Genome analysis for the identification of genes involved in phenanthrene biodegradation pathway in Stenotrophomonas indicatrix CPHE1. Phenanthrene mineralization in soils assisted by integrated approaches.

Genome analysis for the identification of genes involved in phenanthrene biodegradation pathway in Stenotrophomonas indicatrix CPHE1. Phenanthrene mineralization in soils assisted by integrated approaches Alba Lara-Moreno 1 , 2 *, Francisco Merchán 2 , Esmeralda Morillo 1 , Jessica Zampolli 3 , Patrizia Di Gennaro 3 and Jaime Villaverde 1 1 Department of Agrochemistry, Environmental Microbiology and Soil Conservation, Institute of Natural Resources and Agrobiology of Seville, Spanish National Research Council (IRNAS-CSIC), Seville, Spain, 2 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, Seville, Spain, 3 Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy Phenanthrene (PHE) is a highly toxic compound, widely present in soils. For this reason, it is essential to remove PHE from the environment. Stenotrophomonas indicatrix CPHE1 was isolated from an industrial soil contaminated by polycyclic aromatic hydrocarbons (PAHs) and was sequenced to identify the PHE degrading genes. Dioxygenase, monooxygenase, and dehydrogenase gene products annotated in S. indicatrix CPHE1 genome were clustered into different trees with reference proteins. Moreover, S. indicatrix CPHE1 whole-genome sequences were compared to genes of PAHs-degrading bacteria retrieved from databases and literature. On these basis, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis pointed out that cysteine dioxygenase (cysDO), biphenyl-2,3-diol 1,2-dioxygenase (bphC), and aldolase hydratase (phdG) were expressed only in the presence of PHE. Therefore, different techniques have been designed to improve the PHE mineralization process in five PHE artificially contaminated soils (50 mg kg −1 ), including biostimulation, adding a nutrient solution (NS), bioaugmentation, inoculating S. indicatrix CPHE1 which was selected for its PHE-degrading genes, and the use of 2-hydroxypropyl-β-cyclodextrin (HPBCD) as a bioavailability enhancer. High percentages of PHE mineralization were achieved for the studied soils. Depending on the soil, different treatments resulted to be successful; in the case of a clay loam soil, the best strategy was the inoculation of S. indicatrix CPHE1andNS(59.9%mineralizedafter120days).Insandysoils(CRandR soils) the highest percentage of mineralizationwasachievedinpresenceofHPBCD and NS (87.3% and 61.3%, respectively). However, the combination of CPHE1 strain, HPBCD, and NS showed to be the most efficient strategy for sandy and sandy loam soils(LLandALCsoilsshowed35%and74.6%, respectively). The results indicated a high degree of correlation between gene expression and the rates of mineralization. KEYWORDS phenanthrene, Stenotrophomonas indicatrix CPHE1, mineralization, phenanthrene biodegradation pathway, genes expression OPEN ACCESS EDITED BY Datta Madamwar, Charotar University of Science and Technology, India REVIEWED BY Inna Solyanikova, Belgorod National Research University, Russia Qing Hong, Nanjing Agricultural University, China *CORRESPONDENCE Alba Lara-Moreno, [email protected] RECEIVED 03 February 2023 ACCEPTED 14 April 2023 PUBLISHED 04 May 2023 CITATION Lara-Moreno A, Merchán F, Morillo E, Zampolli J, Di Gennaro P and Villaverde J (2023), Genome analysis for the identification of genes involved in phenanthrene biodegradation pathway in Stenotrophomonas indicatrix CPHE1. Phenanthrene mineralization in soils assisted by integrated approaches. Front. Bioeng. Biotechnol. 11:1158177. doi: 10.3389/fbioe.2023.1158177 COPYRIGHT © 2023 Lara-Moreno, Merchán, Morillo, Zampolli, Di Gennaro and Villaverde. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Bioengineering and Biotechnology frontiersin.org01 TYPE Original Research PUBLISHED 04 May 2023 DOI 10.3389/fbioe.2023.1158177 1 Introduction Phenanthrene (PHE) is a low molecular weight polycyclic aromatic hydrocarbon (PAH) constituted by three fused benzene rings. It is mainly produced from anthropogenic sources such as oil mining, accidental discharge, pipeline vandalism, and poor municipal waste management causing adverse effects on public health and the environment (Prabhu and Phale, 2003;Varjani and Upasani, 2017). PHE is characterized by its ubiquity in the environment, and it is a pollutant of concern due to its persistence and toxicity, causing detrimental biological and environmental effects (Abdel-Shafy and Mansour, 2016); indeed, it has been included in the hazardous substances lists of the United States Environmental Protection Agency and the European Union (EPA, 2022). In the environment, PAHs are removed in different ways such as photo-oxidation, chemical oxidation, bioaccumulation, volatilization, or adsorption to soil particles (Parab and Phadke, 2020). However, biological strategy is the main approach to removing PAHs in terrestrial and aquatic systems (Churchill et al., 2008;Nwankwegu et al., 2022), since microbial degradation is a cost-effective and eco-friendly technology (Brown et al., 2017). Several studies showed PHE biodegradation in soil fostered by the presence of different bacterial genera: Bacillus (Mandree et al., 2021), Massilia (Gu et al., 2021), Sphingomonas (Waigi et al., 2015), or Rhodococcus (Wang et al., 2021). Additionally, bacterial strains from Stenotrophomonas genus have been described as PHE degraders (Kumari et al., 2017;Elufisan et al., 2020), such as S. maltophilia (Gao et al., 2013;Zafra et al., 2014;Xiao et al., 2021). However, as far as we know, the study of Lara-Moreno et al. (2021) was the first which demonstrated the ability of S. indicatrix to degrade PHE in solution. Stenotrophomonas indicatrix is a Gramnegative species that can be isolated from sunflower roots (Adeleke et al., 2021), soils (Lara-Moreno et al., 2021;Lozano-Andrade et al., 2021), oligotrophic water ponds (Friedrich et al., 2021), or from food-associated sources (Weber et al., 2018). The degradation of PAH by bacteria is usually catalyzed by oxygenase and dehydrogenase enzymes. For instance, these enzymes include naphthalene dioxygenase (NahAc) and naphthalene monooxygenase (NdoB) (Pseudomonas putida G7, Cébron et al., 2008); pyrene dioxygenase (Mycobacterium sp. strain PYR-1, Stingley et al., 2004); dihydrodiol naphthalene dehydrogenase (NahB)(Pseudomonas stutzeri AN10, Bosch et al., 1999)or naphthalene dehydrogenase (NahB)(P. putida AK5, Izmalkova et al., 2013). Nevertheless, little is known about the genes responsible for PHE degradation in Stenotrophomonas genus. As far as we know, only Kumari et al. (2017) and Elufisan et al. (2020) have conducted research about the genes involved in PAHs biodegradation pathway in Stenotrophomonas genus and there are no scientific studies on Stenotrophomonas indicatrix species. In the environment, the natural ability of soil is often exploited to remove contaminants. However, since natural attenuation is a very slow biological process for the restoration of contaminated soils (Zabbey et al., 2017), (bio)remediation techniques can be applied, including biostimulation, application of availability enhancers, and bioaugmentation. Biostimulation requires the addition of nutrients or electron acceptors into the soil. This option optimizes the strategy and critical factors for the remediation of polluted sites, including restricted oxygen, nutrient transfer, and competition between microorganisms (Masy et al., 2016). Regarding availability enhancers, PHE is considered a pollutant highly persistent in soil due to its strongly hydrophobic character (Humel et al., 2020). Therefore, the high adsorption could cause a decline in the PHE bioavailable fraction, which means a slowdown in the bioremediation progress (Humel et al., 2017). To counter these negative influences, bioavailability enhancers such as cyclodextrins (CDs), can be applied to the soil (Madrid et al., 2019;Madrid et al., 2022). The use of CDs is a greener alternative to organic solvents or synthetic surfactants used by several authors to remediate contaminated soils by PAHs (Sun et al., 2014;Liu et al., 2018). CDs have the capacity to form inclusion complexes with hydrophobic contaminants enhancing their water solubility and improving their elimination from soils (Morillo et al., 2020). Bioaugmentation is considered a promising tool to enhance pollutant biodegradation. Bioaugmentation consists of the addition of exogenous or endogenous pollutant degraders into the contaminated site to accelerate contaminant removal (Tyagi et al., 2011). It has been recommended as the most suitable alternative to be used in bioremediation (Nwankwegu et al., 2022), using bacterial strains isolated from large-scale polluted areas where endogenous microbiota is generally adapted (Mrozik and Piotrowska-Seget, 2010). For this reason, in the present study, a bacterial strain isolated in our lab from highly contaminated industrial soil was selected for its capacity to degrade and mineralize PHE in an aqueous solution (Lara-Moreno et al., 2021). In the previous paper, the isolated bacterial strain was classified as Stenotrophomonas maltophilia CPHE1 (NCBI number: MT138842), but later, a more in-depth genetic study revealed that its classification corresponded to S. indicatrix (Lara- Moreno et al., 2023). Weber et al. (2018) classified for the first time this species as S. indicatrix, although they had previously classified it also as S. maltophilia due to their high genomic similarities. Up to now, no work has shown PHE degradation and mineralization to CO 2 and H 2 0 in the soil in the sole presence of S. indicatrix. Therefore, this study aimed to characterize S. indicatrix CPHE1 at the genomic level to classify genes annotated as dioxygenases, monooxygenases, and dehydrogenases into phylogenetic trees to detect putative PHE-degrading genes. Moreover, S. indicatrix CPHE1 whole-genome sequences were compared to genes of PAHs-degrading bacteria retrieved from databases and literature. The expression of selected genes such as bphC, and phdG genes (encoding for an extradiol dioxygenase, and a hydratase-aldolase) was studied by RT-PCR for their involvement in the PHE degradation pathway. On this basis, several eco-friendly bioremediation treatments were conducted using biostimulation, bioaugmentation with S. indicatrix, and the use of CDs to enhance the extent and rate of PHE biodegradation and mineralization in five contaminated soils with different physicochemical properties. 2 Materials and methods 2.1 Materials PHE (C 14 H 10 , purity 98%) was purchased from Sigma-Aldrich (Madrid, Spain) and radiolabeled compound 14 C-PHE Frontiers in Bioengineering and Biotechnology frontiersin.org02 Lara-Moreno et al. 10.3389/fbioe.2023.1158177 (36 mCi·mmol −1 , purity 99.9%, and radiochemical purity 100%) was acquired from the Institute of Isotopes (Budapest, Hungary). HPBCD was obtained from CycloLab Cyclodextrin Research and Development Laboratory Ltd. (Budapest, Hungary). Mineral Salt Medium (MSM) composition (g L −1 ): 4.0 Na 2 HPO 4 ; 2.0 KH 2 PO 4 ;0.8MgSO 4 ;0.8NH 4 SO 4 . The micronutrients solution (SNs) was composed of (mg L -1 ): 12.5 NiCl 2 6H 2 O; 25.0 SnCl 2 2H 2 O; 12.5 ZnSO 4 7H 2 O; 12.5 Al 2 (SO 4 ) 3 18H 2 O; 75.0 MnCl 2 4H 2 O; 12.5 CoCl 2 2H 2 O; 37.5 FeSO 4 7H 2 O; 10.0 CaSO 4 2H 2 O; 3.75 KBr; 3.75 KCl; 2.50 LiCl (Fenlon et al., 2011). The mixture of MSM and SNs (50:1) was named NS in the study. Five soils (PLD, LL, ALC, CR, R) with diverse physicochemical properties were collected from different points in the South of Spain. PLD soil is an agricultural soil located in Los Palacios y Villafranca—Seville (37°10′20.0″N5 °55′21.9″W), where wheat, cereals, and vineyard crops are mainly cultivated. The agricultural soil LL from Vejer de la Frontera—Cádiz (36°17′52.6″N 5°52′45.2″W) is devoted to intensive agriculture of carrot, cotton, leek, etc. ALC soil was taken from Alcornocales Natural Park (36°20′54″N5 °36′14″O) and is characterized by high organic matter (OM) content. CR soil is an agricultural soil from an area of olives within the experimental farm La HAMPA (IRNAS-CSIC) (37°17′28.3″N, 6°3′55.4″W). R soil, collected from a palm trees zone in Conil de la Frontera—Cádiz (36°18′32.4″N, 6°08′58.6″W), has been managed with a huge number of insecticides. The soil samples collection was conducted from the superficial horizon (0–20 cm), air-dried for 24 h at room temperature, and sieved (2 mm). Table 1S shows the physicochemical properties of the studied soils and their textural classification. The pH was determined in a proportion of 1: 2.5 soil/water extract. The particle size distribution was evaluated using Bouyoucos densimeter; the calcination or muffling method was used to estimate the OM content of the soil weight loss on ignition (LOI) or calcination, the quantification of OM was determined by K 2 Cr 2 O 7 oxidation, and the manometric method was used to measure the total carbonate content. The PHE-degrading microorganism selected, S. indicatrix CPHE1, corresponds to S. maltophilia CPHE1 (NCBI number: MT138842) published by Lara-Moreno et al. (2021). Subsequent studies indicated greater phylogenetic closeness with the species S. indicatrix rather than S. maltophilia (Lara-Moreno et al., 2023). CPHE1 strain was isolated in our lab from a highly contaminated industrial soil as reported by Lara-Moreno et al. (2021). The sequencing data of S. indicatrix CPHE1 were deposited in the National Center for Biotechnology Information (NCBI), under the BioProject ID PRJNA868539. The whole genome shotgun generated has been deposited in DDBJ/ENA/GenBank under the accession number JANQDV000000000. The version described in this paper is version JANQDV010000000. The raw data was deposited in the Sequence Read Archive (SRA) under the accession number SRR21098193 (Lara-Moreno et al., 2023). 2.2 Methods 2.2.1 Bioinformatic analysis: nucleotide and protein sequence analysis The sequences annotated as monooxygenases, dioxygenases and dehydrogenases were individually aligned against PDB (Protein Data Bank) database (Berman et al., 2000) and BLASTp of NCBI pipeline to identify reference sequences. References proteins were selected based on percentage similarity. Subsequently, the selected reference proteins together with CPHE1 proteins were aligned using Clustal Omega, a multiple sequence alignment program (Sievers et al., 2011). The used format was ClustalW with character counts (alignment format with base/residue numbering). Monooxygenase, dioxygenase, and dehydrogenase phylogenetic trees were constructed using the maximumlikelihood method selected from the package MEGA (Molecular Evolutionary Genetic Analysis) version 7 (Kumar et al., 2016). The employed parameters were the following: JTT matrix, gamma distribution of mutation rates with gamma optimized to 2, and the tree robustness was assessed using 1000 bootstrap replicates. 2.2.2 RNA extraction, DNase treatment, cDNA synthesis, and Real-Time PCR (RT-PCR) analysis from S. indicatrix CPHE1 To evaluate the induction of PHE degrading genes, CPHE1 cells were collected by centrifugation (12,000 rpm for 10 min) at different times (0, 1, 3, 7, 14, and 21 days) of growth, after its cultivation in MSM supplemented with 10 mg L −1 PHE. The supernatant was removed, and RNA was extracted from the bacterial pellet by High Pure RNA Isolation Kit (Roche Molecular System, Switzerland). RNA samples from different times were treated first with DNAse, RNase-free (ThermoFisher Scientific, Massachusetts, EE.UU.), using 3U of DNase for 3 μgofRNA. Then complementary DNA (cDNA) synthesis was conducted using RevertAid First Strand cDNA Synthesis kit (ThermoFisher Scientific, Massachusetts, EE.UU.). The total RNA and the random hexamer primers were denatured at 65 C for 5 min. The remaining reagents (Buffer 5x, 20 U μL −1 of RNase Out, 10 mM dNTPs, and 200 U μL −1 retrotranscriptase enzyme) were added and incubated at 25°Cfor5min,42 °Cfor60min and kept at 70°C for 5 min to inactivate the enzyme. cDNAs were amplified with specificprimers(Supplementary Table S2)for different PCR cycles depending on the analyzed gene (11–13 for 16s rDNA, and 30 cycles for considered degrading genes). 16S rDNA was used as a constitutive control of gene expression in S. indicatrix CPHE1. All RT-PCR experiments were performed in triplicate. Specific PCR primers (Supplementary Table S2) for degrading genes were designed in silico using the program Primer3 (primer3 https://primer3.ut.ee/). Designed primers were checked by primer-BLAST tool. 2.2.3 Phenanthrene degrading-microbial characterization of original soils The total viable PHE degrading-bacteria in the studied soils were enumerated using the unit of measure, colony forming units per Gram of soil (CFU g −1 soil). 1 g of soil was added to 5 mL of MSM, and then 100 µL of the suspension was serially diluted (1: 10). Aliquot (100 µL) of the resultant solutions were spread over petri dishes with MSM agar supplemented with 50 mg L −1 of PHE and incubated at 30°C ± 1. The plate count was conducted after 7days. Frontiers in Bioengineering and Biotechnology frontiersin.org03 Lara-Moreno et al. 10.3389/fbioe.2023.1158177 2.2.4 Inoculum preparation of S. indicatrix CPHE1 for phenanthrene mineralization in soils Stenotrophomonas indicatrix CPHE1 grown in LB agar plate was transferred to Luria−Bertani (LB) broth. The culture was incubated under orbital shaking (160 rpm) at 30°C. After 20 h (at the end of the exponential phase), CPHE1 strain was centrifuged and then washed twice using MSM to remove any LB residues. Then, the cell pellet was resuspended in the necessary volume of MSM to obtain a 10 8 CFU mL −1 density. 2.2.5 Phenanthrene mineralization test in soils Mineralization studies using 14 C-ring-labelled PHE were performed in the five studied soils (PLD, LL, ALC, CR, R) under slurry suspension condition. Tests were performed in triplicate, using respirometers, which consist in a modified 250 mL Erlenmeyers with a soda tramp, containing 1 mL of 0.5 N NaOH. All the microcosm components were sterilized (Matachana steam sterilizer model S100 with one cycle at 120°C, pressure of 101 kPa, for 20 min), except the investigated soil, to preserve their endogenous microbiota. 10 g of each soil were added to Erlenmeyer flasks. Soils were artificially contaminated with a mixture of 14 C-PHE (450 Bq per flask) and unlabeled PHE to obtain a total concentration of 50 mg kg −1 . 0.5 mL of a 1000 mg L −1 PHE stock solution in methanol, which also contained 14 C-PHE (450 Bq), was added to 2.5 g of soil (25% of the total soil) and was stayed under a fume hood for 24 h at room temperature to evaporate the methanol. Subsequently, the remaining soil (7.5 g) was mixed, to avoid damage to soil endogenous microbiota. Finally, 50 mL MSM and 1 mL of SNs were added to Erlenmeyer flask and incubated at 30°C±1 °C, under continuous slow stirring for 120 days. Mineralization studies were conducted as follows: (A) Control: soil without treatment; (B) Biostimulation: soil + NS; (C) Bioaugmentation: soil + NS + S. indicatrix CPHE1 (10 8 CFU g −1 ); (D) Bioavailability enhancer: soil + NS + HPBCD solution (10 times the initial molar concentration of PHE); (E) Mixing techniques of biostimulation, bioaugmentation and bioavailability enhancer: soil + NS + S. indicatrix CPHE1+ HPBCD. Simultaneously, abiotic controls were set adding 200 mg L −1 of HgCl 2 to the soil, to monitor abiotic PHE dissipation. Samples were periodically taken to monitor the formation of 14 CO 2 trapped in the alkali trap. NaOH solution from the trap was mixed with 3 mL of a liquid scintillation cocktail (Ready safe from PerkinElmer, Inc., United States). Samples were kept in darkness for 24 h to dissipate the chemiluminescence. A liquid scintillation counter (Beckman Instruments Inc., Fullerton, California, model L55000TD) was used to measure the radioactivity. The mineralization curves were adjusted to three kinetic models: a simple first-order model (SFO), a biphasic first-order sequential model (Hockey-Stick, HS), and a first-order multi-compartment model (FOMC). These models were selected according to the FOCUS (2006) work group. The Solver tool (included in Microsoft Excel statistical software) and the equations described in Lara-Moreno et al. (2022a) were used, depending on the kinetic model that fits the best to each experimental set of data. Kinetics parameters were obtained from kinetic models: k 1 ,k 2 ,α,β (mineralization rate constants), tb (time at which rate constant changes), and DT 50 (time taken for 50% of substance to disappear by dissipation processes). The chi-square (χ2) test was calculated as an indicator of the goodness of fit(χ2 values should be <15 to mean a good fit). 2.2.6 Phenanthrene availability in soil NS and HPBCD solutions were used to determine their effect on soil PHE bioavailability. For this purpose, 1 g of soil contaminated with 50 mg kg −1 of PHE was added to corex glass centrifuge tubes. Samples were extracted with 5 mL of NS (MSM + SNs) or NS + HPBCD (10 times the molar concentration of PHE previously added in soil). The corex tubes were agitated on an orbital shaker for 72 h at 20°C±1 °C and subsequently centrifuged (10 min, 7000 rpm) to separate the supernatant from the soil (Villaverde et al., 2012). The supernatant was filtered through a 0.22 μm Millipore glass fiber membrane. Then, 3 mL of the sample was taken and placed into a glass tube with 2 mL of hexane, which was shaken using a vortex device for 1 min and for 5 min in an ultrasonic bath. The hexane phase was placed in 2.5 mL glass vials. PHE concentration was measured using a gas chromatographer (Agilent GC 6890N) equipped with a mass spectrometer (MS, Agilent MD 5975B). The analytical method was described by Lara-Moreno et al. (2021). 3 Results and discussion 3.1 Bioinformatic analysis of genes involved in phenanthrene degradation pathways Lara-Moreno et al. (2023) provided relevant information about the genome. The genome of S. indicatrix CPHE1 deposited in NCBI (JANQDV000000000) (Lara-Moreno et al., 2023) consists of 163 contigs, with a size of 4.553.664 bp, 66.1% of Guanine- Cytosine (G-C) content, and 4137 genes described. Genes potentially associated with PHE biodegradation were bioinformatically predicted in S. indicatrix CPHE1 genome by two diverse strategies: (i) searching for specific annotation of gene products by RAST server and subsequently clustering them into phylogenetic trees using reference sequences; a bibliographic and database search of enzymes of other bacteria has been carried out to identify reference sequences. Data obtained allowed the improvement of the genome automatic annotation of the CPHE1 strain genes. Similarities between the whole set of selected enzymes have been analyzed by Clustal Omega obtaining a phylogenetic analysis. (ii) Analyzing the sequence homology with respect to reference sequences of bacterial strains able to biodegrade PHE (Cébron et al., 2008;Izmalkova et al., 2013) or bacteria frequently detected in the water systems by other studies (Stingley et al., 2004;Elufisan et al., 2020). Considering the first approach, Supplementary Figure S1 shows the distribution of the annotated genes in different subsystems of S. indicatrix CPHE1 genome. Particular attention was focused on the subsystem of genes that participate in the aromatic metabolism compounds (boxed in red); these annotated genes could take part in the central catabolism of the metabolites formed during the degradation of aromatic compounds. Beta-ketoadipate pathway is an aromatic compound degradation route widely distributed in soil bacteria and fungi. On the one hand, catechol 1,2-dioxygenase converts catechol generated from aromatic hydrocarbons into Frontiers in Bioengineering and Biotechnology frontiersin.org04 Lara-Moreno et al. 10.3389/fbioe.2023.1158177 intermediates of the tricarboxylic acid cycle (TAC). On the other hand, protocatechuic acid is turned into intermediates of the TAC catalyzed by protocatechuate 3,4-dioxygenase. Both enzymes act by cleaving aromatic ring. Catechol-1,2-dioxygenase, has been studied in species belonging to Pseudomonas genus due to its ability to cleave the aromatic ring of catechol molecule (Rodríguez-Salazar et al., 2020). Other annotated genes in the S. indicatrix CPHE1 genome could encode enzymes that participate in the salicylate and gentisate pathway, compounds that have been identified as intermediaries of PAH catabolism (Grund et al., 1992). The salicylic acid pathway has been described for several PHE-degrading bacterial genera or other PAHs, such as Pseudomonas (Lin et al., 2014), Streptomyces (Ishiyama et al., 2004), or Stenotrophomonas (Huang et al., 2013). Several authors have described monooxygenases (Medić et al., 2020) and dioxygenases (Moody et al., 2005;Gao et al., 2013;Kumari et al., 2017) as enzymes responsible for catalyzing the initial reaction of PAHs degradation pathway to produce hydrodiol or dihydrodiol. For this reason, we initially focused on the study of genes annotated as monooxygenases in the genome of S. indicatrix CPHE1. A phylogenetic analysis was carried out using the proteins reported in Table 1 (Figure 1). The phylogenetic tree demonstrated no similarities between the annotated CPHE1 sequences and the reference monooxygenase (AlkB,UniProtKB: A4XPE8), described in Pseudomonas aeruginosa P6 as responsible for petroleum hydrocarbon degradation (Wang et al., 2019;Medićet al., 2020). Since the other reference sequences were not involved in PAH degradation pathways, it has been ruled out that monooxygenases encode the first step in the PHE degradation. A similar approach was then undertaken for another enzyme class since the initial reaction of PAH biodegradation in S. indicatrix CPHE1 could be catalyzed by a multicomponent system of a dioxygenase, producing dihydrodiol (Kumari et al., 2017). This hypothesis is supported by two fundamental facts; first, the presence of similar dioxygenases has been detected in several PAHs degrading bacteria such as Pseudomonas (Prabhu and Phale, 2003;Cébron et al., 2008), Mycobacterium (Stingley et al., 2004;Moody et al., 2005), and Stenotrophomonas (Kumari et al., 2017). Second, the metabolite dihydroxyphenanthrene was identified during the PHE biodegradation of S. indicatrix CPHE1 (Lara-Moreno et al., 2021). These data would suggest the intervention of dioxygenase in PHE biodegradation. Forthisreason,ninedioxygenasesannotatedinthegenomeof S. indicatrix CPHE1 have been exhaustively analyzed and compared with similar dioxygenases of bacterial genomes deposited in several databases. All dioxygenases have been compared by multiple alignments with reference proteins (Table 2), including two dioxygenases known for their involvement in naphthalene biodegradation (NAHAc of P. putida G7 and NIDA of the only naphthalene dioxygenase described in Stenotrophomonas)(Cébron et al., 2008;Kumari et al., 2017).AsshowninthetreeinFigure 2, the enzyme DO2 is the closest phylogenetically to the reference proteins (NAHAc and NIDA). However, annotated dioxygenases have been considered for the subsequent RT-PCR expression experiments since there are no conclusive results to exclude any of them as putative responsible for deoxygenation. After the analysis of the possible dioxygenases, a thorough analysis of the annotated dehydrogenases of S. indicatrix CPHE1 was performed, since the dihydrodiol dehydrogenase enzyme catalyzes the second step of the PHE biodegradation route described by Stingley et al. (2004). This enzyme removes TABLE 1 Monooxygenases annotated in S. indicatrix CPHE1 genome and reference monooxygenases (literature, pdb, NCBI, etc.). Name Function Strain References MO1 Putative monooxygenase Stenotrophomonas indicatrix CPHE1 - F_MO Flavin-dependent monooxygenase ArsO associated with arsenic resistance Stenotrophomonas indicatrix CPHE1 - F_MO1 Flavin-dependent monooxygenase ArsO associated with arsenic resistance Stenotrophomonas maltophilia Jensen et al. (2012) F_MO2 Flavin-dependent monooxygenase ArsO associated with arsenic resistance Stenotrophomonas maltophilia Jensen et al. (2013) FAD_MO monooxygenase, FAD-binding Stenotrophomonas indicatrix CPHE1 - KYN_MO Kynurenine 3-monooxygenase Stenotrophomonas indicatrix CPHE1 - KYN_MO Kynurenine 3-monooxygenase Pseudomonas fluorescens KMOs Kim et al. (2018) NMOA_MO Nitrilotriacetate monooxygenase component A Stenotrophomonas indicatrix CPHE1 - NMOA_MO Nitrilotriacetate monooxygenase component A Mycolicibacterium smegmatis MC2 155 Zhang et al. (2011) NMOA_MO Nitrilotriacetate monooxygenase component A Aminobacter aminovorans ATTCC 29600 Knobel et al. (1996) LUX_MO Luciferase-like monooxygenase YhbW Streptomyces bottropensis Maier et al. (2014) LUX_MO_1 Luciferase-like monooxygenase YhbW Stenotrophomonas indicatrix CPHE1 - LUX_MO_2 Luciferase-like monooxygenase YhbW Stenotrophomonas indicatrix CPHE1 - NP_MO 2-nitropropane monooxygenase Stenotrophomonas indicatrix CPHE1 - NP_MO 2-nitropropane monooxygenase Pseudomonas aeruginosa PA01 Salvi et al. (2014) ALKB Alkane-1-monooxygenase Pseudomonas aeruginosa P6 Wang et al. (2019) Frontiers in Bioengineering and Biotechnology frontiersin.org05 Lara-Moreno et al. 10.3389/fbioe.2023.1158177 TABLE 2 Dioxygenases annotated in S. indicatrix CPHE1 genome and reference dioxygenases (literature, pdb, NCBI, etc.). Name Function Strain References CYS_DO Cysteine dioxygenase Stenotrophomonas indicatrix CPHE1 - 3-MPA_DO 3-Mercaptopropionato dioxygenase Pseudomonas aeruginosa Fellner et al. (2016) TPH_DO Tryptophan 2,3-dioxygenase Stenotrophomonas indicatrix CPHE1 - TPH_DO Tryptophan 2,3-dioxygenase Xanthomonas campestris TDO Forouhar et al. (2007) HPP_DO 4-hydroxyphenylpyruvate dioxygenase Stenotrophomonas indicatrix CPHE1 - HPP_DO 4-hydroxyphenylpyruvate dioxygenase Streptomyces avermitilis Brownlee et al. (2004) HPP_DO 4-hydroxyphenylpyruvate dioxygenase uncultured soil bacterium Lee et al. (2008) HPP_DO 4-hydroxyphenylpyruvate dioxygenase Stenotrophomonas maltophilia SVIA2 Elufisan et al. (2019) HPP_DO 4-hydroxyphenylpyruvate dioxygenase Pseudomonas putida KT2440 Kim et al. (2006) HAO_DO 3-hydroxyanthranilate 3,4-dioxygenase Stenotrophomonas indicatrix CPHE1 - HAO_DO 3-hydroxyanthranilate 3,4-dioxygenase Pseudomonas fluorescens strain KU-7 Muraki et al., 2013 HMG_DO Homogentisate 1,2-dioxygenase Stenotrophomonas indicatrix CPHE1 - HMG_DO Homogentisate 1,2-dioxygenase Pseudomonas putida KT2440 kim et al., 2006 DO2 Putative dioxygenase Stenotrophomonas indicatrix CPHE1 - ARD_DO 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase Stenotrophomonas indicatrix CPHE1 - ARD_DO 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase Klebsiella pneumoniae Dai et al. (2001) ɣBBH_DO Gamma-butyrobetaine dioxygenase Stenotrophomonas indicatrix CPHE1 - NP_DO 2-nitropropane dioxygenase Stenotrophomonas indicatrix CPHE1 - NP_DO 2-nitropropane monooxygenase Pseudomonas aeruginosa PAO1 Salvi et al. (2014) NIDA naphthalene dioxygenase Stenotrophomonas IITR87 Kumari et al. (2017) NAHAc naphthalene dioxygenase Pseudomonas putida G7 Cébron et al. (2008) FIGURE 1 Maximum Likelihood tree based on the monooxygenase protein sequences annotated in the genome of S. indicatrix CPHE1 and on the reference monooxygenases involved in the degradation of polycyclic aromatic hydrocarbons. Bootstrap values (>30%) are expressed as percentages of 1000 replicates. Same branches representation was recovered by the neighbor-joining and maximum-parsimony algorithms. The scale bar indicates 1 substitution per nucleotide position in protein sequence tree. Phylogenetic tree was built using MEGA 6. Frontiers in Bioengineering and Biotechnology frontiersin.org06 Lara-Moreno et al. 10.3389/fbioe.2023.1158177 two hydrogen atoms from the molecule, oxidizing it, the electrons and protons from the hydrogen atoms are captured by the oxidized form of the coenzyme, which is reduced. Among 119 CPHE1 proteins annotated as dehydrogenases, nine were selected based on their function in relation to the biodegradation of PAHs (Table 3). These amino acid (aa) sequences were analyzed in a phylogenetic tree using diverse dehydrogenase references, including dehydrogenases known as catalysts of the second step of the PAHs biodegradation pathway (NahB, BphB, and NidD). Two different trees were obtained based on the considered aa references (Figure 3); the first tree (Figure 3A) comprises dihydrodiol naphthalene dehydrogenase (NahB) described for P. stutzeri AN10, naphthalene dehydrogenase (NahB) of P. putida AK5, and cis-2,3-dihydrobiphenyl-2,3-diol dehydrogenase (BphB) of P. putida 9816-4. Figure 3B includes naphthalene dehydrogenase (NidD) described in Stenotrophomonas sp. IITR87 which resulted in phylogenetically close to five (out of nine) CPHE1 dehydrogenases. Although PABA DH (betaine aldehyde dehydrogenase) and SUCB (oxoglutarate dehydrogenase dihydrolipoamide succinyltransferase component) were the closest CPHE1 sequences to the respective reference proteins (for each tree), it should be noted that reference proteins belong to different bacterial genera, consistent in a wide protein sequences variability (Keshavarz-Tohid et al., 2019). 3.2 Identification of the involvement of dioxygenases of S. indicatrix CPHE1 in phenanthrene degradation by RT-PCR experiments Studies of gene expression have been carried out by RT-PCR experiments to know which annotated dioxygenase of S. indicatrix CPHE1 was expressed throughout the PHE biodegradation process after inoculating CPHE1 strain. Validation of the samples is reported in Supplementary Figure S2 showing the PCR amplification of 16S rDNA gene using genomic DNA (positive control) from S. indicatrix CPHE1, cDNA obtained from RNA extracted from CPHE1 strain without contact with PHE (t 0 ) and RNA samples taken at different times from the PHE biodegradation assay (t 1 ,t 3 ,t 7 ,t 14 , and t 21 ). Several authors have used the semi-quantitative RT-PCR to confirm the involvement of genes in the biodegradation pathway of various pollutants. For example, Thanh et al. (2019) observed the expression of dbfA1A2RBC genes that share 99%–100% identity with Paenibacillus sp. YK5 genes (putatively involved in dibenzofuran degradation). The induction of four of these genes was observed after treatment with dibenzofuran through RT-PCR. Di Canito et al. (2018) studied the role of Rhodococcus opacus R7 oxygenases in the o-xylene degradation. The important involvement of akb genes was demonstrated using RNA obtained from R7 cells in the presence of o-xylene by RT-PCR. The FIGURE 2 Maximum Likelihood tree based on the dioxygenase protein sequences annotated in the genome of S. indicatrix CPHE1 and in the reference dioxygenases involved in the degradation of polycyclic aromatic hydrocarbons. Bootstrap values (>30%) are expressed as percentages of 1000 replicates. Same branches representation was recovered by the neighbour-joining and maximum-parsimony algorithms. The scale bar indicates 1 substitution per nucleotide position in protein sequence tree. Phylogenetic tree was built using MEGA 6. Frontiers in Bioengineering and Biotechnology frontiersin.org07 Lara-Moreno et al. 10.3389/fbioe.2023.1158177 result showed that the transcription was induced by the contaminant. In another study, Churchill et al. (2008) checked the ability of Mycobacterium sp. CH-2 to mineralize PHE, pyrene, and FLT. Primers were designed based on the dioxygenases nidAB and pdoA2B2 and an alkane monooxygenase. RT-PCR analysis indicated that alkane monooxygenase is constitutively expressed, however, nidAB, and pdoA2B2 were expressed only in the presence of PAHs. The results of the CPHE1 dioxygenase gene expression are shown in Figure 4 obtained with specific primers designed on CPHE1 genes reported in Table 2. At the top of the figure are presented the results of the PCR when the 16S rRNA gene was amplified under non-limiting conditions, which serves as a reference gene. Signal intensity quantification (ImageJ.JS) of PCR results indicated that the cDNA of the t 0 ,t 1 ,t 3 , and t 21 samples are the least concentrated, followed by the t 14 sample. The t 7 sample was twice more concentrated. These data will allow for assessing the expression levels of the rest of the studied genes. A previous study carried out by Lara-Moreno et al. (2023) showed that the gene annotated as cysteine dioxygenase (cysDO)wasexpressed in the presence of PHE from the third day of treatment. The KEGG database indicates that the cysDO enzyme is involved in different biodegradation pathways of aromatic compounds, including naphthalene (M00534), salicylic acid (M00638) and phthalic acid (M00623). In addition, Wang et al. (2017) studied the participation of cysDO enzyme in the degradation of aromatic compounds. However, in the currently work, gene expression study has been expanded, all dioxygenases annotated in the genome of CPHE1 strain were analyzed with the aim to know their involvement in PHE biodegradation pathway. The electrophoresis gel obtained is shown in Figure 4. Gene expression of 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase (ardDO), gamma-butyrobetaine dioxygenase (bbhDO) or putative dioxygenase (DO2) was not detected. In the case of tryptophan-2,3-dioxygenase (tphDO), homogentisate dioxygenase (hmgDO), 4-hydroxyphenylpyruvate dioxygenase (hppDO), and nitropropan dioxygenase (npDO) genes, a repression of expression was observed in the presence of PHE. And in case of 3- hydroxyanthranilate 3,4-dioxygenase a constituent expression was detected. It concludes that cysDO was the only one expressed in the TABLE 3 Dehydrogenases annotated in S. indicatrix CPHE1 genome and reference dehydrogenases (literature, pdb, NCBI, etc.). Name Function Strain References MDH Malate dehydrogenase Stenotrophomonas indicatrix CPHE1 - MDH Malate dehydrogenase Pseudomonas putida ATCC12633 Muramatsu et al. (2004) MDH Malate dehydrogenase Aquaspirillium arcticum Kim et al. (1998) SUC_B Dihydrolipoamide succinyltransferase component (E2) of 2- oxoglutarate dehydrogenase complex Stenotrophomonas indicatrix CPHE1 - SUC_B Dihydrolipoamide succinyltransferase component (E2) of 2- oxoglutarate dehydrogenase complex Pseudomonas putida NBRIC19 Mishra et al. (2001) SUC_B Dihydrolipoamide succinyltransferase component (E2) of 2- oxoglutarate dehydrogenase complex Escherichia coli k12 Spencer et al. (1984) NAD_FAD_DH NAD(FAD)-utilizing dehydrogenases Stenotrophomonas indicatrix CPHE1 - NAD_FAD_DH NAD(FAD)-utilizing dehydrogenases Streptococcus oligofermentas Molla et al. (2014) NAH_B Cis-dihydrodiol naphthalene dehydrogenase Pseudomonas stutzeri AN10 Bosch et al. (1999) NAH_B naphthalene dehydrogenase Pseudomonas putida AK5 Izmalkova et al. (2013) BPH_B Cis-2,3-dihydrobiphenyl-2,3-diol dehydrogenase Pseudomonas putida 9816-4 Dennis and Zylstra (2004) ARO_E Shikimate 5-dehydrogenase I alpha Stenotrophomonas indicatrix CPHE1 - ARO_E Shikimate 5-dehydrogenase I alpha Escherichia coli K12 Michel et al. (2003) ARO_E Shikimate 5-dehydrogenase I alpha Pseudomonas putida KT2440 Peek et al. (2014) ALDH_1 Aldehyde dehydrogenase Stenotrophomonas indicatrix CPHE1 - NID_D Naphtalene dehydrogenas Stenotrophomonas sp. IITR87 Kumari et al. (2017) ALDH_2 Aldehyde dehydrogenase Stenotrophomonas indicatrix CPHE1 - ABALDH Gamma-glutamyl-aminobutyraldehyde dehydrogenase Stenotrophomonas indicatrix CPHE1 - ABALDH Gamma-glutamyl-aminobutyraldehyde dehydrogenase Pseudomonas syringae DC3000 McClerklin et al. (2018) PABADH Betaine aldehyde dehydrogenase Stenotrophomonas indicatrix CPHE1 - PABADH Betaine aldehyde dehydrogenase Pseudomonas aeruginosa Gonzalez-Segura et al. (2009) PHD Pyruvate dehydrogenase Stenotrophomonas indicatrix CPHE1 - PHD Pyruvate dehydrogenase Pseudomonas putida Frank et al. (2005) Frontiers in Bioengineering and Biotechnology frontiersin.org08 Lara-Moreno et al. 10.3389/fbioe.2023.1158177 presence of PHE. These results suggest the involvement of a deoxygenation in the PHE degradation pathway when S. indicatrix CPHE1 was inoculated. In addition, as indicated above, the presence of dihydrodiol metabolites was detected after inoculating CPHE1 strain (Lara-Moreno et al., 2021). These results suggest the involvement of a dioxygenation in the PHE degradation pathway when S. indicatrix CPHE1 was inoculated. 3.3 Phenanthrene degrading-genes expression Beside the dioxygenase gene expression, the expression of other genes involved in PHE biodegradation route was investigated relying on the second bioinformatic approach based on the comparison of S. indicatrix CPHE1 genes with sequences belonging to bacterial strains known for their involvement in PHAs biodegradation. Indeed, Stingley et al. (2004) described the most common PHE biodegradation pathways for Mycobacterium vanbaalenii strain PYR-1. These genes were compared to the only two genomic studies (Kumari et al., 2017;Elufisan et al., 2020) that have been published on PHE degradation route in Stenotrophomonas genus. For instance, the genes involved in this degradation pathway showed at least 97% identity compared with Stenotrophomonas IITR87 genome (Kumari et al., 2017). Thus, CPHE1 genes showing a high identity with respect to the genes described in the M. vanbaalenii PYR-1 genome were taken into consideration as references for the gene expression studies in S. FIGURE 3 Maximum Likelihood tree based on the dehydrogenase protein sequences annotated in the genome of S. indicatrix CPHE1 and reference dehydrogenases NAHB, BPHB, and NIDD (A), and NIDD (B) involved in the degradation of polycyclic aromatic hydrocarbons. Bootstrap values (>30%) are expressed as percentages of 1000 replicates. Same branches representation was recovered by the neighbour-joining and maximum-parsimony algorithms. The scale bar indicates 1 or 0.5 substitution per nucleotide position in protein sequence tree. Phylogenetic tree was built using MEGA 6. Frontiers in Bioengineering and Biotechnology frontiersin.org09 Lara-Moreno et al. 10.3389/fbioe.2023.1158177 different mineralization results depending on soil properties.It is remarkable that S. indicatrix has never been described as PHE mineralizing bacterium in soils. Several authors have isolated PHE-degrading bacteria; however, PAHs contamination remains a major global concern, being biostimulation and bioaugmentation the most useful strategies to conduct decontamination of PAHs polluted environment. In soils with an active microbiota to degrade PHE (soils PLD and R), the only addition of NS was sufficient to achieve PHE mineralization. On the contrary, in the cases of LL, ALC, and CR soils, the inoculation of S. indicatrix CPHE1 was crucial to achieving an improvement in PHE mineralization kinetics. HPBCD application provoked an improvement in PHE biodegradation rate in LL, ALC, CR, and R soils, and it was observed that HPBCD was able to achieve better results in the mineralization of PHE in soils with a high OM content. However, the combined application of HPBCD and S. indicatrix CPHE1 evidenced a negative effect presumably due to CPHE1 strain preference for CD instead of PHE as a primary carbon source. In general, selecting the best bioremediation strategy based on the physicochemical and microbial properties of each treated soil has been crucial. Data availability statement The sequencing data generated were deposited in the National Center for Biotechnology Information (NCBI), under the BioProject ID PRJNA868539. The whole genome shotgun of S. indicatrix CPHE1 has been deposited in DDBJ/ENA/GenBank under the accession number JANQDV000000000. The version described in this paper is version JANQDV010000000. The raw data was deposited in the Sequence Read Archive (SRA) under the accession number SRR21098193. Author contributions AL-M: conceptualization, methodology, investigation, formal analysis, data curation, writing—original draft. FM: methodology, resources, supervision. EM: writing—review and editing, funding acquisition. JZ: investigation, writing—review and editing. PD: supervision, writing—review and editing. JV:methodology, supervision, writing—review and editing. Funding This work was supported by the Spanish Ministry of Economy and Competitiveness under the research project CMT 2017-82472- C2-1-R (AEI/FEDER, UE) and by Junta de Andalucía (Council of Economic Transformation, Industry, Knowledge, and University) under the research project PY20_01069 (FEDER Andalucía PO 2014–2020). Acknowledgments AL-M acknowledges the Spanish Ministry of Education, Culture and Sports for her FPU fellowship (FPU15/03740), the Erasmus + traineeship program and University of Seville for her Margarita Salas grant funded by the European Union’sNext- Generation EU. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe.2023.1158177/ full#supplementary-material FIGURE 9 PHE extracted from PLD, LL, ALC, CR, R soil in presence of H20, nutrient solution (NS) and HPBCD. Frontiers in Bioengineering and Biotechnology frontiersin.org16 Lara-Moreno et al. 10.3389/fbioe.2023.1158177 References Abdel-Shafy, H. I., and Mansour, M. S. M. (2016). A review on polycyclic aromatic hydrocarbons: Source, environmental impact, effect on human health and remediation. Egypt. J. Pet. 25 (1), 107–123. doi:10.1016/j.ejpe.2015.03.011 Adeleke, B. S., Ayangbenro, A. S., and Babalola, O. O. (2021). Genomic assessment of Stenotrophomonas indicatrix for improved sunflower plant. Curr. Genet. 67, 891–907. doi:10.1007/s00294-021-01199-8 Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., et al. (2000). The protein Data Bank. 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