P. aeruginosa LV strain was cultured in nutrient broth at 28°C for 24 h in the presence or absence of copper chloride (0.1 g L⁻¹), according to patent (PI0803350-1 – INPI 12/09/20092008; http://www.inpi.gov.br). The cells were recovered by centrifugation in a Jouan CR3 equipment (9,000 rpm/20 min, 4°C), resuspended in TRIZOL (INVITROGEN) and kept at −80°C. The confirmation of the antibiotic activity of the supernatants was confirmed as described elsewhere (De Oliveira et al., 2016). Both cultures were performed in biological triplicate (three samples for each condition, corresponding to three experiments separated spatially and temporally).

The total RNA was isolated using the illustra RNAspin kit™ (Ge Healthcare, UK) and ribosomal RNA depletion was performed using the RiboMinus™ transcriptome Kits (TermoFisher Scientific), followed the manufacturer's recommendation. The integrity of RNA was checked in agarose gel (1.5%) in a denaturant condition and the amount of RNA was determined using a spectrophotometer (Figures S7, S8) (BioTek Sinergy HT).

The construction of library was carried at Biotechnology and Molecular Markers Laboratory in the Universidade Federal de Minas Gerais (Belo Horizonte, MG, Brazil). The rRNA-depleted RNA (1 μg) was used to build cDNA library using the kit TruSeq® RNA Sample Preparation v.2 (Illumina, USA). The libraries were quantified with KAPA Library Quantification Illumina® (KAPA Biosystems) by quantitative PCR. The libraries were normalized to the concentration of 4 nM and sequencing was performed with MiSeq sequencer (Illumina) using MiSeq Reagent v3 (600 cycles) paired-end 2 × 300.

The transcripts differentially expressed in presence of copper were analyzed using BLAST (Basic Local Alignment Search Tool), INTERPRO (Protein Sequence Analysis and Classification), UNIPROT (Universal Protein Resource) e PFAM (Protein Domain) databases, separately and manually classified according to the similarity of sequences description and function.

The generated RNA-Seq library was composed by 30 million reads, obtained by RNA extraction and depletion and synthesis and sequencing of resulting cDNA (Figure 1). The reads were separated in two groups, corresponding to the P. aeruginosa strain LV culture conditions (presence or absence of copper chloride). The first group (absence of copper) was composed of 14,783,618 reads, and the second group (presence of copper) contained 15,091,252 read (Table 1). The triplicates of each group were analyzed together and separately, generating similar results.

The mean covering of each treatment was greater than 100× on the reference genome. Approximately 63% of reads from group one and 65% of group two were mapped (Table 1). The quantification of gene expression used the values of RPKM. The mapping data obtained between the different growth conditions were compared for determining the levels and differences in the gene expression. Nine genes were significantly upregulated (Figure 2), suggesting their relation with the biosynthesis of the bioactive organometallic compound, which only occurs in the presence of copper. On the other hand, four genes related to copper efflux were downregulated in cells cultured in the presence of copper.

Upregulated genes were filtered and classified in functional categories, based on the similarity of sequence description for reference-guided genome analysis (Table 2) and de novo assembly (Table 3). Downregulated genes were also identified (Table 4).

Three upregulated genes (PA2691, PA4141, and PA4782) were not functionally characterized in all data banks used and were classified as hypothetical proteins. Other three upregulated genes were related to the transport of metallic ions (PA3521, PA3523, and PA3920) and one gene (PA3574a) was associated with chaperones group linked with copper. The gene (PA4872) was classified as a transcript regulator and gene (phzA2) was related to the phenazine biosynthesis.

Three hyperexpressed genes were related to the efflux system (PA3521, PA3523, and PA3920) and one gene (PA3574a) was related to a copper-chaperone, the CopZ. The PA3920 gene is related to the transport of heavy metals in Pseudomonas sp. and belongs to the P1B-1 subgroup of the carrying family P-type ATPases. In this system, the copper efflux occurs in the presence of ATP, when copper chaperone translocate the cation and link in a carrying protein, moving the complex through the membrane, to outside the bacterial cell (González-Guerrero et al., 2010). The P. aeruginosa LV strain efflux system was triggered by the presence of copper chloride, with hyperexpression of genes related to this mechanism.

The PA4878 gene belongs to a MerR family, which includes regulator proteins involved in the activation of the efflux system, in response to an inductor (Liao et al., 2013). The high expression values of this gene observed in copper-stimulated P. aeruginosa LV strain may be related to a set of mechanisms working to eliminate the metal from within the cell.

The downregulated genes were related to the ABC transporter (PA203), ABC transporter permease (PA204 and PA205) and an amidase (PA202). The ABC transporter system is composed of an outer membrane carrier, a membrane-fusion protein and an outer-membrane porin. These three components assemble into a complex spanning both membranes and providing the conduit for the translocation of unfolded polypeptides, as well as ions and various other substances into bacteria (Morgan et al., 2017). The downregulation of the ABC transporter and permease in the P. aeruginosa LV strain reveals a strategy for blocking the copper influx. Similar data were found by Li et al. (2009) in copper-resistant Pseudomonas spp. The reduction of the amidase gene is related to the decrease of the expression of porins, since they are involved in the entrance of polysaccharides into the bacterial cell.

The phenazine family are nitrogenous heterocycle compounds with redox activity, produced by fluorescent pseudomonads. The phenazines are small molecules that can penetrate different types of cells, including prokaryotes. Many phenazines present antimicrobial activity, suppressing pathogenic microorganisms of humans, animals and plants, also playing an important role in the survival strategies of bacteria in the environment (Chen et al., 2014; Briard et al., 2015; Dasgupta et al., 2015; Garrison et al., 2016; Morrison et al., 2016). The P. aeruginosa LV strain is a great producer of phenazine carboxylic acid (PCA).

The biosynthesis of phenazine in P. aeruginosa is realized by two seven-gene operons, phz1 (phzA1B1C2D1E1F1G1) and phz2 (phzA2B2C2D2E2F2G2). According to Li et al. (2011), there is a combined regulation of these two operons in the biosynthesis of PCA. The first one (phzA2-G2 gene) produces small amount of PCA and the second one (the phzA1-G1 gene) produces a large amount of PCA. The expression of two operons working together optimizes the PCA biosynthesis, through a positive feedback mechanism. The operon phz2 produces a basal amount of PCA, self-inducing the same operon to keep the biosynthesis process and triggering the transcription of operon phz1, amplifying PCA production.

In the present study, the hyperexpression of phzA2 gene belonging of phz2 operon was observed. Among the nine-upregulated genes identified, the phzA2 is the only one related to a biosynthesis pathway. The proposition is that the augmented expression of phzA2 gene stimulates the production of PCA and, once there is PCA in the cytoplasm, it activates its own biosynthesis process.

Many studies carried out in the Microbial Ecology Laboratory demonstrated that, in absence of copper, the P. aeruginosa LV strain produces pyoverdine (PYO) from PCA, suggesting that the PCA is a precursor for PYO biosynthesis. However, when copper is present, the LV strain produces PCA and the PYO formed helps the bacteria to bioremediate the copper, forming the organometallic compound (Figures S1–S6) with C, Cu, S and O (Figure S9) and N (Figure S10). Possibly, the three hypothetical proteins detected are involved in the biosynthesis of PYO, using PCA as precursor (Figure 3). Further studies will be carried out to better understand such process and to confirm this hypothesis.

In this analysis, twelve upregulated genes and two downregulated genes were identified. In relation to upregulated genes, one gene was not functionally characterized and was classified as a hypothetical protein. Seven upregulated genes were related to increased protein synthesis (RPII, MaK, agaA, PA474, atg4, TIM21, mug8) and two genes related to oxidative stress (FAM120A and AFUA_3G00590). The PA2853 gene was related to an outer membrane lipoprotein (OprI) and the BAV0296 gene (glutamyl-tRNA reductase) was related to the synthesis of tetrapyrroles compounds. From the downregulated genes, one was related to homocitrate synthase, LYS20, and one was an uncharacterized protein, KUJ92327.

Copper is a cofactor for many important enzymes, in eukaryotes and prokaryotes and it is essential for almost all organisms. However, the excess copper can also be harmful due to its redox activity and the ability of binding to sites of other metals, particularly iron-sulfur clusters, leads to an increased microbial metabolism, related to the stress generated by the ion inside the organism (Vita et al., 2016). Seven genes related to increased protein synthesis were detected in this study. From those, five can be detected in prokaryotes: the RPII gene (a RNA-polymerase); MaK (protein kinase) related to many cellular processes, including metabolism, transcription, cell cycle progression, cytoskeletal rearrangement and cell movement, apoptosis, and differentiation; agaA (carbohydrate binding module family 6), which binds to specific ligands, such as cell-surface-attached carbohydrate substrates for galactose oxidase and sialidase; PA474 (preprotein translocase subunit SecG), involved in protein export and translocation and atg4 (peptidase family C54), a proteolytic enzyme that hydrolyses peptide bonds using the thiol group of the cysteine residue as a nucleophile.

Another two genes may be related to increased protein synthesis and cell division, TIM21 and mug8. These genes have been described in eukaryotes and are responsible for the translocation of transit peptide-containing proteins across the mitochondrial membrane and role in bud formation, respectively. This may help the bacteria to maintain the cellular division in the presence of cooper.

The gene FAM120A (oxidative stress-associated Src activator), a critical component of the oxidative stress-induced survival signaling in human, and AFUA_3G00590 (asp-hemolysin), expressed during bacterial sporulation, were also detected. Furthermore, the OprI, the major outer membrane lipoprotein of P. aeruginosa, presented increased expression (Basto et al., 2014). The upregulation of these genes can be explained. High levels of copper are toxic to bacteria and lead to cell damage, by generating reactive oxygen species, affecting the function of proteins or inactivating the enzymes (Li et al., 2009) and hence those overexpressed proteins act on protecting the cell against such damage.

The tetrapyrrole compounds are molecules that have four rings of the pyrrole type, generally linked together by single-atom bridges, between the alpha positions of the five-membered pyrrole rings. They function as a metal-binding cofactor in many important enzymes, proteins and pigments, such as heme, chlorophyll, cobalamine (vitamin B12), siroheme and cofator F430 (Schulze et al., 2006; Schalk and Cunrath, 2016) may suggest its participation in cooper insertion in the bioactive compound produced by LV strain. The Glutamyl-tRNA reductase is an important enzyme involved in this process. The super-expression of this enzyme detected in our study reaffirms our hypothesis that the compound with antimicrobial activity comes from the biosynthesis of pigments produced by P. aeruginosa strain, since the increase of tetrapyrrole can be related to the binding of copper on these pigments.

The overexpressed genes detected were different in the two methods applied in this study. Still, most of them are related to the stress in the cell caused by copper and its elimination. Furthermore, in the reference-guided genome analysis, an increase in phzA2 gene expression was found (related to phenazine biosynthesis) whilst an increased expression of the BAV0296 gene was detected in the de novo analysis (related to the production of tetrapyrrole compounds). Although the results are different, there is a complementarity between them, since the increase in expression of these genes, related to the pigments produced by the P. aeruginosa strain, can also be related to the production of the organometallic compound with antimicrobial activity, as a mechanism of bioremediation of intracellular copper. Additional studies are being carried out with these genes to confirm such hypothesis.

Regarding the down regulated gene LYS20, related to homocitrate synthase, it is known that this enzyme participates in lysine biosynthesis and pyruvate metabolism. The homocitrate synthase catalyzes the first step in lysine biosynthesis in many fungi and certain Archaea (Bulfer et al., 2009). The down expression of this gene can be related to a decreased cell anabolism, caused by cellular stress derived from excess intracellular copper.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.