Draft genome sequence of Pseudomonas extremaustralis strain USBA-GBX 515 isolated from Superparamo soil samples in Colombian Andes

Here we present the physiological features of Pseudomonas extremaustralis strain USBA-GBX-515 (CMPUJU 515), isolated from soils in Superparamo ecosystems, > 4000 m.a.s.l, in the northern Andes of South America, as well as the thorough analysis of the draft genome. Strain USBA-GBX-515 is a Gram-negative rod shaped bacterium of 1.0–3.0 μm × 0.5–1 μm, motile and unable to form spores, it grows aerobically and cells show one single flagellum. Several genetic indices, the phylogenetic analysis of the 16S rRNA gene sequence and the phenotypic characterization confirmed that USBA-GBX-515 is a member of Pseudomonas genus and, the similarity of the 16S rDNA sequence was 100% with P. extremaustralis strain CT14–3T. The draft genome of P. extremaustralis strain USBA-GBX-515 consisted of 6,143,638 Mb with a G + C content of 60.9 mol%. A total of 5665 genes were predicted and of those, 5544 were protein coding genes and 121 were RNA genes. The distribution of genes into COG functional categories showed that most genes were classified in the category of amino acid transport and metabolism (10.5%) followed by transcription (8.4%) and signal transduction mechanisms (7.3%). We performed experimental analyses of the lipolytic activity and results showed activity mainly on short chain fatty acids. The genome analysis demonstrated the existence of two genes, lip515A and est515A, related to a triacylglycerol lipase and carboxylesterase, respectively. Ammonification genes were also observed, mainly nitrate reductase genes. Genes related with synthesis of poly-hydroxyalkanoates (PHAs), especially poly-hydroxybutyrates (PHBs), were detected. The phaABC and phbABC operons also appeared complete in the genome. P. extremaustralis strain USBA-GBX-515 conserves the same gene organization of the type strain CT14–3T. We also thoroughly analyzed the potential for production of secondary metabolites finding close to 400 genes in 32 biosynthetic gene clusters involved in their production.


Introduction
The genus Pseudomonas, subclass Gammaproteobacteria, is an ubiquitous and metabolically versatile bacterial genera and is currently the genus of Gram-negative bacteria with the largest number of species [1]. Since it first description in 1894 [2], an increasing number of species has been described in diverse environments [3][4][5]; and now this genus comprises 255 validly named species, and 13 subspecies, according to the list published in the Namesforlife Database [6]. Psychrophilic environments are the common habitats of the Pseudomonas genus. There are several isolated pseudomonads bacteria from water, freshwater and soils at low temperatures, such as psychrophilic strains of P. aeruginosa, P. fluorescens, P. putida, P. syringae, P. antarctica, P. meridiana and P. proteolytica [5,[7][8][9] and recently P. extremaustralis [10]. The type strain of the species P. extremaustralis was isolated from a temporary pond in Antarctica [10,11]. This species presents high levels of oxidative stress and cold resistance along with production of high levels of polyhydroxybutyrate (PHB) [11][12][13]. It is also able to tolerate and to degrade hydrocarbons, allowing it to be used in extreme environments for hydrocarbon bioremediation [14]. The polyhydroxyalkanoate synthase genes are located within a genomic island, which was probably acquired by horizontal gene transfer [11,12,15]. Furthermore, P. extremaustralis grows under microaerophilic conditions and forms well developed biofilms that degrades long-chain and branched alkanes, while only medium-chain length alkanes are degraded by planktonic cells [14][15][16].
The type strain CT14-3 T (DSM 17835) of P. extremaustralis, and its natural derivative, the strain 14-3b (DSM 25547) have been studied for a long time, but no other strains of this species have been reported to our knowledge. We have been involved on microbial diversity studies in the Nevados National Natural Park (Nevados NNP) that harbors different extreme environments, such as permanent snows, superparamo, paramo and thermal springs associated to volcanic activity [17,18]. These studies aim to isolate and analyze culture collections of different microbes present in these habitats.
Here we present the physiological features of P. extremaustralis strain USBA-GBX-515, isolated from soils in Superparamo ecosystems, in the northern Andes of South America, as well as its draft genome. A genomic comparison with the type strain is also presented.

Organism information
Classification and features Samples were collected in 2010 from Superparamo soil samples within the Nevados NNP at >4000 m.a.s.l with soil temperature of 9.8°C, and pH of 5.2. Paramo and superparamo are Andean ecosystems in the neotropical high mountain biome [19].
Enrichment was initiated by resuspending 10 g of rhizospheric soil samples into M9 basal medium (BM) during 30 m at 150 r.p.m. Then, the cultures were serially diluted, inoculated into M9 BM (10 −2 to 10 −6 ) and amended with 10 mM tributyrin at pH 6.0, and then incubated at 30°C for two weeks. The M9 basal medium contained: 0.5 g NaCl, 3 g KH 2 PO 4 , 6 g Na 2 HPO 4 , 1.0 g NH 4 Cl, and 0.05 g yeast extract. We obtained pure colonies using agar plates with the same medium. Several of the pure isolates obtained were morphologically similar and 16S rRNA genes were 99% similar among them (data not shown). One strain, designated strain USBA-GBX-515, was selected for this study. The isolated bacterium was stored since the collection date at the Collection of Microorganisms of Pontificia Universidad Javeriana as P. extremaustralis strain CMPUJU 515 (CMPUJ, WDCM857). The general features of the strain are reported in Table 1. Growth was assayed at different pHs (4.5 to 8.5) following the protocols described by Rubiano et al. (2013) [20], with the optimal growth pH being 7.0. Also, different growth temperatures (from 4°C to 35°C) were tested and although growth was observed at all temperatures, the optimum temperature was determined as 30°C. Strain USBA-GBX-515 is a Gram-negative rod shaped bacterium of 1.0-3.0 μm × 0.5-1 μm (Fig. 1), aerobic, motile and unable to form spores. Cells present one single flagellum. Colonies are small, smooth, circular and they did not show pigments on Luria Bertani (LB) medium but fluorescent pigments were observed on Centrimide and King B agar. Using the API ZYM strip (BioMérieuxMarcy l'Etoile, France) positive reactions were observed for catalase and oxidase. The API50CH and API 20 (BioMérieux) tests showed positive reactions for L-arginine, sodium citrate and nitrate, nitrite, and negative for starch, casein, urea, indole, D-mannitol, L-arabinose and gelatin. Strain USBA-GBX-515 exhibited alkaline phosphatase and phosphohydrolase activities. This strain presents susceptibility to imipenem, piperacilin, ticarcilin, meropenem, levofloxacin, ceftriaxone, cefoxitin and ceftazidime. On the other hand, strain USBA-GBX-515 T showed resistance to penicillin, colistin or polymyxin E, and nitrofurantoin.
Due to our particular interest on lipase enzymes, we also evaluated the lipolytic activity of strain USBA-GBX-515, following the protocols described in [21]. We observed growth on Tween 80, olive oil, triolein, tricaprylin and tributyrin when these compounds were used as carbon sources. We measured the lipolytic activity using p-nitrophenyl butyrate during its growth for 42 h at 30°C, using tributyrin as carbon source. We detected the maximum activity, 2.0 UL μmol/L/min at 15 h at the end of the exponential phase, as previously reported for the species of the genus [22,23]. Additionally, we observed the higher activity in the extracellular fraction than in the intracellular fraction.
Analysis for initial phylogenetic inferences was done using universal amplification primers 27F (5′ CAG AGTTTGATCCTGGCTCAG 3′) and 1492R (5′ TACG GYTACCTTGTTACGACTT 3′). PCR products were sequenced using Sanger technology with an eight capillary Applied Biosystems GA-3500 sequencer. Neighborjoining phylogenetic tree reconstruction was done using MEGA 7.0.25. Phylogenetic analysis of the 16S rRNA gene sequence (Fig. 2) confirmed that USBA-GBX-515 is a member of Pseudomonas genus. The most closely related strain was P. extremaustralis CT14-3 T and then, our isolate was assigned to P. extremaustralis, by comparison of the 16S rRNA sequence. Strain USBA-GBX-515 T exhibited a 100% 16S rRNA sequence identity (e-value = 0.0) with P. extremaustralis CT14-3 T .
P. extremaustralis strain USBA-GBX-515 was stored at the Collection of Microorganisms of Pontificia Universidad Javeriana (CMPUJ, WDCM857) (ID CMPUJ U515) with the ID USBA-GBX 515, growing aerobically on the same medium as mentioned above. Cells were preserved at −20°C in BM supplemented with 20% (v/v) glycerol.

Genome project history
The strain was selected to sequencing on the basis of its metabolic versatility and the biotechnological potential as revealed by previous studies [10][11][12]. This work is part of the bigger study aiming at exploring the microbial diversity in extreme environments in Colombia. More information can be found on the Genomes OnLine database under the study Gs0118134. The JGI accession number is 1,094,800 and consists of 69 scaffold. Table 2 depicts the project information and its association with MIGS version 2.0 compliance [24]. The USBA-GBX-515 T draft Genome has the ENA accession number FUYI01000001-FUYI01000069, and is also available through the Integrated Microbial Genomes system under the accession 2,671,180,025.

Growth conditions and genomic DNA preparation
Pseudomonas extremaustralis strain USBA-GBX-515 grew aerobically on LB medium at 30°C. A 1 mL of overnight culture was centrifuged for 2 min at 13000 g. the pellet was immediately used for DNA extraction using the Wizard SV GEnomic DNA purification kit (Promega, USA). The integrity and quality of the DNA was verified using agarose gels (Sigma-Aldrich, St. Louis, USA) 0.8% (w/v) and using the NanoDropTM system (Thermo Scientific). The genomic DNA concentration was measured by the Qubit® dsDNA by fluorometric quantitation (Invitrogen, USA). Evidence codes: IDA inferred from direct assay (first time in publication); TAS traceable author statement (i.e., a direct report exists in the literature); NAS nontraceable 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 codes are from the Gene Ontology project [69] Genome sequencing and assembly Genomic DNA for Pseudomonas extremaustralis strain USBA-GBX-515 was sequenced on a HiSeq 2500 sequencer (Illumina, SanDiego, CA, USA) with a pairedend strategy (PE150) of 300-bp reads. The sequencing platform generated 10,817,988 reads. After trimming a total of 10,000,000 paired end reads were obtained and assembled into 77 contigs and 69 scaffolds using ALL-PATHS [25] and Velvet [26] softwares. All samples were processed using BUSCO [27], which offers a measure for quantitative assessment of genome assembly and annotation quality based on evolutionarily informed expectations of gene content. With the raw data (FastQ read files), the estimated genome size was calculated using different k-mer sizes in Kmergenie [28]. Finally, to obtain assembly metrics of the different genomes, QUAST [29] was run. The draft genome of P. extremaustralis strain USBA-GBX-515 consisted of 6,143,638 Mb with a G + C content of 60.9% mol. Table 3 contains all the genome statistics.

Genome annotation
Genes were identified using Prodigal [30] as part of the DOE-JGI Annotation pipeline [31,32]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes (IMG-ER) [33]. Biosynthetic clusters were predicted running anti-SMASH [34], BAGEL3 [35] and NaPDoS [36]. Anti-SMASH was run using the GenBank file generated during annotation from the IMG-ER as the input. Before running the antiSMASH server tool, ClusterFinder algorithm [37], whole-genome PFAM analysis [38] and Enzyme Commission (EC) number prediction were selected. BAGEL3 is a tool specialized in predicting RiPPs and Bacteriocins using as FASTA file as the input.  Finally, the NaPDoS tool was run four times per genome; first with a FASTA nucleotide file as the input and seeking KS domains and second with the same input but seeking C domains. The third and fourth runs were with FASTA amino acid files, seeking KS and C domains respectively. A list of all biosynthetic clusters is available through IMG and IMG-ABC systems [32,39].

Genome properties
The genome of Pseudomonas extremaustralis strain USBA-GBX-515 is 6,143,638 bp -long with a G+ C content of 60.9 mol%. A total of 5665 genes were predicted and of those, 5544 were protein coding genes and 121 RNA genes. The properties and statistics of the genome are summarized in Table 3, of the total CDSs, 72.2% represent COG functional categories. The distribution of genes into COGs functional categories is presented in Table 4. Most genes were classified in the category of amino acid transport and metabolism (10.5%), followed by transcription (8.38%) and signal transduction mechanisms (7.3%).

Insights from the genome sequence
We performed taxonomic genome comparisons between Pseudomonas USBA-GBX-515 and P. extremaustralis strain CT14-3 T . The average nucleotide identity (ANI) calculated with the MiSI (Microbial Species Identifier) method [40] is 98.9% with an Alignment Fraction (AF) of 0.91. Using GGDC web server version 2.1 [41], the DNA-DNA hybridization was calculated, and it showed 96.7% of similarity; the difference in G + C content was less than 1% (0.27 of difference) within both strains. Finally, a pairwise genome alignment performed with Mauve [42] between our strain and the type strain CT14-3 T of P. extremaustralis (17835 T ) was performed, showing the similarity and conserved synteny of genes (Fig. 3). There are few regions that were unassembled in our genome and those remain in small separated contigs. All analyses corroborate the affiliation of our strain to the species P. extremaustralis.
This isolate was screened for lipolytic activities, and the genome analysis showed two genes lip515A and est515A related to a triacylglycerol lipase and carboxylesterase, respectively. Both genes showed a conserved α/β hydrolase motif which is common in lipolytic enzymes [43,44], and are required for the lipids and fatty acid metabolisms. Particularly, the deduced amino acid sequence (296aa) from Gene lip515A showed an identity of 49% (E value 3e-80) with a triacylglycerol lipase from Pseudomonas fragi [45], while gene est515A had a 68% identity (E value 2 e-85) with a hypothetical protein from Colwellia sp. TT2012.
Ammonification genes were also observed, mainly nitrate reductase genes (narG,H,I,J,L,X and napA). Markers of nitrifying bacteria, norB and nosZ reductases were found, both genes were described previously in Pseudomonas stutzeri, a nitrate respiring bacterium [46]. We found a norVW gene, which has a role in protection against reactive nitrogen intermediates [47,48]. A total of 732 genes were identified to play a role in amino acid transport and metabolism, which depends on nitrogen fixation metabolism.  The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome The presence of proline operon proHJ and proA gene demonstrate the response to high osmolarity due to the de novo synthesis of proline as a stress protectant of the cell [49]. Cell protection from toxic effects of hydrogen peroxide was determined by the presence of the catalase (katE) gene.
Similar to previously observed on P. extremaustralis CT14-3 T , we found genes related with the synthesis of polyhydroxyalkanoates (PHAs). Especially poly-hydroxybutyrates genes (PHBs), were detected in the bacterial genome using BlastP (e-value <0.05 and 90% coverage of the gene). The phaABC operon was present, containing the PHA synthase (phaC), β-ketothiolase (phaA), and NADP-dependent acetoacetyl-CoA reductase (phaB). The phbABC operon is also present into the genome, corresponding to the same enzymes.
In order to gain knowledge about the strain USBA-GBX-515 T , we explored the potential production of secondary metabolites by data mining (Fig. 4). The genes responsible for the secondary metabolites were organized in 32 biosynthetic gene clusters using IMG tools. Those contained approximately 400 genes, the 78% of clusters were designed as putative, whilst 22% were related to NRPS and bacteriocin, but it was not possible to identify known metabolites.
Using antiSMASH 3.0 platform we detected 57 cluster of biosynthetic genes. The 56% of clusters were classified as putative. Two biosynthetic clusters (classified as putative) were assigned to fengycin and alginate clusters. Fengycin is a cyclic lipopeptide acting against phytopathogenic viruses, bacteria, fungi, and nematodes. The lipopeptides are synthesized at modular multienzymatic templates [50,51]. The polymer alginate had been identified mainly in the genus Pseudomonas as an exopolysaccharide involved on biofilm formation and pathogenicity [52]. A total of 24.5% of the clusters were classified as saccharides. Five of the biosynthetic clusters included in this category were related to lipopolysaccharide, pseudopyronine, colonic acid, O-antigen and glidobactin. The proteins coded by the cluster associated to lipopolysaccharide (LPS) (Fig. 5) are secreted to the outer surface and the cluster is expressed as a mechanism of resistance to detergents and hydrophobic antibiotics [53]. Colanic acid is an extracellular polysaccharide related to desiccation resistance [54] and to adhesion as pathogenic factor [55] (Fig. 6). O-antigen is a lipopolysaccharide which is associated to adhesion [56] (Fig. 7). The last saccharide cluster is related to glidobactin (Fig. 8), a PKS/NPRS cytotoxic compound which is an antifungal and antitumor antibiotic complex [57]. The   structure of this cluster shows a 36% similarity to the cluster of "Pseudomonas batumici" strain UCM B-321. A PKS/NRPS compound has been founded from "Pseudomonas batumici" named batumin which exhibits potent and selective antibiotic activity against Staphylococcus species [58]. A biosynthetic gene cluster related to arylpolyene-saccharide was detected (Fig. 9), and this metabolite has a similar structure to a pigment produced by members of the genus Xanthomonas and Flexibacter, which is involved on Gram negative bacteria protection against exogenous oxidative stress [37]. Other clusters were associated to coronatine (Fig. 10) and mangotoxin (Fig. 11) compounds. Both are antimetabolites related to phytotoxins. The coronatine acts as a virulence factor and induces hypertrophy, inhibits root elongation, and stimulates ethylene production [59]. The mangotoxin is a small peptidic molecule, which inhibits the biosynthesis of essential amino acids, resulting in an amino acid deficiency [60]. These toxins could be used as herbicides such as glufosinate and bialaphos, two commercial herbicides that mimic bacterial toxins [60]. Finally, we found a cluster related to pyoverdine (Fig. 12), a nonribosomal peptide siderophore [61].
According to NapDOs program [36], several genes were related to fatty acid biosynthesis, particularly two genes fat478 and fat3803 (related to proteins FabB and FabF, respectively); those proteins are chain elongation condensing enzymes (synthases) that control fatty acid composition and influence the rate of fatty acid production [37].
Using BAGEL3 we found the cluster class III related to S-type Pyocin, a compound with a killing activity causing cell death by DNA breakdown through endonuclease activity [62].