**Figure 2.** (**A**) Relative abundance of isolated oil tolerant bacteria using the diffusion sandwich system. (**B**) Percentage of strains displaying plant-growth promotion (PGP) properties, specifically Acetoin, Organic acid, 1-aminocyclopropane-1-carboxylate (ACC)-deaminase, siderophore and indole-3-acetic acid (IAA) productions. (**C**) neighbour-joining phylogenetic tree of all isolated taxa and their PGP and diesel-degrading properties. Coloured blocks and coloured stars indicate that the strain scored positive for the tests, non-coloured (empty) blocks and stars indicate the strain should not produce/utilize the compound evaluated by the corresponding assay.

The RAST Server indicated that the most closely related strains of VI4.1 were *Pseudomonas fluorescens* Pf0-1 (205922.3) and *Pseudomonas* sp. GM18, while for VI4T1 these were *Pseudomonas fluorescens* SBW25 and *Pseudomonas extremaustralis* 14-3. Phylogenetic analyses based on the full-length 16S rRNA gene sequences shows the most closely related strains based on the Ribosomal database (Figure 4). *Pseudomonas* sp. VI4.1 clusters with *Pseudomonas fluorescens* (AY538264), while VI4T1 is closely related to the *Pseudomonas veronii* type strain AF064460. The DDH between VI4.1 and its closest genome sequenced relative, *Pseudomonas silesiensis* sp. nov. strain A3T (CP014870), shows a distance of 61.90% (probability DDH >70%, 49%), suggesting that VI4.1 is a new species. The DDH estimate between VI4T1 and *Pseudomonas veronii* 1YdBTEX2 (LT599583) was 87.9%, with a probability of DDH >70% of 95.03%, indicating that VI4T1 belongs to the *Pseudomonas veronii* group.

**Figure 3.** Cluster analyses of BOX-fingerprint PCR products of indole-positive isolates. Strains VI4.1 and VI4T1 were chosen as representative isolates with positive naphthalene dioxygenase large subunit (*nahAc*) gene PCR amplification. *Pseudomonas putida* f1 and *Pseudomonas aeruginosa* WatG were included as reference strains with naphthalene dioxygenase activity.

To gain insights into the genome organisation, the genome assemblies were visualised in Bandage (Figure S4A and Figure S4b). Because of the draft genome completeness, it is still difficult to determine whether one or two chromosomes are present, though both strains contain one plasmid: VI4T1 has a plasmid of 40,201 bp and VI4.1 one of 81,970 bp. Comparative analyses show matches of VI4T1 plasmid with a conjugative plasmid in *Pseudomonas putida* pBI709 and in *Pseudomonas fragi* strain NMC25. The plasmid of VI4.1 shows high similarity with *Pseudomonas fluorescens* strain PC20 plasmid pNAH20, *Pseudomonas putida* NCIB 9816-4, *Pseudomonas frederiksbergensis* AS1, and *Pseudomonas putida* ND6 plasmid pND6-1, all encoding a naphthalene degradation operon (Figure S4).

The 40 kb plasmid of VI4T1 (pVI4T1-1) contains 48 CDSs, while the 81 kb plasmid of VI4.1 (pVI4.1-1) encodes 103 CDSs (Figure S4). pVI4.1-1 has the full aerobic naphthalene degradation operon (*nah1: nahAaAbAcAdBFCED*) which converts naphthalene to salicylaldehyde and salicylate (Figure 5). The enzymes involved are naphthalene 1,2-dioxygenase (*nahAa-d*), which hydroxylates the two-ring compound, and then *cis*-1;2-dihydro-1,2-dihydroxynaphthalene-1,2-dehydrogenase (*nahB*) catalyses the transformation into naphthalene-1,2-diol, with oxygenation by 1,2-dihydroxynaphthalene dioxygenase (*nahC*). Decisive are the actions of the isomerase (*nahD*) and hydratase-aldolase (*nahE*) to obtain salicylaldehyde further dehydrogenated by the enzyme salicylaldehyde (*nahF*). At this point the molecule is metabolized via the catechol cleavage pathway encoded by the lower naphthalene operon (*nah2: nahGTHINLOMKJ*). Salicylate monooxygenase (*nahG*) oxidises salicylate to catechol, and then further transformations are catalysed by the enzymes: ferredoxin (*nahT*), catechol-2, 3-dioxygenase (*nahH*), 2-hydroxymuconic semialdehyde dehydrogenase (*nahI*), hydrolase (*nahN*), hydratase (*nahL*), acetaldehyde dehydrogenase (*nahO*), aldolase (*nahM*), oxalocrotonate decarboxylase (*nahK*), and oxalocrotonate tautomerase (*nahJ*). Furthermore, the typical elements of a conjugative plasmid are found such as the transfer genes (*traA-D*), replication site (*repA*), and a complete type IV secretion system (*mpfABCDEFGHIJ*). These latter components form the secretion machinery including the membrane proteins (VirB6, VirB8, VirB10, VirB9 and VirB7), with three ATPases that form the power unit (VirD4, VirB11, VirB4), and VirB11 and VirB4 are also required for the biogenesis of the T4S pilus. T4SS can mediate in this case the conjugative transfer of the naphthalene operon to other bacteria. Conjugation is an important strategy employed by bacteria to promote bacterial genome plasticity, and to spread a variety of functions, such as the degradation of anthropogenic toxic compounds or the detoxification of heavy metals, but also bacteriocin and toxin production to ward o ff predators. Having part of the degradation pathways encoded on a plasmid is an interesting property, as the strain can transfer its degradative plasmid via horizontal gene transfer to native soil or plant-associated bacteria, a strategy that has been utilised by our group to clean-up groundwater polluted with toluene and trichloroethylene.

**Figure 4.** 16S rDNA phylogenetic tree of *Pseudomonas veronii* VI4T1 and *Pseudomonas* sp. VI4.1 compared to closely-related strains and with *Pseudomonas gessardii* as an outgroup. Closely-related strains were evaluated against the Ribosomal Database and the phylogenetic tree was built in MEGA7. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site.

**Figure 5.** pVI4.1-1 80 kb plasmid encoding a full naphthalene operon, *nahAaAbAcAdBFCED* to convert naphthalene to salicylate, and the lower *nahGTHINLOMKJY* operon to convert salicylate via catechol to tricarboxylic acid (TCA) cycle intermediates.

The 40-kb pVI4T1 plasmid does not contain a naphthalene nor aromatic compound degradation operon, though it does contain partial T4SS proteins (MpfA-E), and ParA and B which are bacterial proteins involved in plasmid replication and partitioning, which suggests this was a functional conjugative plasmid but it might have lost some essential genes (Figure S5). For the many CDS annotated as "hypothetical proteins", we ran a conserved domain search and this revealed the presence of: amidase expression regulating protein, glutathione permease protein (GsiD), histidine/lysine/arginine/ornithine ABC transporter, transposases, cation efflux system (CzcD), transporters (MntH), and components involved in secretion and vesicular transport. It is a subject of speculation as to whether this plasmid is a rudimentary copy of the pVI4.1-1, or a part was not assembled; additional long-read sequencing can confirm this.

#### *3.4. Physiological and Biochemical Properties*

Strains VI4T1 and VI4.1 are aerobic, heterotrophic, motile, Gram-negative, and non-sporulating strains. The FAME profile of *P. veronii* VI4T1 consisted of 50.39% 16:1 7c/16:1 w6c, 0.15% 18:0 ante/18:2 w6,9c, 0.11% 19:1 w7c/19:1 w6c, 25.51% 18:1 w7c, 0.54% 14:0, 22.6% 16:0, 0.54% 18:0, and 0.15% 18:1 w7c 11-methyl. For VI4.1, the FAME profile was 42.73% 16:1 w7c/16:1 w6c, 0.61% 18:0 ante/18:2 w6,9c, 3.92% 18:1 w7c, 0.55% 10:0 3OH, 4.56% 14:0, 45.39% 16:0, 1.53% 18:1 w9c, and 0.72% 18:0.

The morphology of stain VI4.1 is smooth, creamy and round, while VI4T1 grows as slightly yellow, smooth and irregular colonies on 869 rich medium. The optimal growth temperature was 30 ◦C, but the strains showed also good growth at 4 ◦C. The strains grew preferably on nutrient-rich agar, but they proliferate as well in mineral BH medium and supplemented with diesel oil as the sole carbon source. The strains could grow in 869 medium with NaCl concentrations in the range of 1–4% (w/v) showing salt stress tolerance, and they survived in medium with slight acidic pH, at 5–6.

The ability to capitalize on a variety of carbon sources is an important feature for soil and plant-associated microorganisms. Strain VI4T1 and VI4.1 respired numerous carbon sources as reported in Supplementary Table S1.

Chemical sensitivity of the strains was also tested with the GEN-III array, and this showed tolerance of VI4T1 to grow at pH 5-6, in the presence of 1% NaCl, 1% sodium lactate, fusidic acid, D-serine, troleandomycin, rifamycin SV, minocycline, lincomycin, niaproof 4, vancomycin, tetrazolium violet, tetrazolium blue, nalidixic acid, potassium tellurite, aztreonam, sodium butyrate, and sodium bromate. Strain VI4.1 was also capable of growing at pH 5-6, up to 4% NaCl, in the presence of 1% sodium lactate, fusidic acid, D-serine, troleandomycin, rifamycin SV, minocycline, lincomycin, guanidine HCl, niaproof 4, vancomycin, tetrazolium violet, tetrazolium blue, nalidixic acid, lithium chloride, potassium tellurite, aztreonam, sodium butyrate, and sodium bromate.

#### *3.5. Degradation of Hydrocarbons*

The genome of VI4T1 encodes 45 dioxygenases and 20 monooxygenases, VI4.1 has 50 diand 27 mono-oxygenases. The dioxygenases comprised naphthalene, biphenyl, phenylpropionate, nitropropane, halobenzoate, and catechol dioxygenase, while for monooxygenases, alkanesulfonate, cyclohexane, alkanal, nitrilotriacetate, and l-ornithine were present. The high number and diversity of dioxygenases and monooxygenases suggests that the strains have versatile metabolic capabilities and hydrocarbon degradation potential. Other strains such *Paraburkholderia aromaticivorans* BN5, recently isolated from an oil polluted site in South Korea, have shown to harbour multiple aromatic ring hydroxylating enzymes, facilitating the use of naphthalene and BTEX as sole nutrient source [46]. For efficient phytoremediation activities, it is important to understand the underlying genetics structures of the many degradation pathways to better assess their activity and use in the field. In the following paragraphs we therefore focus on the bioinformatic prediction of degradation pathways of naphthalene, BTEX, and aliphatic hydrocarbons, and their experimental validation, followed by PGP prediction, motility, and emergen<sup>t</sup> contaminant degradation.

Previous studies have shown that aerobic naphthalene degradation in *Pseudomonas* spp. can occur via (1 *<sup>R</sup>*,2*S*)-1,2-dihydronaphthalene-1,2-diol, salicylaldehyde, and salicylate, to be converted to catechol and further degraded to tricarboxylic acid (TCA) cycle intermediates. Bioinformatics analyses showed at least two copies of the upper naphthalene operon *nahAaAbAcAdBFCED* for *P. veronii* VI4T1, similar to *P. veronii* YdBTEX2, and one copy for strain VI4.1, encoded on the pVI4.1-1 plasmid (Figure 6). This confirms that strain VI4.1 has obtained the degradation capabilities of naphthalene through plasmid transfer. Also for VI4T1 one copy of the *nah1* operon seems to be plasmid encoded, since plasmid stabilization proteins and transposases are found flanking the operon. For the downstream *nah2* operon (*nahGTHINLOMKJ*) one complete cluster is present in each strain, based salicylate hydroxylase (*nahG*, in green) and 2.3-catechol *nahH* (in blue) as the key enzymes (Figure 7). Organization of this operon between the strains di ffers considerably, and many of the enzymes are present in multiple copies, such as 2-hydroxymuconic semialdehyde dehydrogenase (*nahI*). The upper and lower pathways are controlled by a LysR family transcriptional regulator (Figures 6 and 7).

Aerobic toluene degradation can occur via five di fferent pathways as described in MetaCyc. Strain VI4.1 harbours the *tomAo12345tomB* operon to degrade toluene via o-cresol (Figure S6). A comparative analysis shows the similarity of the operon structure with strain *Burkholderia cepacia*. Strain VI4.1 has a partial *tbuA1A2BCUVE* monooxygenase operon (missing *tbuU* and *tbuV*) and also a partial *tomABCDE* operon, lacking a homologue for *tomB* (BLAST E-value <0.001, >50% query coverage). Strain VI4T1 does not have homologues to any of the *tom* operons, but seems to use route IV and V. Strain VI4T1 harbours the complete toluene monooxygenase operon *xylMBCXYZL* in two copies to convert toluene and xylene to catechol, and downstream *meta-*cleavage of catechol via *xylEGHFIJK* to actyl-coA which enters the TCA cycle. The operon structures are very similar to the *xyl* operon of the reference

strain *Pseudomonas veronii* 1YDBTEX2. The lower catechol ortho-cleavage pathway contains the genes (Figure S7B) catechol 2,3-dioxygenase (*xylE*), 2-hydroxymuconic semialdehyde dehydrogenase (*xylG*), 4-oxalocrotonate isomerase (*xylH*), 4-oxalocrotonate decarboxylase (*xylI*), 2-oxopent-4-enoate hydratase (*xylJ*), 2-oxo-4-hydroxypentanoate aldolase (*xylK*), and an acetaldehyde dehydrogenase (*todI*). The toluene degradation V route (*todC1C2ABCDEF*) to catalyse toluene degradation via toluene-cis-diol is also present in both strains (Figure S8).

**Figure 6.** Comparative analyses of the upper naphthalene operon, physical map of the degradation genes and proposed biochemical pathways for naphthalene degradation for strains VI4T1 and VI4.1.

**Figure 7.** Comparative analyses of the lower naphthalene operon for strains VI4T1 and VI4.1, physical map of the degradation genes, and proposed biochemical pathways for salicylate degradation to catechol or to gentisiate and further breakdown to TCA cycle intermediates.

Strain VI4T1 encodes also the genes to degrade xylene (*xylBCM*), VI4.1 is lacking the *xylM* homologue (e-value <0.001, coverage of 50%) (Figure S7A). Both strains carry the genes to degrade benzene (*bedC1C2AB*) (Figure S9). Strain VI4T1 has additionally an anthranilate degradation operon *antAaAbAc*, downstream of a tryptophan 2,3-dioxygenase gene, as anthranilate is an important intermediate of tryptophan metabolism [47].

Furthermore, genes encoding for enzymes involved in the linear hydrocarbon degradation were investigated. The complete *alkB* operon was present for VI4T1 (Figure 8) but VI4.1 lacks this alkB operon. The 1-alkane hydrolase subunit (*alkBGT*) introduces molecular oxygen in the terminal carbon atom of the hydrocarbons at the expense of NADH to yield primary alcohols, and these are further catabolized by an octanol dehydrogenase (*alkJ*), an aldehyde dehydrogenase (*alkH*) and a medium-chain acyl-CoA synthetase (*alkK*). Finally, the octanoyl-CoA enters the beta-oxidation-cycle and can be utilized as a carbon and energy source (Figure 8). Instead of the complete *AlkB* operon, VI4.1 encodes a homologue of the two-domain *AlkB* system with rubredoxin and rubredoxin-NAD(+) reductase. Both are essential electron transfer components needed for alkane hydroxylation by AlkB. In fact, AlkB-type alkane

hydroxylases fused to rubredoxin protein have been shown to hydroxylate n-alkanes with chain lengths up to C32. VI4.1 and VI4T1 also have a homolog of the cytochrome P450 CYP153 family, this is another type of alkane hydroxylase for the degradation of short- and medium-chain-length *n*-alkanes. Other enzymes important in aliphatic hydrocarbon degradation are the alkanesulfonate genes, of which 11 are present in VI4T1 and 15 in VI4.1.

**Figure 8.** Comparative analyses of the pathways involving alkane-monooxygenase in different *Pseudomonas* strains included *Pseudomonas veronii* VI4T1.

To confirm the degradation potential, experimental evidence of aromatic hydrocarbon compound degradation was performed with PTR-TOF-MS. VI4T1 and VI4.1 were both capable of respiring 15 μg/<sup>L</sup> naphthalene and 3 μg/<sup>L</sup> each of benzene, toluene, and xylene as sole carbon source within seven days (Figure 9). Metabolites such as salicylic acid or catechol were not identified with PTR-TOF-MS in the headspace.

#### *3.6. Motility and Chemotaxis*

Motility of a strain in its environment influences its survival and competence to colonize to find nutrient sources and colonize plant root surfaces [48]. Additionally, motility allows bacteria to move away from high concentrations of toxic compounds. Bacterial motility was experimentally tested in sterile glass tubes previously filled with 869 semi-solid agar medium. Bacteria were inoculated with a straight wire to about half the depth of the medium. Tubes were incubated at 30 ◦C and observed for bacterial growth at regular intervals (one until seven days). Non-motile bacteria generally grow into the stab-line, while motile bacteria, such as strains VI4T1 and VI4.1, typically grow by differently diffusing through the medium (Figure S10).

Genomic analyses confirmed the presence of genes coding for the flagellar motor complex: *motA*, *motB*, *flhA*, *flhB*, *fliH*, *fliI*, *fliJ*, *fliO*, *flipP*, *fliQ*, *fliR*, *flgB*, *flgC*, *flgG*, *flgH*, *flgI*, *fliE*, *fliF*, *fligA*, *flgD*, *flgN*, *fliK*, *fliS*, *flgE*, *flgK*, *flgL*, *fliC*, *fliD*, *fliG*, *fliM*, *fliN*, *flgJ*, *flhF*, and *fliL*. Furthermore, chemotaxis proteins CheA

and CheY are coded. They are involved in the transmission of sensory signals from the chemoreceptors to the flagellar motors.

**Figure 9.** Concentration of naphthalene in the gas phase remaining after seven days of incubation in vials inoculated with *Pseudomonas veronii* VI4T1 and *Pseudomonas* sp. VI4.1. Negative controls are vials containing sterile sand. All vials were spiked in the headspace with 15 μg/<sup>L</sup> naphthalene or with 3.5 μL/L benzene, toluene, or xylene. 200 μL of bacteria suspension (OD600 = 1) were added to the all the vials except for the negative controls. Error bars represent standard error.

#### *3.7. Plant Growth Promotion Potential*

In vitro tests showed that VI4T1 and VI4.1 scored positive for indole-3-acetate (IAA) production, siderophore production, acetoin, organic acid and phytate mineralization, but negative for ACC-deaminase and nitrogen fixation.

In both genomes, indole-3-acetamide hydrolase was identified converting indole-3-acetamide to IAA. This pathway is operative in several genera of plant-associated bacteria amongs<sup>t</sup> which many *Pseudomonas* spp. [49]. Additionally, genes of the tryptophan biosynthesis gene cluster *trp* were present: *trpD*, *trpG*, <sup>t</sup>*rpE*, *trpC*, *trpF*, *trpD*, *trpB*, *trpA*. Strain VI4.1 has also the ability to produce IAA via an additional nitrilase-driven pathway converting indole-3-acetonitrile in IAA. The ability to produce siderophores was confirmed by the presence of the pyoverdin pathway genes, 48 genes for 4T1 and 29 for VI4.1. We found also the fundamental genes for the enzymes converting pyruvate in (R)-acetoin, with acetolactate synthase (*bud*B) and diacetyl reductase (*bud*A). Strain VI4.1 also carries the two genes responsible for the (S)-acetoin biosynthesis (*bud*C). The absent production of some organic acids such as citric, lactic, succinic, gluconic, itaconic, acetic, propionic, tartaric, malonic, malic, oxalic, fumaric, and lactobionic acid was confirmed after the genome annotation.

Growth and development of both, plants and bacteria require macro- and micro-nutrients, some of which are available only from external sources like the soil in which they grow. Phosphorous is such an essential macronutrient, and is usually absorbed and utilized by the plants in the form of phosphate. The role of genes as Purple Acid Phosphatases (PAPs) in strain VI4T1 has not ye<sup>t</sup> been fully clarified. Experimental evidence suggests that the enzyme is induced by a lack of phosphate and excreted from bacterial cells, suggesting that it may be involved in phosphate acquisition. The presence of the gene coding for the acid phosphatase confirmed the in vitro potential of strain VI4T1 to convert a phosphate monoester in more bioavailable phosphate in the presence of water.

No enzymes were detected involved in the biological fixation of atmospheric nitrogen such as *nif* genes or nodulation genes (*nod*), confirming the negative response on the nitrogen fixation test. We did not detect any homologue for 1-aminocyclopropane-1-carboxylate deaminase; that outcome confirmed the results of the corresponding in vitro test.

The aminopeptidase gene (*pepA*) that is involved in seed germination process was detected in both genomes [50]. The enzyme hydrolyses peptide bonds in tissues as the aleurone layer of the endosperm, the scutellum and the growing tissues of the seedling. The activity of these proteins is reported for several plant species.

Furthermore, both strains can synthesize jasmonic acid a member of the jasmonate class of plant hormones which is involved in the regulation of quite a number of processes as embryo and generative organs development, ageing, sex determination, seed germination, root growth, adaptation to stress factors [51].

#### *3.8. Tolerance to Abiotic Stress*

Plants are often exposed to abiotic stresses such as heat, drought, metal pollution, high salinity and acidic pH. In such circumstances, inoculating plants with stress alleviating microorganisms may offer a biological alternative to the existing agrochemicals in agriculture [52]. Both strains carry the *speA* gene involved in the survival of the bacterium in acidic conditions via the arginine-dependent pathway, conferring potential acid resistance by exchanging external arginine for internal agmatine [53]. This effectively consumes protons within the cytoplasm, raising the pH.

Abiotic stress can create osmotic deficiencies in plant cells. In this context, the presence of trehalose can act as an osmoprotectant, and strains VI4T1 and VI4.1 possess several genes coding for proteins involved in trehalose anabolism, including pathway IV (trehalose synthase converting b-maltose in trehalose), and pathways VI and VII (trehalose synthase producing trehalose from ADP-alpha-D-glucose and glucose) [54]. Trehalose accumulation may act as a biosurfactant and in this way enhancing the biodegradation of hexachlorocyclohexane.

Interestingly, both strains carry genes for salt tolerance including *betA* choline dehydrogenase, alcohol dehydrogenase (*yiaY*, *adhA),* and betaine aldehyde dehydrogenase (*betB*). Glycine betaine (N,N,N-trimethylglycine) is a very efficient osmolyte found in a wide range of bacteria and plants, where it is accumulated at high cytoplasmic concentrations in response to osmotic stress, to act as an osmoprotectant [55].

All organisms living in an aerobic environment are exposed to reactive oxygen species (ROS). Strains VI4T1 and VI4.1 carry genes coding for superoxide dismutases (*sodB*, *sodM*) and catalases (*katE*, *katB*) converting superoxide molecules into oxygen via hydrogen peroxide formation. Furthermore, the genomes encode for glutathione reductase (*gor*) converting two molecules of glutathione in glutathione disulfide with a co-production of reduced glutaredoxin. Oxidative damage to proteins often results in the formation of mixed disulfides within the polypeptides. A primary defence against this damage is mediated by the action of GSH-dependent thiol-disulfide oxidoreductases, also called thioltransferases and best known as glutaredoxins (Grx). These proteins reduce the protein disulfide groups back to their native form.

Heavy metal transporters are involved in acquisition, metal absorption and detoxification. Both strains possess the genes coding for a cadmium-transporting ATPase and a copper-transporting ATPase

(*copA*). Copper is an important element that participates in a high number of enzymatic reactions; in photorespiration, electron transport, in ethylene signalling and many other metabolic processes that have copper-containing enzymes catalysing various reactions.

Furthermore, an arsenic resistance system was detected: arsenical resistance protein ArsH; HTH ArsR-type DNA-binding domain; arsenical-resistance protein Acr3/Arsenical pump membrane protein/ArsBArsenate reductase. In addition, several cation e fflux system proteins are present: cation efflux system protein (CusF), cation e fflux system protein (CusA), cation e fflux system protein (CusC), Cation e fflux system protein (CusB).

#### *3.9. Tolerance to Micro-Pollutants and Emerging Contaminants*

During the last decades, there has been an increasing concern about the presence of micro-pollutants including pharmaceutical, human health care products, medicines, endocrine disruptors, fluorinated chemicals, and microplastics that are found in the soil and waters in increasing concentrations [56]. There are 2700 such compounds listed by the Joint Research Centre of the European Commission.

Although the novelty of these investigations translates to a lack of information in prokaryotic databases, the genome sequencing of our two *Pseudomonas* spp. allows us to investigate the presence of genes involved in the degradation of endocrine disruptors. We identified in *Pseudomonas* strain VI4.1 the genes androsterone 3-deydrogenase and testosterone dehydrogenase, respectively involved in the transformation of androsterone and testosterone in the common intermediate metabolite androst-4-ene-3,17-dione [57]. This compound links this catabolic pathway to the one of the androstenedione degradations until mineralization via the propanoyl CoA degradation pathway I or TCA cycle. *Comamonas testosterone* was the first strain characterized for its capability to degrade steroids, and its capacity was compared with *Pseudomonas* spp. after incubation in testosterone sewage in the work of Chen et al. (2016) [57].

Concerning fluorinated chemicals, strain VI4.1 carries the gene coding for the enzyme haloacetate dehalogenase, the only known enzyme that can specifically hydrolyse the carbon-fluorine bond. The enzyme was first described from a *Pseudomonas* spp. in 1965 and since then has been described in multiple bacterial strains. Fluorinated organic compounds have widespread applications as pesticides, herbicides, pharmaceuticals, flame-retardants, refrigerants and foam-blowing agents, and are consequently accumulating in the environment. Only a handful of carbon-fluorine bonds from biological origin are known, such as fluoroacetate, a toxin found in the leaves and seeds of a variety of tropical plants, often in high concentrations. The extreme toxicity of fluoroacetate stems from its similarity to acetate. Fluoroacetate combines with coenzyme A to form fluoroacetyl-CoA, which can substitute for acetyl CoA in the tricarboxylic acid cycle. Fluoroacetyl-CoA reacts with citrate synthase to produce 2-fluorocitrate, a metabolite of which then binds very tightly to aconitase, stopping the cycle.

Interestingly, strain VI4.1 possesses the genes terephthalate 1,2-dioxygenase (*tphA*) and terephthalate dihydrodiol dhydrogenase (*tphA*) converting terephthalate to 3,4 dihydroxybenzoate. Terephthalate is the major precursor for polyester fibres and coatings. Fibres from clothing constitute a major problem in our waterways, also polluting drinking water [58]. Strains able to degrade these fibres are an important resource for the plastics recycling/upcycling industry. In relation to this, *Pseudomonas* VI4T1 carries genes (*bph*A) coding for the enzyme biphenyl dioxygenase subunit A. This enzyme is involved in the first step that synthesizes 2-hydroxy-2,4-pentadienoate and benzoate from biphenyl. Biphenyl is an aromatic hydrocarbon and an important precursor for the production of polychlorinated biphenyls (PCBs).
