Pseudomonas putida CSV86: a candidate genome for genetic bioaugmentation.

Pseudomonas putida CSV86, a plasmid-free strain possessing capability to transfer the naphthalene degradation property, has been explored for its metabolic diversity through genome sequencing. The analysis of draft genome sequence of CSV86 (6.4 Mb) revealed the presence of genes involved in the degradation of naphthalene, salicylate, benzoate, benzylalcohol, p-hydroxybenzoate, phenylacetate and p-hydroxyphenylacetate on the chromosome thus ensuring the stability of the catabolic potential. Moreover, genes involved in the metabolism of phenylpropanoid and homogentisate, as well as heavy metal resistance, were additionally identified. Ability to grow on vanillin, veratraldehyde and ferulic acid, detection of inducible homogentisate dioxygenase and growth on aromatic compounds in the presence of heavy metals like copper, cadmium, cobalt and arsenic confirm in silico observations reflecting the metabolic versatility. In silico analysis revealed the arrangement of genes in the order: tRNA(Gly), integrase followed by nah operon, supporting earlier hypothesis of existence of a genomic island (GI) for naphthalene degradation. Deciphering the genomic architecture of CSV86 for aromatic degradation pathways and identification of elements responsible for horizontal gene transfer (HGT) suggests that genetic bioaugmentation strategies could be planned using CSV86 for effective bioremediation.

Introduction

Pseudomonas exhibits diverse metabolic capacities; which allow it to survive in different ecological niches, including sites contaminated with pollutants such as aromatic compounds. The required metabolic attributes are reflected by its large size genome (generally >6 Mb). Pseudomopan class="Chemical">nads have been reported for their ability to exchange genetic information through horizontal gene transfer (HGT) via phages, plasmids, transposons and genomic islands (GIs), thus aiding in dissemination as well as evolution of new diversified metabolic pathways [1]. These processes allow sustained survival of genetic resources. Of these mobile genetic elements (MGEs), GIs have especially been reported to code for genes which render metabolic versatility, pathogenicity and heavy metal resistance to microbes [2], [3]. These capacities could be exploited through genetic bioaugmentation for in situ breeding of native population which not only ensures the survival of novel genetic determinants, but also helps in enhancing the bioremediation process.

Pseudomonas putida CSV86 (hereafter referred to as pan class="Species">CSV86), a soil isolate, utilizes aromatic compounds like naphthalene, 1- and 2-methylnaphthalene, phenylacetic acid (PA) and p-hydroxyphenylacetic acid (4-HPA), salicylate, benzylalcohol, benzoate and p-hydroxybenzoate as the carbon source [4], [5], [6], [7]. Strain CSV86 showed a novel property of preferential utilization of aromatic compounds over glucose and co-metabolism of aromatics and organic acids [8], [9], [10], [11]. Though CSV86 lacks plasmid, the naphthalene degradation property could be transferred by conjugation which was found to be integrated in to the chromosome of the transconjugants [12].

In the present study, the reported pathways for the utilization of aromatic compounds have been annotated using the draft genome sequence of pan class="Species">CSV86 (6.4 Mb) [13]. Genome analysis revealed additional catabolic pathways for aromatic compounds as well as pan class="Chemical">heavy metal resistance. These observations were further validated by phenotypic (cell-growth and enzyme activity) experiments. Based on these analyses, bioremediation and bio-augmentation strategies can be developed for the effective remediation of ecosystems polluted with aromatic compounds.

Materials and Methods

CSV86 draft genome assembly, ordering and annotation

The genome of Pseudomonas putida CSV86 was sequenced using Roche 454 pan class="Disease">GS (FLX Titanium) platform. The 867,565 high quality reads were assembled into 228 contigs with Newbler Ver2.0, 454 assembly tool with sequence coverage of 61.08 fold and average read length of 428 bp. Ordering of contigs was performed using a tool, Mauve Contig Mover (MCM) [14] available in Mauve software (http://gel.ahabs.wisc.edu/mauve.) using P. putida S16 complete genome (NC_015733) as the reference. P. putida S16 was also used as a reference for contig scaffolding by using SIS program [15].

The genome was annotated using Rapid Annotations using Subsystems Technology (RAST) v4.0 [16] and NCBI PGAAP (Prokaryotic Genomes Automatic Annotation Pipeline) (http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html). In NCBI pan class="Chemical">PGAAP, the 228 contigs were trimmed down to 209 due to quality check and preprocessing of sequences. These contig were later processed for annotation. The annotation by both RAST and NCBI PGAAP tool was used to describe the genome of CSV86 in this paper.

This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no. AMWJ00000000. The version described in this paper is the first version, AMWJ01000000.

Comparative genomics and phylogenetic relationship

Taxonomic relationship

Phylogenetic relationship of CSV86 was established using 16S rRNA gene sequences of 38 completely sequenced pan class="Species">Pseudomonas species from NCBI database. The alignment was carried out using ClustalW and the phylogenetic tree was constructed using the maximum likelihood algorithm (Hasegawa-Kishino-Yano model) with MEGA 5.2 [17]. In addition, MEGA 5.2 was also used for alignment and constructing phylogenetic tree using promoter sequences of naphthalene degradation genes.

Sequence variation in metabolic genes

Primary DNA and protein sequences of CSV86 were compared with other closely related species for similarity in catabolic pathways using NCBI blast tools such as megaBLAST and BLASTp, respectively. Genes involved in the degradation of aromatic compounds in pan class="Species">CSV86 were identified using RAST and NCBI PGAAP annotation along with available literature, KEGG [18] and Metacyc [19] databases.

Comparative genome analysis using BRIG and Mauve

BRIG (BLAST Ring Image Generator) [20] software was used for the circular representation of multiple genome comparison. The draft genome of CSV86 was used as the reference genome and was compared with genome of pan class="Species">P. putida S16 (NC_015733), P. putida KT2440 (NC_002947), P. entomophila L48 (NC_008027) and P. stutzeri CCUG 29243 (NC_018028).

Progressive alignment function of Mauve software with default settings was used to compare the homology among pan class="Chemical">naphthalene degradation pathway genes reported from various Pseudomonas genomes. The draft genome of CSV86 was aligned against complete genome of P. stutzeri CCUG 29243 (NC_018028), Pseudomonas sp. ND6 plasmid pND6-1 (NC_005244), P. putida plasmid NAH7 (NC_007926) and P. fluorescens strain PC20 plasmid pNAH20 (AY887963).

Prediction of GIs and mobile genetic elements

To predict the GIs, GC-profile, a web based tool [21] was used to compute the GC content variation in DNA sequences. These islands are marked by certain features such as the presence of mobility genes, difference in the G+C content as compared to the rest of genome, codon usage, tRNA genes and direct repeats [22]. Some of these features were manually identified in the genome for the validation of GIs. Also, conserved insertion sequences (IS) elements in pan class="Species">CSV86 genome were identified using IS Finder (http://www-is.biotoul.fr/) to further support the presence of GI [23].

Validation of selected genotype by wet experiments

Growth

Strain CSV86 was grown on 150 ml minimal pan class="Chemical">salt medium (MSM) [5] in 500 ml capacity baffled Erlenmeyer flasks at 30°C on a rotary shaker (200 rpm) supplemented aseptically with vanillin, veratraldehyde, ferulic acid, phenylalanine or tyrosine (0.1%). The cell growth was observed spectrophotometrically at 540 nm.

Preparation of cell-free extracts

CSV86 cells grown on pan class="Chemical">phenylalanine (0.1%) or glucose (0.25%) till late-log phase were harvested and washed twice with Tris-malaete buffer (200 mM, pH 6.0). Cells were re-suspended in ice-cold Tris-malaete buffer (1∶4 [wt/vol]) and sonicated at 4°C with four cycles of 15 pulses each (1 s pulse, 1 s interval, cycle duration 30 s, output 15 W) using an Ultrasonic processor (GE130). The cell lysate was centrifuged at 37,000 ×g for 30 min. The clear supernatant obtained was referred to as the cell-free extract and used as the source of enzyme. Protein was estimated by the method of Bradford [24] using bovine serum albumin as the standard.

Enzyme assay

Homogentisate 1,2-dipan class="Chemical">oxygenase activity was monitored by measuring the rate of O2 consumption at 30°C using an oxygraph (Hansatech) fitted with a Clarke's O2 electrode. The reaction mixture (1 ml) contained Tris-malaete buffer (200 mM, pH 6.0), homogentisate (2.5 mM) and an appropriate amount of enzyme. The enzyme activity was calculated as nmol of O2 consumed per min. The specific activity is reported as nmol of O2 consumed min−1 mg−1 protein.

Heavy metal resistance

CSV86 was grown on 150 ml modified minimal pan class="Chemical">salt medium (MSM, medium contained Tris, 8 g; KH2PO4,0.2 g; NH4NO3, 1 g; MgSO4.7H2O, 100 mg; MnSO4.H2O, 1 mg; CuSO4.5H2O, 1 mg; FeSO4.7H2O, 5 mg; H3BO3, 1 mg; CaCl2.2H2O , 1 mg; NaMoO4, 1 mg; pH 7.5) in 500 ml capacity baffled Erlenmeyer flasks at 30°C on a rotary shaker (200 rpm) supplemented aseptically with naphthalene (0.1%) or glucose (0.25%) and appropriate concentration of heavy metals such as copper, cadmium or cobalt (0.5 or 1 mM) and the growth was monitored.

Results and Discussion

Pseudomonas putida CSV86 genome features and comparative genomics

The 6,469,780 bp draft genome of CSV86 is almost close to sequenced pan class="Species">Pseudomonas genomes (Table S1); and assembled into 209 contigs that have been annotated by NCBI PGAAP into 5,836 coding sequences (CDSs) as shown in Table 1. RAST analysis divided CSV86 genome into different metabolic subsystems including catabolic pathways for various aromatic compounds (Figure S1). The phylogenetic tree of 16S rRNA gene of CSV86 showed taxonomic relationship with Pseudomonas putida S16 sharing 98% homology (Figure 1). This was further supported by SIS program, wherein CSV86 draft genome was assembled into 8 scaffolds (around 0.3 Mb of the genome was unmapped in the scaffold) against P. putida S16. The analysis of ordered draft genome of CSV86 with BRIG software showed ∼70% identity with P. putida S16, P. putida KT2440 and P. entomophila L48 except P. stutzeri CCUG 29243 (<70%) (Figure 2); with gaps observed in the region 6100–6500 kbp. Similarity search of the gapped region using BLASTn with default parameters, revealed genes coding for chromosome replication initiator protein dnaA and other proteins involved in replication. A gene cluster with dnaA gene was identified i.e. rnpA-rpmH-dnaA-dnaN-recF-gyrB. The oriC (replication origin) has been reported to be present in this intergenic region [25], [26]. Therefore, it may be postulated that oriC region is located in this region of CSV86 genome.

Table 1

Features associated with genome of P. putida CSV86 according to NCBI PGAAP.

FEATURE	CHROMOSOME
Length (bp)	6,469,780 bp
Number of contigs≠	209
GC content (%)	61.85
Sequencing coverage	61.08×
t-RNA genes	60
CDSs*	5,836
Hypothetical proteins	1,689

CDSs: coding region, coding sequence.

There are 228 contigs according to Newbler Ver2.0, 454 assembly tool.

Figure 1

Phylogenetic neighbor-joining tree of Pseudomonas putida CSV86.

The tree is constructed from 16S rRNA gene sequences from 38 completely sequenced Pseudomonas spp. The phylogenetic analysis was performed using MEGA 5.2 and the resultant Maximum Likelihood tree shows close taxonomic relationship of P. putida CSV86 to P. putida S16.

Figure 2

BLAST comparison of draft genome of Pseudomonas putidaCSV86 against four Pseudomonas species, using BRIG.

The innermost rings depict GC content (Black) and GC Skew (purple/green) followed by concentric rings of query sequences colored according to BLAST identity. The outermost rings depict genomes of the following microbes- P. putida S16 (Red), P. putida KT2440 (Pink) P. entomophila L48 (Blue), and P. stutzeri CCUG 29243 (Green).

The tree is constructed from 16S rRNA gene sequences from 38 completely sequenced Pseudomonas spp. The phylogenetic analysis was performed using MEGA 5.2 and the resultant Maximum Likelihood tree shows close taxonomic relationship of pan class="Species">P. putida CSV86 to P. putida S16.

The innermost rings depict GC content (Black) and GC Skew (purple/green) followed by concentric rinpan class="Disease">gs of query sequences colored according to BLAST identity. The outermost rings depict genomes of the following microbes- P. putida S16 (Red), P. putida KT2440 (Pink) P. entomophila L48 (Blue), and P. stutzeri CCUG 29243 (Green).

There are 228 pan class="Chemical">contigs according to Newbler Ver2.0, 454 assembly tool.

Mining of aromatic compound degradation pathways in CSV86

The industrial revolution has led to the introduction of new pollutants in the environment; which also ushered the evolution of new catabolic pathways [27]. The absence or withdrawal of such selective pressures often leads to the loss of the catabolic property, if it is pan class="Species">plasmid mediated; and even in cases of genome organization, where it is associated with MGEs such as GIs. These features play a significant role in the evolution of community where these evolved microbes can be ideal candidates for effective bioremediation either alone or in consortium [28].

The in-silico analysis of the pan class="Species">CSV86 genome revealed the genes coding for enzymes involved in the metabolic pathways which are biochemically characterized earlier from pan class="Species">CSV86 (Figure 3) and their arrangement on the genome (Figure 4) as well as newly identified pathways for the catabolism of aromatic compounds (Figure 5).

Figure 3

The metabolic pathways for aromatic compounds in Pseudomonas putidaCSV86.

Enzymes involved are: a, naphthalene dioxygenase; b, 1,2-dihydroxynaphthalene dioxygenase; c, salicylaldehyde dehydrogenase; d, salicylate hydroxylase; e, catechol 2,3-dioxygenase; f, catechol 1,2-dioxygenase; g, benzyl alcohol dehydrogenase; h, benzaldehyde dehydrogenase; i, 3,4-dihydroxybenzoate-3,4-dioxygenase; j, 4-hydroxyphenylacetic acid hydroxylase; k, 3,4-dihydroxyphenylacetic acid dioxygenase; l, homogentisate 1,2-dioxygenase; m, phenylacetyl-CoA ligase. Enzymes with wide-substrate specificity involved in various pathways in CSV86 are indicated in square bracket (4, 5, 6, 7).

Figure 4

Gene organization of aromatic degradation pathways reported to be functionally characterized from Pseudomonas putida CSV86.

A. Naphthalene pathway (Contig 105), B. Salicylate pathway (Contig 69), C. Benzoate pathway (Contig 175), D. Aromatic alcohol degradation pathway (Contig 119), E. Phenylacetic acid pathway (Contig 88), F, Hydroxylphenylacetic acid pathway(Contig 7) and G–I, 4-Hydroxybenzoate (Contig 107, 99, 118). For details refer to Figures S2, S3, S6, S7, S8, S9, S10.

Figure 5

New aromatics degradation pathways genes identified in Pseudomonas putida CSV86 by genome analysis.

A–B. Phenylpropanoid pathway genes (Contig 115, 220), C–E. Homogentisate pathway genes (Contig 27, 99, 177), F–H. Copper resistance genes (Contig 19). For details refer to Figures S11, S12, S12.

Enzymes involved are: a, naphthalene dipan class="Chemical">oxygenase; b, 1,2-dihydroxynaphthalene dioxygenase; c, salicylaldehyde dehydrogenase; d, salicylate hydroxylase; e, catechol 2,3-dioxygenase; f, catechol 1,2-dioxygenase; g, benzyl alcohol dehydrogenase; h, benzaldehyde dehydrogenase; i, 3,4-dihydroxybenzoate-3,4-dioxygenase; j, 4-hydroxyphenylacetic acid hydroxylase; k, 3,4-dihydroxyphenylacetic acid dioxygenase; l, homogentisate 1,2-dioxygenase; m, phenylacetyl-CoA ligase. Enzymes with wide-substrate specificity involved in various pathways in CSV86 are indicated in square bracket (4, 5, 6, 7).

A. Naphthalene pathway (pan class="Chemical">Contig 105), B. Salicylate pathway (Contig 69), C. Benzoate pathway (Contig 175), D. Aromatic alcohol degradation pathway (Contig 119), E. Phenylacetic acid pathway (Contig 88), F, Hydroxylphenylacetic acid pathway(Contig 7) and G–I, 4-Hydroxybenzoate (Contig 107, 99, 118). For details refer to Figures S2, S3, S6, S7, S8, S9, S10.

A–B. Phenylpropanoid pathway genes (pan class="Chemical">Contig 115, 220), C–E. Homogentisate pathway genes (Contig 27, 99, 177), F–H. Copper resistance genes (Contig 19). For details refer to Figures S11, S12, S12.

Naphthalene degradation pathway

CSV86 can utilize pan class="Chemical">naphthalene and its derivates such as 1- and 2-methylnaphthalenes as the sole source of carbon and energy via ring-hydroxylation pathway (Figure 3), while side-chain hydroxylation pathway leads to its detoxification [4], [5]. In CSV86, naphthalene catabolic pathway is initiated by naphthalene 1,2-dioxygenase (a three-component system) which catalyzes the hydroxylation of the aromatic ring to yield 1,2-dihydroxynaphthalene as a upper pathway (contig 105). This diol is further sequentially oxidized to catechol via lower pathway (contig 69), which enters the tricarboxylic acid cycle (TCA) after meta ring-cleavage (Figure 3) [4]. Using BLASTp, amino acid sequences of CSV86 naphthalene degrading upper and lower pathway genes (Table S2) were compared with that of other reported bacteria. It was observed that upper pathway amino acid sequences shares higher homology and hence are more conserved than that in lower pathway (Table S3).

Both the nah and sal operon of pan class="Species">CSV86 showed similarity with Pseudomonas putida NCIB 9816-4 plasmid pDTG1 and Pseudomonas sp. ND6 plasmid pND6-1 (Figure 4A & 4B; Figures S2 & S3). Interestingly, in contig 105, the arrangement of genes observed was tRNAGly, integrase followed by nah operon in the order nahAa,Ab,Ac,Ad, BFCED. This arrangement is a characteristic feature of a GI for e.g. clc element [29]. The sal operon consists of 9 genes organized as nahGTHILMOKJ with the regulatory gene nahR present downstream of nahG gene. The regulation of nah genes is controlled by nahR, which is in turn induced by salicylate [30], [31]. In CSV86 a transposase encoding gene is present upstream of nahR gene which is missing in the plasmids being compared (Figure 4B & Figure S3).

Regulation of nah operon

The genome sequence analysis of CSV86 revealed that the naphthalene pathway is under the control of LysR family of transcription regulators (LTTRs) [32], [33]. We have analyzed the differential regulation of naphthalene degradation pathway in CSV86 with P. stutzeri CCUG 29243 (NC_018028) (chromosomally coded) and Pseudomonas putida plasmid NAH7 DNA, strain G7 (AB237655). NahR protein is essential for the activation of both the upper and lower operon of naphthalene pathway in the presence of salicylate [34]. The binding site for NahR with the promoter for Pnah and Psal are reported to be located at 60 bp upstream to transcriptional start site [34], [35]. Therefore, the promoter data for nahAa (upper pathway), nahG (lower pathway) and also the coding sequences for NahR protein was analyzed. The consensus binding sequences of NAH7 promoter has two cis-acting elements situated 6 bp apart that interact with NahR protein [36]. The nahAa promoter of CSV86 and P. stutzeri are identical with the reported NAH7 binding site for NahR protein. Both these promoters have an additional cis-acting element with one base pair substitution and 4 bp spacing between the cis-acting elements. Whereas, nahG promoter has a base pair substitution (TGAT is changed to TAGT) in both the chromosomal promoters, with 4 bp separating the two cis-acting elements (Table 2). A phylogenetic tree of nahR, nahAa and nahG promoter sequences was also constructed. In all the three promoter sequence comparisons, CSV86 and P. stutzeri were grouped in same cluster (Figure S4).

Table 2

Consensus sequence of nahR binding site in nahAa and nahG gene obtained from P. putida plasmid NAH7.

Promoter	NahR binding site
pNah7	-CGCAnTATTCAyGyTGuTGAT nnAnnAnnTnnn-
PnahAa
CSV86	-GACAT TATTCATATTAGTGAT ACTAA TATTCA TTTATGGT TTATTGAC-
P. stutzeri	-GACAT TATTCATATTAGTGAT ACTAA TATTCATTTATGGT TTATTGAC-
PnahG
CSV86	-TAGTG TATTTATCAATAGT TATGGCTTCGCTACTGTT-
P. stutzeri	-TAGTG TATTTATCAATAGT TATGGCTTCGCTACTGTT-

Table shows consensus sequence of NahR binding site form P. putida plasmid NAH7 as reported by Schell et al., 1989. The sequences used in Figure S4, shown the homologous cis-acting NahR regulated elements of nahAa and nahG genes (nah and sal operons, respectively) in case of P. putida CSV86 and P. stutzeri CCUG 29243 genomes. The bold type face alphabets indicate nucleotides required for NahR activation of NahR-regulated promoters. (n: no nucleotide preference, Y: pyrimidine; U: purine).

Table shows consensus sequence of NahR binding site form pan class="Species">P. putida plasmid NAH7 as reported by Schell et al., 1989. The sequences used in Figure S4, shown the homologous cis-acting NahR regulated elements of nahAa and nahG genes (nah and sal operons, respectively) in case of P. putida CSV86 and P. stutzeri CCUG 29243 genomes. The bold type face alphabets indicate nucleotides required for NahR activation of NahR-regulated promoters. (n: no nucleotide preference, Y: pyrimidine; U: purine).

The NahR of pan class="Species">CSV86 interestingly showed 100% identity with protein from P. stutzeri (chromosomally located) as compared to 81% identity with NAH7 (plasmid encoded) protein. The substitution of methionine to isoleucine in NahR protein of NAH7 altered the specificity of protein to salicylate and allowing salicylate analog like benzoate to act as an inducer [37]. In CSV86 and P. stutzeri, NahR protein at 116th position has isoleucine (Figure S5). However in CSV86, the enzymes responsible for naphthalene and salicylate degradation are inducible in nature. Benzoate does not induce these operons as the benzoate grown cells failed to respire on naphthalene or salicylate and showed no activity of enzymes involved in naphthalene or salicylate degradation [5].

Benzoate degradation pathway

In CSV86 benzoate degradation is initiated with the incorporation of molecular oxygen by benzoate dioxygenase (encoded by benABC genes, a two-component system) to yield catechol which enters the central carbon metabolism via β-ketoadipate pathway after ortho-cleavage (Figure 3) [5], [38]. The details of the genes for benzoate degradation that are present in contigs 175, 103, 118 and 116 are described in Table S2. The CSV86 genome has the presence of complete benzoate utilization system including the regulatory option of the benABC operon i.e., transcriptional activator BenR with benzoate as effector; and utilization of catechol regulated via CatR with cis,cis-muconate as effector [38]. In P. fluorescnes Pf0-1 the catBC and catR genes are located between genes encoding benzoate MFS transporter and catechol 1,2-dioxygenase while, in CSV86 these genes are located upstream to the benzoate cluster. The catBC genes are absent in this cluster of P. putida KT2440 (Figure 4C, Figure S6).

The optional genes for benzoate degradation were also identified in pan class="Species">CSV86 genome, which encode p-hydroxybenzoyl-CoA thioesterase (catalyses the conversion of p-hydroxybenzoyl-CoA to p-hydroxybenzoate) and 5-carboxymethyl-2-hydroxymuconate Delta-isomerase (catalyses the conversion of 5-carboxymethyl-2-hydroxymuconate to form 5-carboxy-2-oxohept-3-enedioate) located in contig 60 and 103, respectively. Besides salicylate hydroxylase in contig 69 (sal operon), contig 103 also showed the presence of an additional salicylate hydroxylase with 23% identity.

Aromatic alcohol degradation pathway

Although the detoxification pathway of methylnaphthalenes closely resembles to the side-chain hydroxylation of pan class="Chemical">toluene degradation, CSV86 failed to utilize toluene or xylene as the sole source of carbon and energy. Interestingly, strain could grow on benzyl alcohol, 2- and 4-hydroxy benzyl alcohol (Figure 3) [5]. The key enzymes of the aromatic alcohol metabolism, benzyl alcohol dehydrogenase (BADH) and benzaldehyde dehydrogenase (BZDH), have been purified and were found to be wide-substrate specific and shown to catalyze the conversion of 1- and 2-hydroxymethylnaphthalene to respective naphthoic acids (dead end products) [5], [6].

In CSV86, the gene cluster encoding pan class="Chemical">BZDH and BADH was located in contig 119. The proposed BZDH or NAD+-dependent aryl aldehyde dehydrogenase gene was located adjacent to BADH, aryl alcohol dehydrogenase gene in CSV86 and shares homology with Pseudomonas putida DOT-T1E (aldehyde dehydrogenase, 87%). In CSV86 gene encoding for transcriptional regulator (AraC family) was located downstream to the gene encoding putative benzaldehyde dehydrogenase oxidoreductase protein, which is absent in the same cluster of Pseudomonas putida GB-1 and Burkholderia sp. (Figure 4D, Figure S7, Table S2).

Phenylacetic and p- hydroxyphenylacetic acid degradation pathway

CSV86 metabolizes pan class="Chemical">phenylacetic acid (PA) and p-hydroxyphenylacetic acids (HPA) using an unconventional ‘aerobic hybrid’ pathway (Figure 3). PA is first activated to its CoA-thioester (phenylacetyl CoA) by phenylacetyl-CoA ligase, which through a series of CoA-thioester intermediates ultimately leads to the formation of succinyl-CoA and acetyl-CoA that enters the central metabolic pathway (Figure 3) [7], [39]. In CSV86 the PA catabolic genes (contig 88, Table S2) were organized in the order as observed in Pseudomonas putida W619 and P. putida GB-1 (Figure 4E, Figure S8).

4-HPA degradation pathway in pan class="Species">CSV86 follows homoprotocatechuate route which involves initial hydroxylation of 4-HPA followed by ring cleavage (Figure 3) [7]. The pathway genes of 4-HPA in CSV86 were present in contig 7, 120, 175 and 216 (Table S2). There were two copies of each hpaD (contig 7 and 120) and homoprotocatechuate degradative operon repressor gene (contig 7 and 216). The genes present in CSV86 contig 7 were organized as hpaAGEDFHI while, in Pseudomonas fluorescens SBW25 homoprotocatechuate degradative operon repressor gene was present upstream of this hpa cluster (Figure 4F, Figure S9).

p-Hydroxybenzoate degradation pathway

In CSV86, pan class="Chemical">p-hydroxybenzoate degradation is initiated by ring-hydroxylating 4-hydroxybenzoate 3-monooxygenase (PobA) to yield 3,4-dihydroxybenzoate (protocatechuate) which is further metabolized by ortho ring-cleavage to yield carboxy-cis,cis-muconate by protocatechuate dioxygenase (PcaGH, Figure 3). The later part of the pathway was catalyzed by the pcaBCDIJF gene products which are also involved in the degradation of benzoate.

The genes for 4-hydroxybenzoate pathway were distributed in pan class="Chemical">contig 99 (pcaHG), 107 (pobA), 118 (pcaRKFTBDC) and 175 (pcaIJ, Table S2) in CSV86. Like Pseudomonas fluorescens Pf-5 and P. putida GB-1, pobA gene in CSV86 (contig 107) was located downstream to the gene encoding transcriptional regulator PobR (AraC family) (Figure 4G, Figure S10A). The pcaHG genes were located in contig 99 and present downstream of zinc metalloprotease superfamily, as observed in P. entomophila L48 and P. putida KT2440 (Figure 4H, Figure S10B). A transporter, PcaK, is involved in the transport of 4-hydroxybenzoate across the membrane and reported to be located in between pcaR and pcaF in P. putida, as can also be seen in contig 118 in CSV86 (Figure 4I, Figure S10C). The expression of pcaK gene has been shown to be repressed by benzoate, suggesting cells prefer benzoate instead of 4-HPA when given together [40]. The pca genes have been shown to be arranged in a single cluster in P. fluorescens [41], while in CSV86 they were segregated in different contigs (Figure 4, Table S2, Figure S10).

Identification of additional aromatic compound degradation pathways

Phenylpropanoid degradation pathway

Although lignin degradation pathways are shown in fungi by enzymes such as pan class="Chemical">lignin peroxidases, manganese peroxidases, laccases etc [42]; aromatic degrading bacterial species are also reported to metabolize lignin [43]. Vanillin, ferulic acid, veratraldehyde, coniferyl aldehyde, β-coniferylether are identified as degradation intermediates of lignin. In Pseudomonas sp. strain HR199, the feruloyl-CoA synthetase (encoded by fcs) activates ferulic acid to its CoA-thioester followed by hydration and non-oxidative cleavage by enoyl-CoA hydratase/aldolase (encoded by ech) to form vanillin and acetyl Co-A [44]. Vanillin is further converted to vanillate and later protocatechuate by vanillin dehydrogenase (encoded by vdh) and vanillin monooxygenase (encoded by vanAB) or vanillate-O-demethylase (encoded by vanA and vanB), respectively [41], [44]. In CSV86, genes involved in the phenylpropanoid degradation were segregated in different contigs (Figure 5A&B, Table S2, Figure S11). The fcs-vdh-ech genes formed a cluster in contig 115 (Figure 5A, Figure S11A), vanAB genes in contig 119; and Vanillate O-demethylase oxygenase subunit, vanA and vanillate O-demethylase oxidoreductase, vanB along with the transcriptional regulator for ferulate or vanillate catabolism in contig 220 (Figure 5B, Figure S11B).

Strain CSV86 showed good growth on MSM supplemented with pan class="Chemical">vanillin, veratraldehyde, or ferulic acid (0.1%). These observations suggest that the lignin degradation intermediates can be used as the sole source of carbon and energy and reflects the existence of functional phenylpropanoid metabolic pathway in CSV86. However, cells failed to utilize lignin (lignin sulphonic acid) as the sole source of carbon and energy (data not shown).

Homogentisate degradation pathway

Homogentisate is a metabolic intermediate of pan class="Chemical">aromatic amino acid pathways. Phenylalanine via tyrosine is converted to 4-hydroxyphenylpyruvate (by PhhABC). The generated 4-hydroxyphenylpyruvate, is then transformed to homogentisate (2,5-dihydroxyphenylacetic acid) by 4-hydroxyphenylpyruvate dioxygenase (encoded by hpd gene) [41]. Homogentisate 1,2-dioxygenase (HmgA) is the first enzyme of the homogentisate pathway which catalyses the transformation of homogentisate to 4-maleylacetoacetate. Isomerization of 4-maleylacetoacetate by maleylacetoacetate isomerase (HmgC) leads to the formation of fumarylacetoacetate, which is finally hydrolyzed by fumarylacetoacetase (HmgB) generating fumarate and acetoacetate [45], [46]. The hpd gene is present along with hmg genes in Pseudomonas syringae, Pseudomonas stutzeri and Pseudomonas mendocina, whereas in P. putida these are scattered; in P. aeruginosa the hpd gene is clustered with phh genes [41]. In CSV86, phh, hpd and hmg genes were segregated in different clusters (Figure 5C, D & E; Figure S12,Table S2). The phh genes were present in the contig 177 with the arrangement of phhR (phenylalanine hydroxylase transcriptional activator) and phhABC genes similar to P. putida KT2440 and P. putida F1 (Figure 5C, Figure S12C). The hpd gene was present in contig 99 (Figure 5D, Figure S12B) and 134. The gene coding for transcriptional regulator, TetR family, was present upstream to hpd gene in CSV86 (contig 99), P. fluorescens Pf-5 and SBW25. The clustering of hmg genes (contig 127) was similar to P. putida F1 and KT2440, with the gene coding for transcriptional regulator (IclR family) being transcribed in reverse direction to hmgABC genes (Figure 5E, Figure S12A).

Strain CSV86 showed good growth on MSM supplemented with pan class="Chemical">phenylalanine or tyrosine (0.1%) as the sole source of carbon and energy. Cell-free extract prepared from the cells grown on phenylalanine showed the activity of homogentisate dioxygenase (specific activity 49.9 nmol min−1 mg−1protein) while glucose grown cells failed to do so. These results suggest that the homogentisate pathway is functional in CSV86 and the enzyme is inducible in nature (data not shown).

Identification of heavy metal resistance genes in CSV86

Bioremediation of soils co-contaminated with heavy metals and organics pose a major environmental challenge. Therefore, bacteria harboring the properties of pan class="Chemical">heavy metal resistance as well as aromatic compound degradation would be highly beneficial. The metal resistance is achieved by employing efflux pumps or enzymatic detoxification or bioaccumulation (intracellular/surface sequestration) or in combinations. Genes involved in heavy metal resistance have been found to be encoded by plasmids [47], [48] or chromosome [49]. Chromosomal coded efflux system for cadmium resistance has been reported in Bacillus as well as for arsenic and antimony resistance in E. coli [50]. Cyanobacterium synechocystis PCC6808 was also found to contain a homolog to Czc (cadmium, zinc, and cobalt resistance system) and genes apparently involved in arsenic and copper transport [51].

The genome of CSV86 was found to harbor pan class="Chemical">heavy metal resistance genes for copper (Figure 5F–H), cadmium, cobalt and arsenic (Table S4). The copper resistance genes were dispersed in CSV86 genome (contig 19, 153, 82) with a majority being in contig 19 (Figure 5F, Figure S13A). The genes encoding copper sensory histidine kinase (cusS), copper-sensing two-component system response regulator (cusR) and copper tolerance were also found to be located in the same contig (Figure 5G and H; Figure S13B and C). Like copper, arsenic resistance genes were also located in contig 153, with the exception of arsenic reductase gene in contig 82. Genes for cobalt, chromate, cadmium, zinc and lead resistance were also mapped during the genome analysis.

Strain CSV86 showed good growth on pan class="Chemical">glucose or naphthalene in the presence of heavy metals like copper, cadmium or cobalt at 0.5 as well as at 1 mM concentration, suggesting the ability of strain to express the tolerance/resistance to these heavy metals (data not shown).

Genetic bioaugmentation

Bioaugmentation using genetically engineered microorganisms or consortia has been reported as an alternative strategy to enhance the bioremediation of contaminated sites [52], [53], [54]; however the bio-safety issues pose a concern with these modified bacteria. A wild isolate with its ability to transfer catabolic genes through natural processes (HGT) such as conjugation, may provide a better solution to contain and remediate these compounds [55], [56]. Pseudomonas putida IncP-9 pan class="Species">TOL plasmid pWW0, has been studied for genetic bioaugmentation of soil, wastewater and aerobic microbial granules [57]. In another example, dissemination of plasmid pGKT2 harboring catabolic genes for hexahydro-1,3,5-trinitro-1,3,5,-triazine (RDX) degradation was studied by means of conjugation between the Gordonia sp. KTR9 and the native population of the contaminated site, so as to enhance the efficiency of bioremediation [58]. Genetic bioaugmentation via self-transmissible catabolic genes by donor bacteria have stability issues in host bacteria. The other options for HGT events are mediated by MGEs such as plasmids, GIs, transposons, integrons and phages [55], [56]. MGEs have been shown to play significant role in supporting various types of genomic rearrangement. HGT through GIs provides a better and desirable advantage over plasmids as these elements are integrated in host chromosome resulting in a stable genotype [3]. Therefore, selective pressure for the survival of genotype is not essential for better bioremediation capability and efficiency. These shuffling introduce new gene clusters in a recipient bacteria guided through stressed conditions of environment. IS elements are also associated with transfer of metabolic loci and are therefore evolutionarily significant in bacterial genomes. They are generally less than 2.5 kb in length and encode a protein that is involved in transposition [59], [60]. Using IS Finder, we report existence of these elements in CSV86 genome (Table S5); however none of these are present in vicinity to degradation pathway genes which are associated with integrase (Figure 4A, Figure S2).

Attempts to isolate plasmid from pan class="Species">CSV86 were unsuccessful. Further, Southern hybridization suggests that naphthalene degradation genes were localized in the genome. The naphthalene degradation property of CSV86 can be transferred by conjugation to Stenotrophomonas maltophila CSV89 with the transconjugants thus obtained preferentially metabolizing aromatic compounds over glucose. However, these properties were found to be unstable when transconjugants were grown on rich medium [12]. These results indicate probable involvement of GI in naphthalene degradation capability of CSV86. Comparative analysis of CSV86 genome with genome of established naphthalene degrading strains like P. stutzeri CCUG 29243, Pseudomonas sp. ND6 plasmid pND6-1, P. putida plasmid NAH7 and Pseudomonas fluorescens strain PC20 plasmid pNAH20 was performed. Analysis revealed that naphthalene and salicylate degrading gene clusters of CSV86 and P. stutzeri CCUG 29243 shares a high degree of homology at nucleotide sequence and showed the presence of genes encoding integrase (Figure S14) and transposase (Figure S15) upstream to both (nah and sal) operons. This feature was found to be absent for naphthalene degrading genes encoded by plasmids (pND6-1, NAH7 and pNAH20). This observation suggests the presence of GI or conjugative element(s). GIs have specific integration site (near or in tRNA gene) and lower G+C content compared to rest of the genome. The GC-profile tool, which calculates the compositional heterogeneity of DNA sequences, also postulates the presence of GI in CSV86 genome. The analysis of contig 105 which contained genes encoding for naphthalene upper pathway revealed that there was a marked difference in G+C content between the region containing naphthalene upper pathway genes (90555–100230) with the rest of the contig DNA (1–90554) suggesting possible insertion of GI in this region (Figure S16). This is supported by the occurrence of genes encoding for tRNA-Gly and integrase, located just upstream to the upper pathway genes of naphthalene degradation. Neither tRNA-Gly nor difference in the G+C content was observed in the contig 69 which encodes salicylate pathway.

Conclusions

The analysis of draft genome of Pseudomonas putida CSV86, which encodes for 5836 ORFs revealed the presence of complete catabolic pathway for pan class="Chemical">naphthalene degradation with more than 95% homology with reported coding sequences for P. stutzeri CCUG 29243. Identification of the GI at tRNAGly containing naphthalene degradation genes supports the ability to transfer the property by conjugation and the stability of this property. The identification of additional degradative and metal resistance genotype supported by phenotypic experiments further displays the diversity of CSV86. The degradative capacities associated with conjugation capabilities make this bacterium a possible donor in safe dissemination of catabolic potential for the process of genetic bioaugmentation.

Subsystem distribution of pan class="Species">Pseudomonas putida CSV86 genome in RAST.

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Organisation and comparison of catabolic genes involved in naphthalene degradation (pan class="Chemical">nah operon) in P. putida CSV86 (contig 105) against P. putida NCIB 9816-4 plasmid pDTG1 (NC_004999), Pseudomonas sp. ND6 plasmid pND6-1 (NC_005244), Acidovorax sp. JS42 (NC_008782) and Leptothrix cholodni SP-6 (NC_010524). Analysis was performed using RAST.

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Organisation and comparison of catabolic genes involved in salicylate acid degradation (sal operon) in pan class="Species">P. putida CSV86 (contig 69) against Pseudomonas sp. ND6 plasmid pND6-1 (NC_005244), P. putida NCIB 9816-4 plasmid pDTG1 (NC_004999), P. putida MT53 plasmid pWW53 (NC_008275) and Dechloromonas aromatica RCB (NC_007298). Analysis was performed using RAST.

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Phylogenetic tree showing comparison of nahR (4A), pan class="Chemical">nahAa (4B) and nahG (4C) promoter sequences of P. putida CSV86 (AMWJ00000000), P.stutzeri CCUG 29243 (NC_018028) and P. putida plasmid NAH7 (NC_007926).

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Alignment of NahR amino acid sequence of pan class="Species">P. putida CSV86 (NZ_AMWJ01000062.1) with that of P. putida plasmid NAH7 (NC_007926) and P.stutzeri CCUG 29243 (NC_018028). The pink highlighted bar indicates residue 116: Methionine in plasmid NAH7 and Isoleucine in both CSV86 and P. stutzeri. Similarly, the blue highlighted bar indicates residue 248 (Arginine in all the three); Green bar indicates residue 132 (Arginine in all the three) and Grey bar indicates residue 169 (Asparagine in all the three).

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Organisation and comparison of catabolic genes involved in the benzoate pathway in pan class="Species">P. putida CSV86 (contig 175) against P. fluorescens PfO-1 (NC_007492), P. putida KT2440 (NC_002947), P. putida F1 (NC_009512) and P. putida W619 (NC_010501). Analysis was performed using RAST.

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Organisation and comparison of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase genes in pan class="Species">P. putida CSV86 (contig 119) against P. putida GB-1 (NC_010322), Burkholderia cenocepacia HI2424 (NC_008544), Burkholderia cenocepacia AU 1054 (NC_008060) and Burkholderia ambifaria AMMD (NC_008392). Analysis was performed using RAST.

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Organisation and comparison of catabolic genes involved in phenylacetic acid degradation in pan class="Species">P. putida CSV86 (contig 88) against P. putida W619 (NC_010501), P. putida GB-1 (NC_010322), P. putida KT2440 (NC_002947) and P. putida F1 (NC_009512). Analysis was performed using RAST.

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Organisation and comparison of catabolic genes involved in 4-hydroxy phenylacetic acid degradation in pan class="Species">P. putida CSV86 (contig 7) against P. fluorescens SBW25 (NC_012660), P. aeruginosa PA7 (NC_009656), P. aeruginosa UCBPP-PA14 (NC_008463) and P. aeruginosa PAO1 (NC_002516). Analysis was performed using RAST.

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Organisation and comparison of catabolic genes involved in phydroxy benzoate degradation in pan class="Species">P. putida CSV86 against P. fluorescens Pf-5 (NC_004129), P. putida GB-1 (NC_010322), P. putida W619 (NC_010501), P. putida KT2440 (NC_002947), P. entomophila L48 (NC_008027) , P. putida F1 (NC_009512), P. fluorescens SBW25 (NC_012660), P. syringae pv. syringae B728a (NC_007005) and P .syringae pv. phaseolicola 1448A (NC_005773). Analysis was performed using RAST.

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Organisation and comparison of catabolic genes involved in phenylpropanoid pathway in pan class="Species">P. putida CSV86 against P. putida F1 (NC_009512), P. putida W619 (NC_010501), P. putida KT2440 (NC_002947), P. syringae pv. tomato str. DC3000 (NC_004578) and P. putida GB-1 (NC_010322). Analysis was performed using RAST.

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Organization and comparison of catabolic genes involved in homogentisate pathway in pan class="Species">CSV86 against P. putida KT2440 (NC_002947), P. putida F1 (NC_009512), P. fluorescens Pf-5 (NC_004129), P. putida W619 (NC_010501), P. fluorescens SBW25 (NC_012660), P. fluorescens PfO-1 (NC_007492), P. putida GB-1 (NC_010322) and P. entomophila L48 (NC_008027). Analysis was performed using RAST.

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Organisation and comparison of catabolic genes involved in copper resistance in pan class="Species">P. putida CSV86 against P. putida GB-1 (NC_010322), P. putida W619 (NC_010501), P. entomophila L48 (NC_008027), P. putida KT2440 (NC_002947), P. putida F1 (NC_009512), P. mendocina ymp (NC_009439) and P. stutzeri A1501 (NC_009434). Analysis was performed using RAST.

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Progressive alignment between the draft genomes of P. putida CSV86 and the complete genome of pan class="Species">P. stutzeri CCUG 29243 (NC_018028), Pseudomonas sp. ND6 plasmid pND6-1, complete sequence (NC_005244) and P. putida plasmid NAH7(NC_007926) and P. fluorescens strain PC20 plasmid pNAH20 (AY887963). Naphthalene degrading upper operon has been aligned with all the 4 genomes. Integrase coding genes has been highlighted by the black vertical bar. Colored blocks outline genome sequence that align to part of another genome, and is presumably homologous and internally free from genomic rearrangement (Locally Colinear Blocks or LCBs). The white colored area indicates regions with no alignment as they may probably contain sequences specific to that genome. The inverted blocks below the centre line indicate regions that align in the reverse complement (inverse) orientation and the height of the colored bars signify the nucleotide sequence similarity.

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Progressive alignment between the draft genomes of P. putida CSV86 and the complete genome of pan class="Species">P. stutzeri CCUG 29243 (NC_018028), Pseudomonas sp. ND6 plasmid pND6-1 (NC_005244) and Pseudomonas putida plasmid NAH7(NC_007926) and P. fluorescens strain PC20 plasmid pNAH20 (AY887963) using MAUVE software. Naphthalene degrading lower operon has been aligned with all the 4 genomes. Transposase coding genes has been highlighted by the black vertical bar.

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Output image of GC Profile software using nucleotide sequence of contig 105 having pan class="Chemical">naphthalene degradation upper pathway gene. The arrow indicates the segmentation point 1 from where the GC content varies as compared to rest of the contig sequence. The GC content of 1–90554 bp region is 63.8, whereas the region after the segmentation point i.e. 90555–100230 bp has GC content of 51.64 (segmentation strength-258.32).

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Summary of pan class="Species">P. putida CSV86 draft genome compared with other complete genome of pan class="Species">Pseudomonas spp. available in KEGG database.

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Pathways present in pan class="Species">P. putida CSV86 genome based on NCBI pan class="Chemical">PGAAP annotation.

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Percentage homology of Naphthalene upper and lower operon pathway proteins in pan class="Species">P. putida CSV86 with homologous proteins of 4 Pseudomonas species reported for naphthalene degradation capability.

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pan class="Chemical">Heavy metal resistance genes identified in pan class="Species">P. putida CSV86 genome annotation in RAST and their percentage homology with the closest respective gene.

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Mobile genetic elements present in pan class="Species">P. putida CSV86 genome located using IS finder.

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