J. Microbiol. Biotechnol. (2018), 28(2), 330–337 https://doi.org/10.4014/jmb.1709.09002 Research Article Review jmb

Genome Analysis of Naphthalene-Degrading Pseudomonas sp. AS1 Harboring the Megaplasmid pAS1 S Jisun Kim and Woojun Park*

Laboratory of Molecular Environmental Microbiology, Department of Environmental Sciences and Ecological Engineering, Korea University, Seoul 02841, Republic of Korea

Received: September 1, 2017 Revised: November 8, 2017 Polycyclic aromatic hydrocarbons (PAHs), including naphthalene, are widely distributed in Accepted: November 21, 2017 nature. Naphthalene has been regarded as a model PAH compound for investigating the First published online mechanisms of bacterial PAH biodegradation. Pseudomonas sp. AS1 isolated from an arsenic- November 25, 2017 contaminated site is capable of growing on various aromatic compounds such as naphthalene,

*Corresponding author salicylate, and catechol, but not on gentisate. The genome of strain AS1 consists of a Phone: +82-82-3290-3067; 6,126,864 bp circular chromosome and the 81,841 bp circular plasmid pAS1. Pseudomonas sp. Fax: +82-2-953-0737; E-mail: [email protected] AS1 has multiple dioxygenases and related enzymes involved in the degradation of aromatic compounds, which might contribute to the metabolic versatility of this isolate. The pAS1 plasmid exhibits extremely high similarity in size and sequences to the well-known naphthalene-degrading plasmid pDTG1 in Pseudomonas putida strain NCIB 9816-4. Two gene clusters involved in the naphthalene degradation pathway were identified on pAS1. The expression of several nah genes on the plasmid was upregulated by more than 2-fold when naphthalene was used as a sole carbon source. Strains have been isolated at different times and places with different characteristics, but similar genes involved in the degradation of S upplementary data for this aromatic compounds have been identified on their plasmids, which suggests that the paper are available on-line only at http://jmb.or.kr. transmissibility of the plasmids might play an important role in the adaptation of the microorganisms to mineralize the compounds. pISSN 1017-7825, eISSN 1738-8872

Copyright© 2018 by Keywords: Pseudomonas frederiksbergensis, naphthalene degradation, polycyclic aromatic The Korean Society for Microbiology hydrocarbons (PAHs), horizontal gene transfer, plasmid and Biotechnology

Polycyclic aromatic hydrocarbons (PAHs) and their contaminated site in Gwangju, Kyunggi-Do, South Korea derivatives are widespread in the natural environment [1- [7]. Pseudomonas sp. AS1 is able to grow on naphthalene, 3] and can contaminate the ecosystem for a long time as a salicylate, and catechol, but not on gentisate (Fig. 1) [7]. In result of their low solubility in water and their absorption our previous study, PCR analysis using degenerate primers to small particles [2, 4]. Various bacterial strains have been showed that partial regions of the genes involved in discovered that degrade low-molecular-weight PAHs as naphthalene degradation, which include nahA (encoding part of their metabolism [5-8]. One of the simplest PAHs is naphthalene dioxygenase), nahG (encoding salicylate naphthalene, which has been widely studied and referred hydroxylase), and nahR (encoding LysR-type regulator) in to as a model compound for investigating the mechanisms Pseudomonas sp. AS1, were identical to those of P. putida of bacterial biodegradation [3, 5]. Microbial naphthalene NCIB 9816-4 [7]. Thus, Pseudomonas sp. AS1 might possess metabolism and the genetic regulations involved in the the classical naphthalene degradation pathway in which degradation pathway are extensively characterized in salicylate can be converted into acetyl coenzyme A and several bacterial strains, particularly the Pseudomonas pyruvate. Salicylate is the main secreted metabolic species [9-13]. intermediate of Pseudomonas sp. AS1 in the presence of Pseudomonas sp. AS1 was first isolated from an arsenic- naphthalene as the sole carbon source, which has been

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Fig. 1. Test of Pseudomonas sp. AS1 growth under various aromatic hydrocarbons. (A) Growth support of naphthalene-degrading Pseudomonas sp. AS1 toward non-naphthalene-degrading A. oleivorans DR1. Cells were inoculated alone or mixed in minimal basal salts (MSB medium containing 0.5% naphthalene as the sole carbon source). Plating on MSB plate with selective substrates (2% glucose for Pseudomonas sp. AS1 and 2% paraffin for A. oleivorans DR1) was conducted to calculate the CFU. (B-L) Growth of Pseudomonas sp. AS1 with various aromatic hydrocarbons. Cells were grown at 30°C in MSB medium supplemented with various compounds and

OD600 values were measured at the indicated time points. shown by NMR spectroscopy and high-performance liquid during commensal metabolic interactions might be passively chromatography [8]. Microbial interaction in naphthalene- taken up by A. oleivorans DR1, where it functions as a amended conditions between Pseudomonas sp. AS1 and growth-supporting factor by inducing salicylate hydroxylase Acinetobacter oleivorans DR1, a bacterial strain that cannot [8]. degrade naphthalene, was found to be mediated by salicylate, To obtain the detailed genetic information involved in which was able to promote the growth of A. oleivorans DR1 naphthalene degradation, we sequenced the whole genome (Fig. 1) [8]. The salicylate generated by Pseudomonas sp. AS1 of Pseudomonas sp. AS1. Genomic DNA was extracted using

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Table 1. General features of the Pseudomonas sp. AS1 genome. Genetic element Chromosome Plasmid (pAS1) Size (bp) 6,126,864 81,841 GC content (%) 58.9 % 56.2 % Total coding DNA sequences 5,634 99 rRNA genes (5S, 16S, 23S) 8, 7, 7 0 tRNA genes 66 0 No. of CRISPR sequences 1 0 No. of genomic islands 40 5 GenBank Accession No. CP018319 CP018320

the Wizard Genomic DNA purification Kit (Promega, USA). Whole-genome sequencing was performed by the Pacific Biosciences RSII Single Molecule Real Time (SMRT) sequencing technology (Pacific Biosciences, USA) with a SMRTbell template library. A total of 184,024 reads with a mean read length of 8,088 bp were generated. The complete genome was assembled using the Hierarchical Genome Assembly Process ver. 3.0, and gene annotation was achieved by the NCBI Prokaryotic Genomes Automatic Annotation Pipeline. The genomic features of Pseudomonas sp. AS1 are summarized in Table 1, and consist of a single circular chromosome with a circular plasmid (Fig. 2). The sizes of the chromosome and plasmid were 6,126,864 bp with a GC content of 58.9%, and 81,841 bp with a GC content of 56.2%, respectively. The Pseudomonas sp. AS1 chromosome contains 5,634 coding sequences (CDS), 66 tRNA genes, 22 rRNA genes, and 158 pseudogenes. There are 99 CDS in the plasmid region. The predicted genes were functionally characterized using Pfam [14], Cluster of Orthologous Groups (COG) [15, 16], and KEGG [17] databases; 82.47%, 70.08%, and 28.28% of the CDS were functionally assigned on the basis of Pfam, COG, and KEGG, respectively. A total of 5,735 CDS were classified using the COG database [15, 16]. The COG functional classification is shown in Fig. S1. The most abundant group is involved in amino acid metabolism and transport (COG category E, 7.4%), followed by a group involved in transcription (COG category K, 6.9%). Fig. 2. Circular map of the chromosome and the plasmid of This pattern of COG classification was commonly found in Pseudomonas sp. AS1. soil or water-derived pollutant-degrading Pseudomonas From the outside to the center: genes on the forward strand (colored strains having genomes approximately 6.1-6.3 Mbp in by COG categories), genes on the reverse strand (colored by COG size (Fig. S2). The clustered regularly interspaced short categories), RNA genes (tRNAs, green; rRNAs, red), GC content, and palindromic repeat (CRISPR) gene sequences were found GC skew. using Integrated Microbial Genomes (IMG; https:// img.jgi.doe.gov/). CRISPRs are elaborate defense strategies Pseudomonas sp. AS1 contained only one possible CRISPR against bacteriophage predation [18]. The genome of consisting of 188 bp in size that possessed two spacers

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without predicted cas gene (Tables 1 and S1). The 45 habitat. Thus, the presence of CRISPR, the large number of genomic islands were identified in the genome sequence of genomic islands, and putative horizontally transferred Pseudomonas sp. AS1 using the online integrated GI genes in the genome of Pseudomonas sp. AS1 suggest that prediction tool (IslandViewer 4; http://www.pathogenomics. complex genetic rearrangements may have occurred as a sfu.ca/islandviewer/). The GC contents of predicted GIs consequence of adaptive evolution of this strain. (44.4% to 62.6%) were quite different from that of the whole The plasmid in Pseudomonas sp. AS1 (hereafter referred chromosome and plasmid of Pseudomonas sp. AS1 (58.9% to as pAS1) contains the IncP-9 plasmid backbone, which and 56.2%, respectively) (Table S2). Predicted GIs were has a region involved in plasmid transfer (tra, position involved in transport, replication, and hydrocarbon 51908-57689), conjugation (mpf, position 60,813-71,545), degradation (Table S2). Many of the GIs carrying genes plasmid partitioning (par, position 70,247-73,535), and encoding mobile elements, such as integrases and plasmid replication including oriV (rep, position 73,536- transposases, provide evidence regarding the genomic 75,269) (Fig. 3). Interestingly, pAS1 exhibits striking plasticity of Pseudomonas sp. AS1 as a result of horizontal similarities in size and sequence to the pDTG1 plasmid that gene transfers and genetic rearrangements in the genome carries naphthalene catabolic genes (nah) from Pseudomonas of Pseudomonas sp. AS1. The IMG database predicted 18 putida strain NCIB 9816-4 [9]. Five mismatch nucleotides in genes to be putative horizontally transferred genes in the the nah upper pathway were identified as well as 1,201 bp genome sequence of Pseudomonas sp. AS1 (Table S3). Most in differences between pDTG1 and pAS1. Five missing of the genes listed in Table S3 were possibly transferred nucleotides corresponding to positions 26,623, 55,994, from other proteobacteria. Pseudomonas sp. AS1 might 58,070, 60,906, and 62,294 of pDTG1 were identified in pAS1. acquire several genes from different donors and finally The most distinct difference between the two plasmids was have the current set of genome contents appropriate for its a 1,196 bp region absent in pAS1 corresponding to position

Fig. 3. (A) Alignment of the linear maps of the plasmids pDTG1 and pAS1 with a focus on the naphthalene degradation pathway and (B) expression analysis of nah genes in Pseudomonas sp. AS1 under naphthalene degradation.

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49,370-50,565 of pDTG1, which has an insertion sequence with P. frederiksbergensis ERDD5:01 (84.6%), P. frederiksbergensis element (transposase) between nahT and nahG (encoding BS3655 (97.3%), and P. frederiksbergensis SI8 (82.2%), salicylate hydroxylase) consisting of the naphthalene suggesting Pseudomonas sp. AS1 is closely related to catabolic pathway genes [9]. A set of genes involved in the P. frederiksbergensis BS3655. P. frederiksbergensis strains are degradation of naphthalene may have been inserted into commonly capable of degrading aromatic compounds [7, each backbone of pDTG1 and pAS1 at different sites 20, 21]. Pseudomonas sp. AS1 can grow in MSB medium because plasmids have been found individually, indicating supplemented with various aromatic compounds, including multiple independent acquisitions of the degradation trait the aforementioned naphthalene as a sole carbon source, as and global distribution of the IncP-9 plasmid backbone in well as catechol, benzene, sodium salicylate, salicylic acid, the environment. salicylaldehyde, benzoic acid, 3-hydroxylbenzoic acid, 4- Naphthalene degradation is organized into upper and hydroxylbenzoic acid, and 3,4-dihydroxylbenzoate (Fig. 1). lower pathways [3]. The upper pathway enzymes are There was no growth in the presence of hexadecane and involved in the conversion of naphthalene to salicylate. dodecane, which might be due to the absence of alkane This pathway comprises 10 genes organized in the order monooxygenase required for alkane degradation. Further nahAaAbAcAdBFCQED. The lower pathway enzymes are studies on alkane degradation by Pseudomonas sp. AS1 are encoded by nahGTHINLOMKJY and are involved in the necessary because alkane degradation generally depends oxidation of salicylate to pyruvate and acetyl coenzyme A on the chain length of alkanes and involvement of [3]. According to our genome sequencing and analysis, cytochrome P450s and hydroxylases with unknown pAS1 has all 21 genes consisting of the upper and lower function. Pseudomonas sp. AS1 has an abundance of genes pathways as well as the nahR-encoding transcriptional encoding hydroxylases and related genes, including regulator required for the induction of the nah genes monooxygenase and dioxygenase, genes that might enable (Fig. 3). To determine the expression of genes involved in the high utilization of various aromatic compounds (Table 2). naphthalene degradation, we compared the mRNA The Pseudomonas sp. AS1 genome revealed 34 dioxygenases, expression levels of Pseudomonas sp. AS1 via quantitative including benzoate dioxygenase (PFAS1_RS22900, reverse transcription-PCR (qRT-PCR) under two growth PFAS1_RS22905, and PFAS1_RS22910), protocatechuate conditions: in the presence of naphthalene as a sole carbon 3,4-dioxygenase (PFAS1_RS18060 and PFAS1_RS18065), and source and under glucose-growing conditions. The qRT- catechol 1,2-dioxgenase (PFAS1_RS22890). Three homologs PCR was conducted as described previously [19]. The cells of genes coding for taurine dioxygenases (PFAS1_RS09965, were incubated until the exponential phase (OD600 ~ 0.3), at PFAS1_RS10310, and PFAS1_RS23020) and eight predicted which point the cells were collected for RNA isolation. monooxygenase genes are also present (Table 2). In Relative gene expression was calculated on the basis of cells addition, the genome encodes 87 putative hydrolases, more grown in minimal salts basal (MSB) medium supplemented than 375 transferases, and 234 dehydrogenases, all of with 10 mM glucose. All of the tested nah genes showed which have uncharacterized functions and belong to a more than a 2-fold expression change when Pseudomonas number of different protein families. Related to the sp. AS1 was grown in the presence of naphthalene (Fig. 3B). bioremediation process, the detected ssuFBCDAE operon Naphthalene cannot be degraded through the gentisate (from PFAS1_RS12590 to PFAS1_RS12615) might allow for pathway in Pseudomonas sp. AS1 because this strain cannot sustainable growth on aromatic and aliphatic sulfonates, utilize gentisate as a carbon source (Fig. 1) owing to the and 14 glutathione S-transferases may be involved in absence of gentisate 1,2-dioxygenase as well as a lack of detoxification processes. In addition, PAHs or toxic expression for hmgA encoding homogentisate 1,2-dioxygenase metabolic intermediates can generate reactive oxygen during naphthalene degradation (Fig. 3B). Therefore, species (ROS) under the biodegradation processes [2, 7, 10]. Pseudomonas sp. AS1 might have a naphthalene degradation The genome of Pseudomonas sp. AS1 contains two oxidative pathway via salicylate as a central intermediate, which can stress response regulators, SoxR and OxyR (encoded by be converted into acetyl coenzyme A and pyruvate. PFAS1_RS02805 and PFAS1_RS11840, respectively) to The 16S rRNA sequence of Pseudomonas sp. AS1 has over protect against and relieve ROS-derived oxidative stress 99% DNA identities with those of Pseudomonas frederiksbergensis through the regulation of the genes. P. frederiksbergensis strains. The average nucleotide identity analysis of strain AS1 strains have been isolated from various environmental with P. frederiksbergensis strains revealed a similarity index conditions, and all genome-sequenced strains showed

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Table 2. Genes encoding hydroxylases and related genes in Pseudomonas sp. AS1. Locus tag Gene product name PFAS1_RS01505 Acireductone dioxygenase apoprotein PFAS1_RS02800 Antibiotic biosynthesis monooxygenase PFAS1_RS03120 4,5-DOPA dioxygenase extradiol PFAS1_RS04745 Intradiol ring-cleavage dioxygenase PFAS1_RS04850 Heme oxygenase PFAS1_RS05325 p-Hydroxybenzoate 3-monooxygenase PFAS1_RS06565 (hmgA) Homogentisate 1,2-dioxygenase PFAS1_RS07555 Glyoxalase/bleomycin resistance protein/dioxygenase superfamily protein PFAS1_RS09965 Taurine dioxygenase PFAS1_RS10310 Taurine dioxygenase PFAS1_RS10360 p-Aminobenzoate N-oxygenase AurF PFAS1_RS10865 DOPA 4,5-dioxygenase PFAS1_RS12600 (ssuD) Alkanesulfonate monooxygenase PFAS1_RS12890 p-Aminobenzoate N-oxygenase AurF PFAS1_RS13525 Gamma-butyrobetaine dioxygenase PFAS1_RS13895 Lysine 2-monooxygenase PFAS1_RS14470 p-Aminobenzoate N-oxygenase AurF PFAS1_RS15220 4-Hydroxyphenylpyruvate dioxygenase PFAS1_RS16755 Nitric oxide dioxygenase PFAS1_RS18060 (pcaH) Protocatechuate 3,4-dioxygenase, beta subunit PFAS1_RS18065 Protocatechuate 3,4-dioxygenase, alpha subunit PFAS1_RS22890 Catechol 1,2-dioxygenase PFAS1_RS22900 Benzoate 1,2-dioxygenase, alpha subunit PFAS1_RS22905 Benzoate/toluate 1,2-dioxygenase beta subunit PFAS1_RS22910 Benzoate/toluate 1,2-dioxygenase reductase subunit PFAS1_RS22915 1,2-Dihydroxycyclohexa-3,5-diene-1-carboxylate dehydrogenase PFAS1_RS23020 Taurine dioxygenase PFAS1_RS23040 Ferredoxin subunit of nitrite reductase or a ring-hydroxylating dioxygenase PFAS1_RS24315 Tryptophan 2-monooxygenase precursor PFAS1_RS24325 Phenylpropionate dioxygenase, large terminal subunit PFAS1_RS24455 Flavin-dependent oxidoreductase PFAS1_RS25260 Nitronate monooxygenase PFAS1_RS25375 (hppD) 4-Hydroxyphenylpyruvate dioxygenase PFAS1_RS25815 (sfnG) Dimethylsulfone monooxygenase PFAS1_RS26480 FMN-dependent oxidoreductase, nitrilotriacetate monooxygenase family PFAS1_RS28660 (nahAc)* Ferredoxin-NAD(P)+ reductase (naphthalene dioxygenase ferredoxin-specific) PFAS1_RS28665 (nahAb)* Naphthalene 1,2-dioxygenase system ferredoxin subunit PFAS1_RS28670 (nahAc)* Naphthalene 1,2-dioxygenase subunit alpha PFAS1_RS28675 (nahAd)* Naphthalene 1,2-dioxygenase subunit beta PFAS1_RS28680 (nahB)* Cis-1,2-dihydro-1,2-dihydroxynaphthalene/dibenzothiophene dihydrodiol dehydrogenase PFAS1_RS28685 (nahF)* Salicylaldehyde dehydrogenase PFAS1_RS28690 (nahC)* 1,2-Dihydroxynaphthalene dioxygenase PFAS1_RS28700 (nahE)* Trans-O-hydroxybenzylidenepyruvate hydratase-aldolase

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Table 2. Continued. Locus tag Gene product name PFAS1_RS28705 (nahD)* 2-Hydroxychromene-2-carboxylate isomerase PFAS1_RS28730* N-Ethylmaleimide reductase PFAS1_RS28815 (nahJ)* 4-Oxalocrotonate tautomerase PFAS1_RS28820 (nahK)* 4-Oxalocrotonate decarboxylase PFAS1_RS28825 (nahM)* 4-Hydroxy-2-oxovalerate aldolase PFAS1_RS28830 (nahO)* Acetaldehyde dehydrogenase PFAS1_RS28835 (nahL)* 2-Oxopent-4-enoate/cis-2-oxohex-4-enoate hydratase PFAS1_RS28840 (nahN)* 2-Hydroxymuconate semialdehyde hydrolase PFAS1_RS28845 (nahI)* 2-Hydroxymuconate semialdehyde dehydrogenase PFAS1_RS28850 (nahH)* Catechol 2,3-dioxygenase PFAS1_RS28865 (nahG)* Salicylate hydroxylase *Located on the plasmid of Pseudomonas sp. AS1.

Table 3. Comparative features of all sequenced Pseudomonas frederiksbergensis genomes. Size GC Plasmid Strain Isolation site Biodegradationa Assembly level Reference/source (Mb) (%) (bp) AS1 Arsenic-contaminated Naphthalene, various 6.22 58.86 1 Complete This study; soil, Korea aromatic hydrocarbon (81,841 bp) [7, 8] compounds ERDD5:01 Glacial stream, India NDb 6.12 58.48 1 Complete NCBI BioProjects: (371,069 bp) PRJNA350793 BS3655 ND ND 6.39 58.90 ND Contig NCBI BioProjects: PRJEB16448 SI8 Desert soil, Kuwait Toluene, ethylbenzene, 6.57 60.50 ND Contig [21] and propylbenzene JAJ28 Coal gasification site, Phenanthrene-degrading ND ND ND ND [20] Denmark aExperimentally determined. bNot determined. differences in genome size, GC content, and plasmids has been deposited in NCBI under the GenBank accession (Table 3). Bacterial cells can obtain exogenous genes via numbers CP018319 (chromosome) and CP018320 (plasmid). horizontal gene transfer, which is indispensable for survival and adaptation to harsh environmental conditions [22-24]. Acknowledgments We expect that Pseudomonas sp. AS1 acquired its plasmid for this reason during adaptation to contaminated soil. To This research was supported by the Basic Science Research explore the genetic evolution among P. frederiksbergensis Program through the National Research Foundation (NRF) strains, it will be necessary to perform comparative of Korea funded by the Ministry of Education, Science and genomics. In that respect, the complete genome sequence Technology (No. NRF-2015R1C1A2A01054058). This work of Pseudomonas sp. AS1 will provide clues for the early was also supported by a grant (NRF-2017R1A2B4005838 to stages of genetic research on P. frederiksbergensis species. W.P.) of the NRF of Korea.

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