Two draft genome sequences of Pseudomonas jessenii strains isolated from a copper contaminated site in Denmark

Pseudomonas jessenii C2 and Pseudomonas jessenii H16 were isolated from low-Cu and high-Cu industrially contaminated soil, respectively. P. jessenii H16 displayed significant resistance to copper when compared to P. jessenii C2. Here we describe genome sequences and interesting features of these two strains. The genome of P. jessenii C2 comprised 6,420,113 bp, with 5814 protein-coding genes and 67 RNA genes. P. jessenii H16 comprised 6,807,788 bp, with 5995 protein-coding genes and 70 RNA genes. Of special interest was a specific adaptation to this harsh copper-contaminated environment as P. jessenii H16 contained a novel putative copper resistance genomic island (GI) of around 50,000 bp.


Introduction
Copper is an essential micronutrient in most organisms and required as a co-factor in biological processes such as redox reactions (electron transport, oxidative respiration, denitrification) [1,2]. However, at higher concentrations copper will become toxic and inhibit or kill cells. Therefore, microorganisms have developed sophisticated copper homeostasis and resistance mechanisms in order to maintain the normal cellular copper supply to essential cuproenzymes while avoiding copper poisoning [3,4]. Some highly copper resistant microorganisms have attracted great interests due to potential biotechnological applications in bio-mining and bioremediation of environments contaminated with copper [5].
Pseudomonas spp. are ubiquitous inhabitants of soil, water and plant surfaces belonging to the Gammaproteobacteria. Pseudomonas spp. has an exceptional capacity to produce a wide variety of secondary metabolites, including antibiotics that are toxic to plant pathogens [6,7]. Pseudomonas jessenii was also found to be an important rhizobacterium conferring protection against a number of soilborne plant pathogens [8]. P. jessenii C2 and P. jessenii H16 were isolated from low-Cu soil and high-Cu soil from an abandoned wood impregnation site in Hygum, Denmark, respectively [9]. The Hygum site was contaminated with copper sulfate from 1911 to 1924, then the area was cultivated until 1993 and has been a fallow field since then [9,10]. P. jessenii H16 was able to grow in medium containing high concentrations of copper, whereas P. jessenii C2 was sensitive to high copper concentrations. Here, we present the genome sequences, a brief characterization and annotation of P. jessenii C2 and P. jessenii H16.

Classification and features
A highly copper contaminated high-Cu soil and a corresponding low-Cu soil were collected (0-20 cm depth) from a well-described Cu gradient field site in Hygum, Denmark. The high-Cu site was contaminated almost exclusively with CuSO 4 more than 90 years ago [9]. The adjacent low-Cu control site was located just outside the contaminated area and had been subjected to the same land use for more than 80 years. The low-Cu and high-Cu soil had similar physicochemical characteristics except for their total Cu contents of 21 and 3172 mg kg -1 , respectively [9,11]. Bacteria were isolated from replicated soil subsamples (n = 3) and diluted, spread-plated on Pseudomonas-selective Gould's S1 agar [11]. For each dilution series, 30 colonies emerging after two days at 25°C were selected and isolated in pure culture by repeated plating [11]. Two of the resulting isolates were selected for further study. P. jessenii H16 was able to grow at high concentration of Cu (2 mM) on onetenth strength LB agar, whereas P. jessenii C2 only grew with up to 0.125 mM Cu.

16S rRNA gene analysis
Comparative 16S rRNA gene sequence analysis using the EzTaxon database [13] indicated that strain C2 and H16 were both most closely related to P. jessenii CIP 105275 T (GenBank accession no. AF068259) with sequence similarities of 99.87 and 99.14 %, respectively. Phylogenetic analysis was performed using the 16S rRNA gene sequences of strains C2, H16 and related species. Sequences were aligned and phylogenic trees were constructed using Maximum Likelihood method implemented in MEGA version 6 [14]. The resultant tree topologies were evaluated by bootstrap analyses with 1000 random samplings (Fig. 2).

Genome project history
Next-generation shotgun-sequencing was performed at the Beijing Genomics Institute (BGI, Shenzhen). The whole genome shotgun project of P. jessenii C2 and P. jessenii H16 has been deposited at DDBJ/EMBL/GenBank under the accession numbers JSAK00000000 and JSAL00000000. The version described in this paper is the first version. A summary of the project and the Minimum Information about a Genome Sequence [15] are shown in Table 3.
Growth conditions and genomic DNA preparation P. jessenii C2 and P. jessenii H16 were aerobically cultivated on Pseudomonas-selective Gould's S1 agar at 28°C [16]. Total genomic DNA was extracted using Puregene Yeast/Bact Kit according to the manufacturer's instructions (QIAGEN). The quantity of the genomic DNA was determined by Qubit® fluorometer (Invitrogen, CA, USA) with Qubit dsDNA BR Assay kit (Invitrogen, CA, USA) and amounted to 55 ng/μL of DNA for P. jessenii C2 and 48.2 ng/μL of DNA for P. jessenii H16.

Genome sequencing and assembly
The genome sequence of P. jessenii H16 and P. jessenii C2 was determined by BGI using the Illumina Hiseq2000 with a 500 bp library constructed [17], generating 1.09 gigabytes of DNA sequence with an average coverage of~160 fold and~170 fold; yielding 1,205,9244 and 1,203,8756 paired-end reads with a 90-bp read length, respectively. The resulting sequence data was quality assessed, trimmed, and assembled de novo as described previously [18] using CLCBio Genomic Workbench 7.0 (CLCBio, Denmark). P. jessenii H16 generated 78 contigs with an n50 value of 279,014 bp. P. jessenii C2 generated 64 contigs with an n50 value of 224,893 bp. Evidence codes -IDA inferred from direct assay, TAS traceable author statement (i.e., a direct report exists in the literature), NAS non-traceable author statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [49]. If the evidence is IDA, the property was directly observed by the authors Evidence codes -IDA inferred from direct assay, TAS traceable author statement (i.e., a direct report exists in the literature), NAS non-traceable author statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [49]. If the evidence is IDA, the property was directly observed by the authors

Genome annotation
The genes in the assembled genome were predicted based on the RAST database [19]. The predicted ORFs were annotated by searching clusters of orthologous groups [20] using the SEED database [21]. RNAmmer 1.2 [22] and tRNAscanSE 1.23 [23] were used to identify rRNA and tRNA genes, respectively.

Genome properties
P. jessenii C2 contained 6,420,113 bp with a G+C content of 59.83 %, 5881 predicted genes, 5814 were proteincoding genes, 63 tRNA genes and 4 rRNA genes. In total, 5179 genes were assigned to biological functions and 635 were annotated as hypothetical proteins. P. jessenii H16 contained 6,807,788 bp, with a GC content of 59.02 %, Fig. 2 Phylogenetic tree of P. jessenii C2 and P. jessenii H16 relative to type strains within the genus Pseudomonas. The strains and their corresponding GenBank accession numbers of 16S rRNA genes are displayed in parentheses. The sequences were aligned using Clustal W, and the maximum likelihood tree was constructed based on Jukes-Cantor model by using MEGA6 [14]. Bootstrap values above 50 % are shown obtained from 1000 bootstrap replications. Bar 0.005 substitutions per nucleotide position   The total is based on the total number of protein coding genes in the genome 6065 predicted genes, and 5995 were protein-coding genes, 65 tRNA and 5 rRNA genes. Among the protein coding genes 5344 were assigned to biological functions, while 651 were annotated as hypothetical proteins. The properties and statistics of those two genomes are summarized in Table 4. The distribution of genes into COG functional categories is presented in Table 5 and Fig. 3.

Insights into the genome
Genes conferring resistances to heavy metals in the two studied strains are listed in Table 6. Copper efflux from the cytosol is mediated by the P 1B -type ATPase family, which is highly conserved from bacteria to humans [24]. Both P.
jessenii C2 and P. jessenii H16 contained genes encoding a copper-transporting P 1B -type ATPase (CopA) with conserved CPCALG motif [25], a copper-responsive metalloregulatory protein CueR, and the multicopper oxidase CueO. In addition, one additional gene encoding a Cu + -ATPase is present on the genome of P. jessenii H16 as part of the GI discussed later. P. jessenii H16 also contained ccoI encoding a Cu + -ATPase catalyzing a slower rate of efflux for copper insertion into cytochrome c oxidase [26]. The presence of a cop operon, comprising copABCDRS had been reported in related P.fluorescens SBW25 and P.putida KT2440 [27,28]. Both P. jessenii strains contained copCDRS probably encoding proteins Fig. 3 Circular map of the chromosome of P. jessenii C2 and P. jessenii H16. From outside to the center: P. jessenii H16 genes on forward strand (color by COG categories), P. jessenii H16 CDS on forward strand, tRNA, rRNA, other; P. jessenii H16 CDS on reverse strand, P. jessenii H16 tRNA, rRNA, other, genes on reverse strand (color by COG categories); P. jessenii C2 CDS blast with P. jessenii H16 CDS; P. fluorescens SW25 (NC_012660) CDS blast with P. jessenii H16 CDS; P. jessenii H16 GC content; P. jessenii H16 GC skew, where green indicates positive values and magenta indicates negative values responsible for copper uptake, however, only P. jessenii H16 also contained copAB as part of the GI. Both P. jessenii C2 and P. jessenii H16 contain an arsenic resistance determinant (arsRBCH) [29] a gene involved in chromate resistance (chrA) [26] ( Table 6). The two strains also contained genes encoding a multidrug efflux system MexEF-OprN regulated by MexT and genes encoding DNA gyrase subunit A and B, and topoisomerase subunit (IV) A and B [30,31].
P. jessenii H16 contained an additional putative metal fitness/pathogenicity island when compared with P. jessenii C2. It encompasses about 50,000 bp beginning at a gene encoding a sulfur carrier protein (KII37703) and ending with genes encoding Tn7 transposition proteins (KII37740-KII37743). This potential pathogenicity/fitness island harbored several copper resistance determinants including the cus determinant encoding CusABCRS (KII37706-37708, KII37711-37712) involved in periplasmic copper detoxification [32,33]. In addition, genes encoding the P-type ATPase CopA, the multicopper oxidase CueO and CopBDG (KII37893, KII37715, KII37716, KII37709, KII37717) could be identified (Fig. 4). We also predicted specific GI for both P. jessenii H16 and P. jessenii C2 using the IsfindViewer [34]. Based on the automatic prediction algorithm two putative regions (coordinates KII37706-KII37717, KII37721-KII37737) were only identified in P. jessenii H16. Similar copper fitness islands could also be detected in P.extremaustralis 14-3b (AHIP00000000), isolated from a temporary pond in Antarctica; Pseudomonas sp.Ag1 (AKVH00000000) isolated from midguts of mosquitoes and P. fluorescens FH4 (AOHN00000000) [35][36][37]. This island also contained genes encoding the nickel efflux transporter NcrA (KII37721) and the transcriptional repressor NcrB (KII37723) [38]. Moreover, genes merTR-CAB (KII37733-37737) encoding a mercury-resistance determinant are present on this island [39]. Many of the various putative GI contain functions related to mobility such as integrases or mobile genetic elements (MGE) which includes transposons and IS elements. As shown in P. jessenii H16, these putative GI have conferred this strain with additional heavy metal resistance capability, which may be transferred to other bacteria via Tn7 transposons and are highly relevant for adaption to this specific copper contaminated niche.

Conclusion
The draft genome sequences of P. jessenii C2 isolated from low-Cu soil and P. jessenii H16 isolated from high-Cu soil were determined and described here. H16 provided an insight into the genomic basis of the observed higher copper resistance when compared with C2. Based on analysis and characterization of the genome, P. jessenii H16 is predicted to be resistant to a number of heavy metal(loid)s, such as Hg 2+ , Ni 2+ Cr 2+ and As 3+ . Comparative genomic analysis of those two strains suggested acquisition of a fitness island encoding numerous genes involved in conferring resistance to Cu and other metals as an important adaptive mechanism enabling survival of P. jessenii H16 in its Cu contaminated habitat. Possibly, P. jessenii H16 may have potential for bioremediation of copper contamination environments.