A Novel Marine Pathogen Isolated from Wild Cunners (Tautogolabrus adspersus): Comparative Genomics and Transcriptome Profiling of Pseudomonas sp. Strain J380

Cunner (Tautogolabrus adspersus) is a cleaner fish being considered for utilized in the North Atlantic salmon (Salmo salar) aquaculture industry to biocontrol sea lice infestations. However, bacterial diseases due to natural infections in wild cunners have yet to be described. This study reports the isolation of Pseudomonas sp. J380 from infected wild cunners and its phenotypic, genomic, and transcriptomic characterization. This Gram-negative motile rod-shaped bacterium showed a mesophilic (4–28 °C) and halotolerant growth. Under iron-limited conditions, Pseudomonas sp. J380 produced pyoverdine-type fluorescent siderophore. Koch’s postulates were verified in wild cunners by intraperitoneally (i.p.) injecting Pseudomonas sp. J380 at 4 × 103, 4 × 105, and 4 × 107 colony forming units (CFU)/dose. Host-range and comparative virulence were also investigated in lumpfish and Atlantic salmon i.p. injected with ~106 CFU/dose. Lumpfish were more susceptible compared to cunners, and Atlantic salmon was resistant to Pseudomonas sp. J380 infection. Cunner tissues were heavily colonized by Pseudomonas sp. J380 compared to lumpfish and Atlantic salmon suggesting that it might be an opportunistic pathogen in cunners. The genome of Pseudomonas sp. J380 was 6.26 megabases (Mb) with a guanine-cytosine (GC) content of 59.7%. Biochemical profiles, as well as comparative and phylogenomic analyses, suggested that Pseudomonas sp. J380 belongs to the P. fluorescens species complex. Transcriptome profiling under iron-limited vs. iron-enriched conditions identified 1159 differentially expressed genes (DEGs). Cellular metabolic processes, such as ribosomal and energy production, and protein synthesis, were impeded by iron limitation. In contrast, genes involved in environmental adaptation mechanisms including two-component systems, histidine catabolism, and redox balance were transcriptionally up-regulated. Furthermore, iron limitation triggered the differential expression of genes encoding proteins associated with iron homeostasis. As the first report on a bacterial infection in cunners, the current study provides an overview of a new marine pathogen, Pseudomonas sp. J380.


Introduction
The ectoparasitic infestation by sea louse (e.g., Lepeophtheirus salmonis) is the most serious threat to both wild and cage-cultured salmonids (e.g., Salmo salar) in the Northern hemisphere [1]. During the infestation, sea lice feed on salmonid tissues leading to skin erosion, osmoregulatory failure, immunosuppression, increased disease susceptibility and, Pseudomonas sp. J380 is a new strain and closely related to the P. fluorescens species complex. Finally, RNA-Seq transcriptome profiling under iron-enriched vs. iron-limited conditions revealed a modulated expression of canonical and unique genes potentially linked to metabolism, environmental adaptation mechanisms, and iron homeostasis. Collectively, our findings provide insights into the biology of a novel marine pathogen, Pseudomonas sp. J380.

Bacterial Growth Conditions
Single colonies of Pseudomonas sp. J380 were grown routinely in 3 mL of Trypticase Soy Broth (TSB, Difco, Franklin Lakes, NJ, USA) at 15 • C in a 16 mm diameter glass tube and placed in a roller for 24 h. To conduct the assays under iron-enriched andlimited conditions, TSB was supplemented with 100 µM FeCl 3 or 100 µM 2,2'-dipyridyl (DIP), respectively. Siderophore synthesis was detected on Chrome Azurol S (CAS) agar plates [25] using bacterial cells harvested at the mid-log phase at an optical density (OD; at 600 nm) of~0.7 (~1 × 10 8 CFU/mL).

Siderophores Synthesis
The secretion of siderophores was tested using CAS plates by previously described assays [28,29]. Pseudomonas sp. J380 was grown under previously described conditions. Briefly, bacterial cells grown in TSB were harvested at the mid-log phase. Thirty microliters of bacterial inoculum were added to 3 mL TSB supplemented with 100 µM of FeCl 3 or 100 µM of DIP and grown at 15 • C for 24 h with aeration. As a control, Vibrio anguillarum J360 was grown under the same conditions but in TSB supplemented with 2% NaCl [30].
Following the incubation period, the cells were harvested (6000 rpm, 10 min), washed twice with PBS, and resuspended in 50 µL of PBS. Ten microliters of the concentrated bacterial pellet were inoculated onto CAS agar plates and incubated at 15 • C for 48-72 h. Following the incubation, the secreted siderophores were visualized as a yellow-orange halo around the bacterial colony. The plates were also observed using a UV trans-illuminator. To photo-document the fluorescent siderophores, bacteria grown under previously described conditions were washed and resuspended in 1 mL of PBS and stained with 4',6-diamidino-2-phenylindole (DAPI; Thermo Fisher, USA) for 30 min in darkness. After washing cells with PBS three times (6000 rpm, 10 min), cells were visualized using DAPI (461 nm) and EtBr (358 nm) filters through a Nikon AR1 laser scanning confocal microscope. Cunner fish (Tautogolabrus adpersus) captured in Conception Bay, NL, Canada ( Figure S1), were transferred to JBARB for 4 weeks of quarantine. Upon arrival, fish were acclimated to~8-10 • C in 500 L tanks supplied with oxygen saturation of 95-110%, UV-treated, and filtered flow-through seawater, with 12-12 photoperiod and illuminance of 10-15 lux. The animals were fed with chopped capelin (Mallotus villosus) two times per week ad libitum. Lumpfish were acclimated to~8-10 • C in 500 L tanks supplied with oxygen saturation of 95-110%, UV-treated, and filtered flow-through seawater, and 12-12 photoperiod with an illuminance of 30 lux. Biomass was maintained at 6.6 kg/m 3 . The fish were fed a commercial diet (Skretting-Europa, BC, Canada) daily at a rate of 0.5% of body weight per day using automated feeders. Farmed Atlantic salmon were held under optimal conditions in 3800 L tanks as described previously [31], with some modifications (at 10 • C, and a 12/12 photoperiod with an illuminance of 40-60 lux). The fish were fed three days per week at a level of 1% body weight per day using a commercial dry pellet (Skretting-Europa).

Tissue Sampling and Analysis
After infection, samples of internal organs (liver, spleen, head kidney, brain, and blood), were taken from a minimum of 5 fish euthanized with an overdose of MS222 (400 mg/L) in every sampling. Tissue samples were aseptically removed at 7, 14, 21, and 35 dpi for cunners; 7, 14, 21, and 28 dpi for lumpfish; and 7 and 14 for Atlantic salmon, based on the progression of the disease. The samples were placed into sterile homogenizer bags. Subsequently, they were weighed and homogenized in PBS in a final volume of 1 mL (weight: volume), serially diluted in PBS (1:10), and plated on TSA plates before incubating at 15 • C for 3-4 days. The following formula was used to determine the CFU of Pseudomonas sp. J380 per g of tissue [34]:

DNA Extraction and Sequencing
Pseudomonas sp. J380 genomic DNA was extracted from cultures grown to midlogarithmic phase as described in Section 2.2. The Wizard DNA extraction high molecular weight kit (Promega, Madison, WI, USA) was used to extract and purify the genomic DNA. Integrity and purity of the DNA were evaluated by gel electrophoresis (0.8% agarose) and spectrophotometry (Genova-Nano spectrophotometer, Jenway, UK). Library preparations and sequencing were conducted by Genome Quebec (ON, Canada) using PacBio RS II and Miseq Illumina sequencers.

Genome Assembly, Annotation, and Mapping
Celera Assembler (August 2013; version v7.0) at Genome Quebec was used to assemble the PacBio reads. Rapid Annotation Subsystem Technology pipeline (RAST) was used for the annotation [35]. The Pseudomonas sp. J380 chromosome was submitted to the National Center for Biotechnology Information (NCBI) and re-annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP; 4.10).
To detect small plasmids, the raw Illumina reads were trimmed using CLC Genomics Workbench v20.0 (CLCGWB; Qiagen, Hilden, Germany) and examined for quality using FastQC version 0.11.9 (Babraham Institute, Cambridge, UK) [36]. High-quality Illumina reads were assembled using de novo tool (CLCGWB) and aligned to the reference Pseudomonas sp. J380 chromosome using the genome finishing module tools with default parameters. Illumina sequences that did not align with the chromosome were analyzed and annotated. Pseudomonas sp. J380 whole-genome illustration was developed by using CGview Server [37].

Multi-Locus Sequence Analysis (MLSA) Using Housekeeping Genes
To infer the phylogenetic history of Pseudomonas sp. J380, several reference genes from different pseudomonads with complete genomes were considered. These genes included 16S ribosomal RNA subunit (rrn), cell-division protein (ftsZ), glyceraldehyde-3-phosphate dehydrogenase (gapA), gyrase beta subunit (gyrB), rod shape-determining protein (mreB), uridine monophosphate (UMP) kinase or uridylate kinase (pyrH), recombinase A (recA), RNA polymerase alpha subunit (rpoA), and topoisomerase I (topA) and were used in MLSA. The gene loci and accession numbers for each species are listed in Table S1. Sequences were aligned using CLCGWB. Concatenation of locus sequence was made using Sequence Matrix software v1.7.8 [38]. The phylogenetic history was estimated using the neighbour-joining (NJ) method [39] with a bootstrap consensus of 1000 replicates using MEGA X.

Whole Genome Comparison and Evolutionary Analysis
The genomes used in this study are listed in Table S2, and the analyses were conducted using the whole-genome analysis tool (CLCGWB) as described earlier [30]. To determine the average nucleotide identity (ANI), genomes were aligned with default parameters (min. initial seed length = 15; allow mismatches = yes; min. alignment block = 100). A correlation matrix was generated with default parameters (Euclidean distance method and complete cluster linkages). The phylogenetic history was determined using NJ method [39] with a bootstrap consensus of 1000 replicates using CLCGWB. Aeromonas salmonicida strain J223 (NZ_CP048223) was used as an outgroup. The analysis was repeated using MEGA X using the same parameters [40]. Whole-genome dot plots between closely related strains were also constructed to visualize and further analyze genomic differences.

Bacterial Growth under Iron-Enriched and -Limited Conditions, and Total RNA Extraction
Triplicate cultures of 50 mL of Pseudomonas sp. J380 grown under iron-enriched and iron-limited conditions at 15 • C with aeration (180 rpm) to mid-log phase were subjected for RNA extraction according to established protocols [31,41]. Briefly, cells were harvested (6000 rpm, 10 min, 4 • C) and washed twice with PBS. The cell pellet was used for total RNA extraction using MirVana following the manufacturer's instructions (Invitrogen, Waltham, MA, USA). To degrade any residual genomic DNA, the RNA samples were treated with 2 U of DNase (TURBO DNA-free™ Kit, Invitrogen) and incubated at 37 • C for 30 min. Then, 2.5 µL of DNase Inactivation Reagent was added, and further incubated for 5 min at room temperature. The supernatant containing clean RNA was harvested by centrifuging samples at 10,000× g for 1.5 min. RNA samples were quantified and evaluated for purity (A260/280 and A260/230 ratios) using a GenovaNano-spectrophotometer (Jenway, UK). RNA integrity was evaluated by agarose gel electrophoresis. The A260/280 and A260/230 ratios of purified RNA samples were 1.9-2.1 and 1.9-2.2, respectively.

RNA-Seq Data Analysis
The RNA-Seq data is available at the NCBI database under the accession number PRJNA717273. Obtained paired-end raw reads were mate-paired and filtered to remove low-quality reads using CLCGWB with default parameters (mate-paired read information, minimum distance = 1; maximum distance = 1000) (Table S3). Adapter trimming was realized by CLCGWB using the trim reads tool with default parameters (quality trimming, trim using quality scores, limit: 0.05, and trim ambiguous nucleotides, maximum number of ambiguities = 2). The number of reads and nucleotides removed are indicated in Table S3. The quality of the reads was checked using FastQC [36] before and after trimming. Trimmed high-quality reads were, then, mapped by CLCGWB against the Pseudomonas sp. J380 genome (NZ_CP043060.1) using the RNA-Seq analysis tool. Reads mapping and transcript counts were performed using the following settings: mismatch cost = 2, insertion and deletion costs = 3, minimum length fraction and minimum similarity fraction = 0.8, maximum number of hits for a read = 10, and strand-specific = both. Gene expression quantification and normalization of the mapped reads were performed by alignmentdependent expectation-maximization (EM) algorithm [42] based on the RESM and eXpress methods [43]. The transcript per million reads (TPM) values were, then, computed from the counts assigned to each transcript, after normalization by the trimmed mean of M-values (TMM) [44]. A global correlation analysis was performed using log 2 -transformed TPM values (x + 1) of each gene under iron-enriched and iron-limited conditions. The correlation was estimated by Pearson method. Abundance data were subsequently subjected to differential expression analyses using the CLCGWB and the differential expression tool based on a negative binomial general linear model (GLM) [45]. A standard selection of biologically significant differentially expressed genes (DEGs) was performed with cut-off values of log 2 fold-change (FC) ≥ |1| and false discovery rate (FDR), p ≤ 0.05.

Analyses of Enriched Gene Ontology (GO) Terms and KEGG Kyoto Encyclopedia of Genes and Genomes Pathways
DEGs identified in this study under standard selection criteria were subjected to Gene Ontology (GO) and KEGG enrichment analyses by ShinyGO version 0.61 [46]. GO terms and KEGG pathways with FDR, p ≤ 0.05 were considered as significantly enriched, and the top 20 elements were selected to construct graphical illustration using ggplot2 version 3.3.1 [47]. A GO term-gene network analysis was performed using ClueGO plug-in [48] in Cytoscape (v3.8.2). Three GO category resources (updated on 23.03.2021) for biological process (BP), molecular function (MF), and cellular component (CC) were used.

In Silico Tools
Pseudomonas Genome DB (PGDB; https://pseudomonas.com/) and homology search by DIAMOND Blast against other pseudomonads were used to determine the identity of unknown genes. The genes encoding the virulence factors were surveyed by homology search at the virulence factor database (VFDB) [49] using other P. fluorescens strains (i.e., pf-5, pf0-1, and SBW25) as references. In addition, the prediction of Fur box sequences in potential target DEGs associated with iron homeostasis was carried out by regulon analysis tool at the Virtual Footprint suite [50] using P. aeruginosa (PAO1) as the reference.

Statistical Analysis
One-way ANOVA followed by a Tukey's post hoc test was used to determine significant differences (p < 0.05) in percent survival. All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA).

Bacterial Isolation
Wild cunners collected during the spring months (April-June) from different locations in Newfoundland have often been found to exhibit a characteristic skin ulcer. Among the wild cunners collected along the Newfoundland seashore in Conception Bay (spring, 2017), several individuals presented with this skin ulcer ( Figure S1). In order to identify the etiological agent associated with this disease, internal organs, including the liver, spleen, and head kidney were sampled from fish showing visible skin wounds. Tissue homogenates were separately plated for microbial isolation onto TSA (+2% NaCl). Colonies developed from all three tissues appeared morphologically identical and exhibited the same characteristics in multiple aspects, including colony morphology, Gram-stain, antibiogram, biochemical profile, and 16S sequencing (Table 1). This suggested that the bacterium causing lesions/ulcers in cunners is potentially a single strain capable of colonizing multiple internal organs, and received the specific strain identifier of 'J380 .

Physiological Characteristics of Pseudomonas sp. J380
Morphological, biochemical, and enzymatic characteristics of Pseudomonas sp. J380 are summarized in Table 1. The bacteriological characterization indicated that Pseudomonas sp. J380 is a Gram-negative motile non-capsulated rod-shaped bacterium, which is oxidasepositive and resistant to vibriostatic agent O-129, and demonstrated mesophilic and halotolerant growth. The API20NE test (profile code 0347554) suggested P. fluorescens as the significant taxa with 99.4% confidence. API ZYM test showed that Pseudomonas sp. J380 synthesizes several enzymes including catalase, urease, alkaline phosphatase, acid phosphatase, esterase, esterase lipase, lipase, and some amino acid modifying enzymes, such as arginine dihydrolase, leucine arylamidase, valine arylamidase, and cystine arylamidase. Pseudomonas sp. J380 was able to assimilate glucose, mannose, and maltose ( Table 1). The antibiogram analysis further showed that Pseudomonas sp. J380 was resistant to SXT, CTX, AMP, and CHL, whereas, it was susceptible to OXA, OTC, and TET (Table 1).
In order to examine the siderophore synthesizing property, Pseudomonas sp. J380 and V. anguillarum J360 (control; [30]) were grown under iron-enriched and iron-limited conditions. Growth studies showed that the iron-limited conditions retarded the growth of both species ( Figure S3A-C). However, Pseudomonas sp. J380 was able to grow relatively better under iron-limited conditions when compared to V. anguillarum J360 ( Figure S3B,C). Hemolysis assay on blood agar plates showed that Pseudomonas sp. J380 and V. anguillarum J360 display β-hemolysis (only at 15 • C) and α-hemolysis (at 28 • C; [30]), respectively ( Figure S3D).
Parallelly-grown cultures were then used in CAS plate assay. Under iron-enriched conditions, there was no siderophore production by both bacteria. In contrast, under iron-limited conditions, both Pseudomonas sp. J380 and V. anguillarum J360 synthesized siderophores and produced yellow halos with almost similar diameters in CAS plates ( Figure 1A). Visualization of CAS plate under UV light revealed that the siderophores synthesized by Pseudomonas sp. J380, but not V. anguillarum J360, are fluorescent ( Figure 1B

Determination of Koch Postulates and Pseudomonas sp. J380's Host-Range in Lumpfish, and Atlantic Salmon
The virulence of Pseudomonas sp. J380 on cunners was evaluated by an i.p. injection with three different bacterial doses (low, 4 × 10 3 ; medium, 4 × 10 5 ; and high, 4 × 10 7 CFU/dose) ( Figure 2A). The PBS-injected control group had no clinical signs and/or mortalities throughout the experiment. Mortality began at 10 and 7 dpi for low and medium dose groups, respectively. However, these two doses caused almost similar mortalities (~12%) in cunners. At 33 dpi,~21% of cunners in the high dose group died ( Figure 2A). Overall, Pseudomonas sp. J380 demonstrated mild virulence in wild cunners. The highest dose used in this study did not kill all the animals and, thus, LD 50 could not be determined (predicted to be LD 50 > 10 8 -10 9 CFU/dose).  Figure S4 for additional details.
The host-range of Pseudomonas sp. J380 was studied in lumpfish and Atlantic salmon ( Figure 2B). In lumpfish infected with 1 × 10 6 CFU/dose of Pseudomonas sp. J380, mortality began at 5 dpi and only 50% of lumpfish survived after 30 dpi. In Atlantic salmon infected with 2.2 × 10 6 CFU/dose of Pseudomonas J380, mortality began at 6 dpi. At 30 dpi, 95% of the fish survived and displayed good health. In contrast to the deceased cunners and lumpfish ( Figure 2C,D), necropsy and bacteriological analysis of the dead Atlantic salmon did not evidence bacterial infection during the infection trials. The percent survival for cunners and salmon was greater than lumpfish ( Figure 2B). A significant difference was found between the percent survival of different host fish species during the Pseudomonas sp. J380-infection (p < 0.0001).
Moribund lumpfish infected with Pseudomonas sp. J380 revealed acute clinical signs, in contrast to cunners, in which the bacterial infection produced chronic disease characteristics ( Figure 2C,D and Figure S4). In contrast, Pseudomonas sp. J380 caused neither apparent clinical signs nor disease in Atlantic salmon. External clinical signs in infected cunners and lumpfish included skin ulcers with hemorrhagic borders, which were superficial at the beginning of the infection, and deeper with muscle compromise at advanced stages ( Figure 2C,D and Figure S4A-D). Abdominal dropsy revealed internal pathological signs in diseased fish that included splenomegaly ( Figure S4E) and hemorrhagic petechiae in liver ( Figure S4F,G). These features were identical characteristics that were observed in naturally infected wild cunners ( Figure S1C,D).

Infection Kinetics of Pseudomonas sp. J380 in Different Host Tissues
The infection kinetics and tissue colonization were determined in the liver, spleen, head kidney, brain, and blood from three infected host species (Figure 3). Cunners injected with 4 × 10 7 CFU/dose Pseudomonas sp. J380 were heavily colonized in all tissues sampled from the majority of the individuals, and in particular, the spleen, head kidney, and liver had >10 6 CFU/g tissue at 7 and 35 dpi ( Figure 3A). In lumpfish injected with 1 × 10 6 CFU/dose, no bacterium was detected at 7 dpi (except in head kidney of one fish). However, two of the five lumpfish presented >10 5 CFU/g tissue for all the tissue specimens at 14 dpi ( Figure 3B). In Atlantic salmon injected with 2.2 × 10 6 CFU/dose, a limited bacterial load was detected in tissues other than blood at 7 dpi (<10 2 CFU/g tissue), but not at 14 dpi ( Figure 3C). In summary, Pseudomonas sp. J380 was able to invade and colonize cunners' tissues. Nevertheless, the degree of invasion and colonization was low in lumpfish and minimal in Atlantic salmon.

Genome of Pseudomonas sp. J380
Genomic DNA of Pseudomonas sp. J380 was sequenced using PacBio RS II and MiSeq (Illumina) platforms and assembled. The genome of Pseudomonas sp. J380 has been deposited at NCBI under the accession number NZ_CP043060, BioProject (acc. no. PR-JNA561239), and BioSample (acc. no. SAMN12612376). The genome of Pseudomonas sp. J380 had a single chromosome with a total length of 6,261,650 bp with a GC content of 59.7% ( Figure 4, Table 2). No plasmids were detected by using gel electrophoresis or MiSeq data. PGAP at NCBI determined a total of 5734 genes, including 5568 genes with CDSs, six complete rRNA operons, four ncRNAs, and 74 pseudogenes (Table 2).
A homology search at VFDB identified 73 putative genes encoding virulence factors in the Pseudomonas sp. J380 genome (Table S4), and were found to be associated with flagella, lipopolysaccharides, phenazines and alginate biosynthesis, phospholipases, and pyoverdines. rganisms 2021, 9,812 14 of 38  A homology search at VFDB identified 73 putative genes encoding virulence factors in the Pseudomonas sp. J380 genome (Table S4), and were found to be associated with flagella, lipopolysaccharides, phenazines and alginate biosynthesis, phospholipases, and pyoverdines.  In order to understand the phylogenic history of Pseudomonas sp. J380, an MLSA was conducted using eight different canonical genes from different bacterial species (Table S1). Pseudomonas sp. J380 demonstrated a closer relationship with two other P. fluorescens strains (i.e., L321 and FW300-N2C) with 100% bootstrap support ( Figure S5).

Whole Genome Alignment and Phylogenomic Analyses
Twenty-five genomes of selected pseudomonads were aligned (Table S2). The ANI analysis was conducted on this alignment in CLCGWB platform to compare the pseudomonad genomes. This genome-based ANI analysis revealed that P. libanensis DMSP-1 and P. lactis SS101 share significantly higher percent identity (~95%) with Pseudomonas sp. J380 ( Figure S6). Moreover, Pseudomonas sp. J380 demonstrated~84-~87% of identity with other P. fluorescens strains analyzed. A correlation matrix showed that Pseudomonas sp. J380 clustered together with P. libanensis DMSP-1 and P. lactis SS101 ( Figure 5A). The genome-based phylogeny analyses using CLCGWB ( Figure 5B) and MEGA ( Figure S7) also positioned Pseudomonas sp. J380 within a sub-cluster together with P. libanensis DMSP-1 and P. lactis SS101 with 100% bootstrap support. In order to study the physiological and environmental adaptation mechanisms of Pseudomonas sp. J380 under iron-stress, we determined the global gene expression profile under iron-enriched and iron-limited conditions using RNA-Seq. Information regarding the sequencing statistics and data quality is provided in Figure S2 and Table S3. A global expression correlation analysis showed a high degree of expression correlation (R 2 = 0.8524; p < 0.0001) between iron-enriched vs. iron-limited samples ( Figure 6A). PCA results and heat map revealed clear segregation of samples based on growth conditions ( Figure 6B,C). PC1 and PC2 explained 76.2% of the total variation in expression data ( Figure 6B). Tight distribution of samples under iron-limited conditions compared to control samples indicated that iron has a global impact on gene expression. A clear separation of samples based on growth conditions as illustrated in the heat map was in agreement with the PCA results ( Figure 6C). The log 2 fold-change (FC) ≥ |1| and false discovery rate (FDR) p-value of ≤0.05 were set as the cut-off criteria for significant differential expression. We found 1159 differentially-expressed genes (DEGs) under iron-limited conditions compared to the iron-enriched condition. These DEGs included 657 up-regulated and 502 down-regulated genes ( Figure 6D). Gene identifier, description/annotation, fold-change, FDR (p-value), and associated GO terms for the DEGs of Pseudomonas sp. J380 under iron-limited conditions compared to the iron-enriched conditions are listed in Table S5.

Analyses of Gene Ontology (GO) Terms and KEGG Pathways
In order to investigate which biological aspects are affected by these DEGs identified under iron-limited conditions, GO term and KEGG pathway analyses were performed. ShinyGO determined a total of 577 GO terms (MF, 135; CC, 44; and BP, 398) with an FDR p-value cut-off of 5% (Table S6). The twenty most significant GO terms from each GO category and KEGG pathways are graphically illustrated in Figure 7. It was evident that several GO terms associated with cellular metabolism, gene expression, and biosynthesis were enriched ( Figure 7A). There were 62 enriched KEGG pathways (5% FDR), where a majority of them were related to metabolic processes (biosynthesis and degradation) ( Figure 7B; Table S7).
Iron limitation altered several genes associated with iron homeostasis (Figure 8 and Table 4). Ferric iron uptake regulator (Fur), the master mediator of iron homeostasis, was slightly down-regulated under iron-limitation, and absent in the DEG list (FXO12_23360; Log 2 FC = −0.954; FDR = 0.019; Figure 8A). Moreover, many of these DEGs harbored putative Fur box sequences in their promoter regions ( Figure 8B,C). There was a Fur box in the promoter of fur itself ( Figure 8C).
A pair of hemolysin secretion/activation proteins (FXO12_25165, FXO12_15125) with putative iron-liberating function demonstrated contrasting transcription profiles under iron-limitation. Putative proteins involved in siderophore-mediated iron piracy, including sigma-70 ECF factor (FXO12_17340), siderophore-interacting protein (FXO12_06740) were transcriptionally up-regulated. Although a TonB-dependent siderophore receptor (FXO12_07150) was less -expressed, a number of transport system involved in iron/ironcomplex transportation (e.g., permease, FXO12_07455; Fe 3+ -transporter (FXO12_08250) and two ABC transporters, FXO12_04205, FXO12_08260) were over-transcribed. Genes encoding a biliverdin-producing heme oxygenase (bpho; FXO12_24140) and an iron-storing bacterioferritin (FXO12_15500) were also found to be highly expressed. In addition, the transcript abundance of several genes encoding proteins related to iron-sulfur cluster (ISC), including hscA (FXO12_24275) and fdx (FXO12_24280), two other putative fdx-like genes (FXO12_11905, FXO12_19935) and cyaY (FXO12_19255) decreased under iron-limited conditions ( Figure 8C and Table 4). Interactive analysis of GO terms and genes involved in iron homeostasis identified three GO clusters including 'ISC binding', 'ISC assembly and ISC-associated functions', and 'iron ion binding and transport' (Figure 8D and File S1). Each GO cluster was composed of specific GO terms that interacted with one or multiple genes ( Figure 8D).
Iron limitation altered several genes associated with iron homeostasis (Figure 8 and Table  4). Ferric iron uptake regulator (Fur), the master mediator of iron homeostasis, was slightly down-regulated under iron-limitation, and absent in the DEG list (FXO12_23360; Log₂FC = −0.954; FDR = 0.019; Figure 8A). Moreover, many of these DEGs harbored putative Fur box sequences in their promoter regions (Figures 8B,C). There was a Fur box in the promoter of fur itself ( Figure 8C).  [50]. This inset table only shows Fur boxes with a score >8.0; b, when a gene contains more than one Fur box, the one with the highest score is shown. (C) Heat map illustration of genes with the putative function in iron homeostasis. †, part of the isc operon ( Figure S12); -, no Fur boxes detected; FC of DEGs is in bold. *, refer to Table 3 footnote. (D) Gene Ontology (GO) term enrichment and pathway term network analysis of genes associated with iron homeostasis. GO enrichment analysis was performed with ClueGO [48]. Inset pie chart summarizes % genes per GO group. The circular pictogram is divided into left and right halves for genes and GO terms, respectively, in which the functionally grouped GO terms share the same color. The shape of the nodes indicates specific GO modules (i.e., ellipse, BP; triangle, MF; rectangle, CC). Names of the up-and down-regulated genes are indicated in red and green, respectively. As shown in the legend, node height reflects the term p-value corrected with Bonferroni step down method. Three genes that are not connected with any GO terms are shown below the circular pictogram. Refer to the File S1 for details.

Discussion
Based on previous studies, cunners have been considered as potential sea lice cleaner fish for the Atlantic salmon aquaculture industry, and the development of a breeding program towards establishing cunner hatcheries in Canada has recently been suggested [51]. Over the past few years, wild-caught cunners captured along the NL coastal waters by the field service team of the DOS, MUN, have often exhibited characteristic skin lesions and ulcers. Unfortunately, infectious diseases impacting cunner fish are not well characterized and documented [52]. In this study, we report the identification and complete characterization of a novel Gram-negative marine pathogen infecting wild cunners.

Characterization of Phenotypic, Physiological, and Biochemical Features
Pseudomonas sp. J380 was isolated from the head kidney, liver, and spleen of infected wild cunners, and we found that all the strains isolated showed identical characteristics (Table 1). This also indicated that Pseudomonas sp. J380 is capable of colonizing multiple internal organs of the cunners during a natural infection.
The halotolerant nature of Pseudomonas sp. J380 indicated that this bacterium is adapted to marine environments. Pseudomonas sp. J380 was capable of tolerating a wide temperature range (4-28 • C). This mesophilic property and inducible pyoverdine synthesis at higher temperatures (i.e., 28 • C) suggested that increasing ocean temperatures due to climate change might favor the infection events caused by Pseudomonas sp. J380.
The API20NE analysis suggested P. fluorescens (99.4% of confidence) as the significant taxa for the strain isolated in the current study. Being one of the diverse groups within the Pseudomonas genus, P. fluorescens species complex comprises more than 50 species, which have been found in various habitats including marine water [53]. In agreement with the phenotypic characterization, the majority of the substrate utilization and enzyme characteristics of Pseudomonas sp. J380 were similar to those reported from other P. fluorescens strains (e.g., CFS215 and 3a) [54,55]. Similar to these strains, Pseudomonas sp. J380 demonstrated detectable activities for catalase, urease, acid and alkaline phosphatases, and arginine dihydrolase, and capacity for exploiting various C-sources such as glucose, mannose, and maltose [54,55].
Pseudomonas spp. have been reported to carry multiple intrinsic and acquired antimicrobial resistance genes [56]. The genome of Pseudomonas sp. J380 was found to harbor putative antibiotic resistance genes associated with the observed phenotypic antibiogram (Table 1). Two copies of dhfr (dfrA; FXO12_02730, FXO12_19880) and a single copy of folP (sul or dhps; FXO12_23425) that encode for dihydrofolate reductase and dihydropteroate synthase, respectively, were identified and could be responsible for SXT-resistance [57,58]. An ampC gene (FXO12_03565) encoding a class C beta-lactamase, mediating AMP-and CTX-resistance, as well as a putative ppqFABCDE operon responsible for pyrroloquinoline quinone biosynthesis ( Figure S9) and implicated with CHL-resistance, were also present in Pseudomonas sp. J380 genome [59,60].
Siderophores are iron-scavenging molecules produced by microbes and the species that produce siderophores survive better in iron-limited environments [61]. In addition, siderophores act as virulence factors in many bacterial pathogens including pseudomonads [62]. We showed that Pseudomonas sp. J380 synthesized fluorescent siderophore, which is known as pyoverdine. These high-affinity fluorescent siderophores are mainly produced by P. fluorescens species complex [63]. In addition to their iron uptake role, pyoverdines have been implicated with the colonization of host tissues and the formation of biofilms [64]. Confocal microscopic observation of Pseudomonas sp. J380 further confirmed the pronounced production of pyoverdines under iron-limited conditions compared to iron-enriched conditions (Figure 1). Based on this pyoverdine-secreting property of Pseudomonas sp. J380, we hypothesized that it could be a novel member of the P. fluorescens species complex.

Characterization of Infectivity and Host-Specificity
We used the Pseudomonas sp. J380 isolated from infected wild cunners to verify the Koch's postulates. At the end of the infection assay, the survival of cunners in the low and medium dose groups was similar (~88%), and it decreased up to 79% in the high dose group. The clinical signs observed during the disease progression and the biochemical characteristics of the re-isolated bacterium were similar to those of the original strain from wild cunner fish. Next, we expanded our infection assays with lumpfish and Atlantic salmon to determine if Pseudomonas sp. J380 could infect different hosts. Although the mortality started at almost the same time (5-6 dpi), percent survival varied between species. Based on the percent survival, it was interesting to note that Pseudomonas sp. J380 presents a more acute virulence in lumpfish when compared with cunners. This observation raised a negative concern over the cohabiting cunner-lumpfish dual-cleaner fish model in sea cages since cunners could horizontally transmit Pseudomonas sp. J380 to lumpfish, which could end up in mass mortality of both cleaner fish species. Our results revealed that Pseudomonas sp. J380 infected both cunners and lumpfish, but not Atlantic salmon.
As a part of determining Koch's postulates as well as to study the infection kinetics, Pseudomonas sp. J380 was re-isolated from multiple internal organs of infected fish. The degree of bacterial colonization varied in terms of host species, organs, and time (dpi). Immune tissues of cunners (liver, spleen, and head kidney) were heavily colonized at initial and later (7 and 35 dpi) phases. Conversely, low relative bacterial load was detected in lumpfish (except for 14 dpi) and Atlantic salmon. Our data suggested that lumpfish is the most susceptible species to Pseudomonas sp. J380 among the hosts examined, regardless of the low bacterial colonization. This was in agreement with our post-mortem findings ( Figure S4C,D,F). In contrast to lumpfish, cunners seem better at confronting the pathogen as revealed by relatively low mortality despite the heavy tissue colonization, and severe damage in multiple organs (Figure S4B,E,G). The low virulence of Pseudomonas sp. J380 combined with its persistence suggests that Pseudomonas sp. J380 and cunners have been interacting for a long time, and Pseudomonas J380 might be an opportunistic pathogen in these animals. However, under stress conditions, for instance, after the hypometabolic dormancy in spring, some cunners may become immune suppressed and susceptible to Pseudomonas sp. J380 resulting in chronic infection. Additionally, we cannot rule out that the wild cunners utilized in our experiments might have developed immunity against Pseudomonas sp. J380, thus less-susceptible.

Characterization of Genomic Features, Comparative, and Phylogenomics
The Pseudomonas sp. J380 genome was composed of a single circular chromosome of 6.26 Mbp, encoding 5568 putative proteins. There were 808 (14.5%) hypothetical genes with no assigned function. Among the ncRNAs found in the Pseudomonas sp. J380 genome, three were encoded by rnpB, ssrS and ffs, and their regulatory roles remain unknown. The size and GC% content of Pseudomonas sp. J380 were comparable to those of different pseudomonads (Table S2). By homology search, we found~70 virulence factors encoded by Pseudomonas sp. J380 genome (Table S4), however, their roles in physiology and pathogenesis require further investigation.
We utilized the genetic information to phylogenetically classify Pseudomonas sp. J380. First, we attempted to confirm the identity of the isolated strain based on the sequences of eight canonical genes from different pseudomonads using MLSA-based phylogeny. Our results suggested that Pseudomonas sp. J380 belongs to the P. fluorescens phylogroup. Next, we extended our analyses with 25 pseudomonad genomes using comparative genomic and phylogenetic approaches to validate our results. The closest relatives of Pseudomonas sp. J380 were P. libanensis DMSP-1 and P. lactis SS101, both of which are members of P. fluorescens phylogroup. In addition, the NCBI database indicates that Pseudomonas sp. J380 belongs to unidentified Pseudomonas species. These outcomes strongly suggest that Pseudomonas sp. J380 is a novel strain belonging to P. fluorescens species complex.

Profile of Iron-Regulated Transcriptome
Iron acquisition strategies play vital roles in the growth, pathophysiology, and virulence of pathogenic bacteria [22]. In order to successfully colonize the host and to establish the infection, pathogenic bacteria have to overcome a transient period of iron limitation by scavenging iron from different host sources [65]. As a part of their nutritional immunity, hosts employ multiple iron sequestering mechanisms to tightly regulate the availability of iron for pathogens. However, bacterial pathogens have evolved several transcriptionally regulated iron acquisition strategies to circumvent these withholding strategies of hosts [22,23]. Identifying the elements of these mechanisms will certainly provide clues about how Pseudomonas sp. J380 colonizes host tissues and further sheds light on its pathogenicity. We examined the global transcriptomic response of Pseudomonas sp. J380 under iron-enriched and iron-limited conditions using RNA-Seq to identify the potential DEGs, and to explore the associated biological processes.
Our RNA-Seq analyses revealed that~1/5 (20.8%; 1159/5568) of the genes were dysregulated by iron-limitation, which included six pseudogenes. There were more overexpressed genes compared to less-expressed ( Figure 6D,E). To explore the significantly impacted biological pathways by the bioavailability of iron, we conducted enrichment analyses using GO terms and KEGG pathways.

Fundamental Cellular Functions
Two inter-related GO terms frequently represented by the DEGs identified in this study are ribosome (GO:0005840) and translation (GO:0006412) ( Figure 7A). An array of genes encoding large (50S, rpl; n = 23) and small (30S, rps; n = 20) ribosomal proteins were all found to be less-expressed under iron-limitation (Table S5). As documented in previous studies [66][67][68][69], the overall down-regulation of ribosomal protein-coding genes, and other elements involved in transcription, such as regulator (NrdR) and terminator (Rho), and translation, including elongation factors (e.g., EF-G and EF-P) and translation initiation factors (IF-1−3), combined with the energy deficiency, could diminish protein synthesis and retard growth of Pseudomonas sp. J380 under iron-limitation. The translation is an energy-demanding process, and therefore protein biosynthesis is strongly correlated with respiration, energy production, and metabolism [66].
Oxidative phosphorylation, the final step of respiratory metabolism in aerobic organisms, typically utilizes iron cofactor-dependent proteins and was present in some enriched GO terms (GO:0019646). Genes encoding members of different cytochrome subunits were found to be differentially expressed under iron-limitation. Two cytochrome-c oxidaserelated genes (FXO12_17965, FXO12_17960), that are arranged in a putative cytochrome c oxidase cluster along with ctaD and coxB, were up-regulated (Log 2 FC = 1.16; Table S5). However, the majority of them were down-regulated (e.g., ccoN, cyoD, ccoP) suggesting that iron limitation hindered energy metabolism in Pseudomonas sp. J380 similar to other species [68,70] (Table 3 and Table S5). Respiratory complex II is composed of multiple succinate dehydrogenase (SDH) iron-sulfur proteins and a vital enzyme participating in both Kreb's cycle and electron transport chain (ETC). Pseudomonas sp. J380 possesses four genes in a putative sdhCDAB cluster ( Figure S10A; sdhB, FXO12_08710, sdhD, sdhC), in which the last two genes were down-regulated by iron-limitation. The atp operon of Pseudomonas sp. J380 is composed of eight genes (atpBEFHAGDC) encoding for subunits of ATP synthase ( Figure S10B), and all of them were down-regulated (log 2 FC −1.11 to −2.23) under iron-limitation. In agreement with previous results, the decreased transcription of genes involved in Kreb's cycle and ETC implied that respiratory metabolism was inhibited by iron-limitation in Pseudomonas sp. J380. However, we also noticed an increased expression of glycolytic enzymes (e.g., glk, pgi; Table S5) involved in alternative ATP production to potentially compensate for the decreased energy generation from Kreb's cycle and ETC [71].

Two-Component Systems (TCS)
TCS are dominant gene control switches in bacteria and play essential roles in signal transduction, physiology, cell-cell communication, adaptation to changing environments, host interaction, and pathogenesis [72]. Classical TCS consist of a sensor HK that phosphorylates the cognate RR upon receiving a signal. RRs are generally DNA-binding transcriptional modulators and could activate or repress their target genes. By controlling the expression of ferrienterobactin receptor that involves in iron uptake, TCS was found to take part in iron homeostasis of P. aeruginosa [73]. Transcriptomic analyses showed the increased expression of 4 out of 7 HKs by log 2 FC > 2 and 8 RRs by log 2 FC > 1 (Table 3). Conversely, an OmpR domain-containing RR was down-regulated (FXO12_02395). Upregulated TCS encoding genes under iron-limitation have also been reported in other pseudomonads [70,74]. The increased expression of multiple TCS components in the current study indicated that Pseudomonas sp. J380 TCSs may govern an array of downstream genes in response to low iron levels. Nevertheless, pairing HKs to their cognate RRs, and their relation to iron-mediated transcriptional regulation (e.g., Fur-mediation) require further functional investigation in Pseudomonas sp. J380.
Although nearly 70 putative virulence factors encoding genes were identified in Pseudomonas sp. J380 genome, majority of them were not differentially expressed (except LPS biosynthesis protein, FXO12_08045; Flp family type IVb pilin, FXO12_15135; cpaB and cpaF) under iron-limitation (Table S4). Over-production of alginate could promote survival and persistence of Pseudomonas spp. by making them a mucoid phenotype and alginate is considered to be a virulence factor [75]. The components of the alginate biosynthetic pathway in P. aeruginosa were encoded by alg operon [76]. We identified the 12-gene alg operon in Pseudomonas sp. J380 genome by homology search ( Figure S11). While alg operon was not differentially expressed, a few regulators of this operon, including algR, (FXO12_19295), algP (FXO12_19335), algB (FXO12_17830), kinB (FXO12_17835) were determined to be up-regulated by iron-limitation.

Hut Pathway
The Hut pathway is associated with histidine catabolism and utilizes several enzymes. The genes encoding these histidine metabolizing enzymes are generally structured in a conserved operon [77]. Pseudomonas sp. J380 genome possessed 6 Hut pathway genes ( Figure S8; two copies of hutH, each one copy of hutU, hutG, hutD, hutC), where both copies of hutH and hutU were induced under iron-limited conditions. Histidine metabolism and Hut pathway have been implicated in bacterial virulence and pathogenesis [78,79]. Production of extracellular proteases in V. alginolyticus was found to utilize the Hut pathway [78]. Moreover, HutC has been shown to directly regulate the expression of virulence-related genes [79]. Although hutC repressor had no significant transcriptional changes in our study, there might be potential changes in virulence of Pseudomonas sp. J380 as previous studies suggested the existence of alternative regulators (e.g., TCS) that could serve as a transcriptional activator of hut genes [80].

Stress and Oxidative Damage-Related Genes
Transcriptional modification occurred in genes encoding proteins that are involved in oxidative stress protection (Table 3). Synergistic induction of two classic redox biomarkers katE (catalase) and sodA (MnSOD) was noticed. These oxidative stress-responsive enzymes counteract against harmful reactive oxygen species (ROS). Iron-limitation induced the expression of dps, a gene that encodes for a stress-mediatory protein with dual functions, including protecting DNA from oxidative damage and ferroxidase activity [81]. Dps oxidizes Fe 2+ at its ferroxidase center and stores the resulting Fe 3+ within the iron core [82]. Hemerythrin (Hr) is a nonheme Fe-containing O 2 carrier-protein found in marine organisms [83]. Hr-like domain-containing proteins in prokaryotes are thought to perform secondary functions including O 2 sensing [84] and protecting from oxidative damage [85]. Meanwhile, elevated expression of an Hr domain-containing protein (FXO12_06285) and a putative iron-containing redox protein (FXO12_02520; member of haem oxygenase (HO) superfamily) was detected under iron-limitation. Modulated transcription of redox genes has also been observed in other bacteria under iron-limited conditions [70,86]. Under low intracellular iron levels, the probability of accumulating harmful levels of ROS is low. However, the increased expression of oxidative stress responders in Pseudomonas sp. J380 suggests that their transcriptional regulation might be under the governance of Fur [70]. Rubredoxin (Rd) is an iron-containing multifunctional protein, and it forms an alternative cytoplasmic oxidative stress protection system along with rubrerythrin [87,88]. Among two putative rd orthologs found in Pseudomonas sp. J380 genome, one was significantly less transcribed under low iron levels in the current study (Table 3).

Iron-Homeostasis
Our DEG profiling revealed the transcriptional modulation of various proteins involved in iron-homeostasis (e.g., acquisition, transportation, and storage) and several iron-containing (e.g., Fe-S) proteins. Proteomic profiling of Yersinia ruckeri under normal and iron-limited conditions revealed differential expression of an array of proteins involved in Fe 3+ capture and transport, and cellular metabolism [69].
The majority of iron acquisition strategies in bacterial pathogens, including pseudomonads, are completely or at least partially regulated by members of the Fur superfamily [89,90]. Fur is a homodimeric metalloregulator that binds to Fur box within the promoter region of target genes when liganded by Fe 2+ , and inhibits the transcription under iron-enriched conditions [22]. Fur repressor dissociates from DNA when Fe 2+ level is low allowing transcription of target genes to occur. As a global regulator of iron uptake, it directly and indirectly controls the expression of associated regulatory RNAs, sigma factors, and various transcriptional regulators [90]. Fur was found to be down-regulated in our study ( Figure 8A) suggesting that many potential Fur-regulated target genes could have actively transcribed under iron-limited conditions. Regulon prediction analysis showed that majority of the DEGs involved in iron homeostasis harbor putative Fur box sequences ( Figure 8B,C). The auto-regulatory feature of Fur has been previously reported in a few species [91]. It was interesting to note the presence of Fur box at the promoter of fur itself, suggesting that Fur might regulate its own expression in Pseudomonas sp. J380; however, this requires further investigation.
The fundamental mechanisms for iron uptake by pathogenic bacteria include (1) sequestering heme-Fe from host hemoproteins utilizing receptors and secreted proteins, (2) capturing Fe 3+ from host iron-storages (e.g., transferrin and lactoferrin) using binding proteins and siderophores, and (3) permeabilizing free iron with the aid of ferric reductases and ferrous permeases [22]. Heme is a preferred host iron source for bacteria that secrete hemolysins to enhance the heme availability. Pseudomonas sp. J380 genome possesses five putative hemolysin-related elements (Table S8), and two of them (FXO12_25165, FXO12_15125; hemolysin secretion/activation protein) were present in the DEG list.
A number of genes involved in the biosynthesis of siderophores including pyoverdines have been reported from different pseudomonads [92]. In P. aeruginosa, pvcABCD operon is involved in pyoverdine synthesis, while pvdD, pvdS and pvdR are involved in other regulatory roles. Based on current annotation, a few genes associated with pyoverdine siderophores were identified in Pseudomonas sp. J380 genome (e.g., FXO12_27210, siderophore synthetase; FXO12_12235, siderophore-interacting protein; FXO12_06740, PvdJ/PvdD/PvdP-like protein; Table S8). Moreover, the Pseudomonas sp. J380 genome has 18 TonB-dependent siderophore receptor genes and three of them were DEGs (Table S8). These receptors facilitate uptake of iron in the form of siderophore-or other proteincomplexes. The sigma ECF factors in different species have been implicated in the regulation of heat-shock response, iron-transport, metal ion efflux system, and alginate secretion [93]. A sigma-70 ECF factor (FXO12_17340) was up-regulated in Pseudomonas sp. J380 under iron-limitation; however, its association with iron-homeostasis remains to be characterized. Unexpectedly, only a few genes involved in putative siderophore biogenesis and uptake were identified as DEGs, including FXO12_12235 and a TonBdependent siderophore receptor (FXO12_07150). This implied that alternate pathways for these molecular events might be existing in Pseudomonas sp. J380. Soluble Fe 2+ can freely enter through the outer membrane of Gram-negative bacteria, and from the periplasm, Fe 2+ transport occurs through metal uptake transporters [94]. Transcript of Fet4-like iron permease (FXO12_07455) was strongly up-regulated (log 2 FC = 3.16). Two up-regulated ABC transporter family members and EC solute binding ferric transporter (FXO12_08250) might take part in iron transportation. In accordance with earlier studies [70,74,95], enhanced transcription of various iron transporter systems in Pseudomonas sp. J380 under iron-starvation suggests that it strives to effectively modulate the intracellular iron levels.
Once the heme reaches the bacterial cytosol, heme oxygenases (HOs) release Fe 2+ by catabolizing the heme to produce biliverdin and CO [96]. Transcript of a BPHO homolog (FXO12_24140) was induced by iron limitation. Similarly, HO orthologs have been shown to be abundantly expressed under iron limitation in different pseudomonads [68,74,97], further suggesting a pivotal iron acquisition role for HOs in bacteria during iron starvation.
There are two types of ferritin-like iron storage molecules found in bacteria: bacterial ferritins (Ftn) and bacterioferritins (Bfr) [98]. We identified three putative bfr genes in the genome of Pseudomonas sp. J380. While one was induced by iron limitation (FXO12_15500), the other two that shared~45% amino acid identity were down-regulated. Protein sequencewise, the upregulated bfr was quite different from down-regulated bfrs. Similar results were reported in P. aeruginosa and P. fluorescens Pf-5 [70,95], suggesting a reduced need for iron storage when iron availability is low. Bacterioferritin-associated ferredoxin (Bfd) is a bacteria-specific physiological cognate partner of Bfr whose function is to mobilize and release iron from Bfr. However, transcription of a bfd ortholog (FXO12_11965) in Pseudomonas sp. J380 remained unaffected under iron limitation, presumably to sustain the iron storage within Bfr.
Fe-S cofactors are utilized in several molecular functions such as electron transfer and activation of substrates to sense ROS. A family of metalloproteases containing Fe-S cluster takes part in vital cellular functions including ribosome function, amino acid and C metabolism, and transcription [99]. To assemble these clusters, bacteria employ Fe-S cluster biogenesis systems, such as the iron-sulfur cluster (ISC) pathway. The enzymatic Fe-S cluster machinery is encoded by isc operon, which is composed of eight genes in the classic Escherichia coli model [100]. We identified a putative iscRSUA-hscBA-fdx-iscX cluster in Pseudomonas sp. J380, which was structurally identical to that of E. coli isc operon ( Figure S12). Although the genes in isc cluster were found to be down-regulated (except iscX), only hscA and fdx were identified as significantly DEGs in our study. While HscA (and its co-chaperon HscB; FXO12_24270) is involved in ISC assembly or transfer [101], Fdx acts as an electron donor in the ISC biogenesis pathway [99]. Two other putative fdx-like genes (FXO12_11905 and FXO12_19935) and an iron-trafficking CyaY protein that supplies iron for ISC biogenesis were also less-expressed under iron-limitation. In contrast, a 2Fe-2S ISC binding domain-containing protein (FXO12_23050) was up-regulated. Not surprisingly, the promoter region of iscR was found to harbor a putative Fur binding site suggesting a potential iron-dependent, Fur-mediated transcriptional regulation of isc operon in Pseudomonas sp. J380. These findings suggest that iron limitation depressed the isc operon resulting in an overall reduction in the biogenesis of Fe-S proteins, which could lead to compromised cellular processes that depend on Fe-S proteins including genome maintenance, protein translation, and energy conversion.
To visualize the interaction between GO terms and genes associated with iron homeostasis, we used ClueGO and determined three GO clusters including 'ISC binding', 'ISC assembly and ISC-associated functions', and 'iron ion binding and transport' ( Figure 8D and File S1). The information on bacterial gene annotation was a significant limiting factor. The identification of more iron homeostasis-related genes will depend on the curated annotation of homologs, and advances in omics resources will help to resolve this.

Conclusions
In this study, the causative agent of a natural infection in wild cunners in Atlantic Canada was isolated and identified as a novel member of Pseudomonas species. Pseudomonas sp. J380 appears to be an opportunistic pathogenic bacterium in cunners and may emerge as an opportunistic pathogen when cunners are immune-suppressed resulting in chronic infection. However, lumpfish were more susceptible than cunners and acutely infected by Pseudomonas sp. J380. Under iron limitation, Pseudomonas sp. J380 produced pyoverdinetype siderophores. A large-scale transcriptomic alteration occurred under iron-limited conditions in various cellular processes, such as metabolism, TCS, Hut pathway, redox balance and, particularly, in iron-homeostasis. This study provides the basis for the biology of Pseudomonas sp. J380 and further studies are required to diagnose, manage and treat this disease.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/microorganisms9040812/s1, Figure S1. The geographical location of sampling site and clinical pathology of wild-caught cunners, Figure S2. Illumina sequencing quality data for the samples (n = 6) used in the transcriptomic profiling of Pseudomonas sp. J380 under iron-enriched and iron-limited conditions, Figure S3. Growth and hemolysis assays for Pseudomonas sp. J380, Figure S4. Gross pathology in cleaner fish (cunners, Tautogolabrus adspersus, and lumpfish, Cyclopterus lumpus) infected with Pseudomonas sp. J380, Figure S5. Evolutionary relationships of taxa based on MLSA, Figure S6. Comparative genomic analyses of Pseudomonas sp. J380 and other pseudomonads, Figure S7. Evolutionary relationships of taxa based on whole genomes, Figure S8. Gene structure of the hut operon in Pseudomonas sp. J380, Figure S9. The ppqFABCDE operon involved in pyrroloquinoline quinone biosynthesis in Pseudomonas sp. J380, Figure S10. The operons involved in respiratory metabolism in Pseudomonas sp. J380, Figure S11. The alg operon and its regulators involved in alginate biosynthetic pathway in Pseudomonas sp. J380, Figure S12. The isc operon in Pseudomonas sp. J380. Table S1. Features of the genes utilized for the Multilocus Sequence Analysis (MLSA), Table S2. Comprehensive information of species and their genomes utilized in whole genome comparison and phylogenomics, Table S3. Summary of the RNA-seq reads, Table S4. Comprehensive summary of putative virulence factors identified in Pseudomonas sp. J380 genome, Table S5. Comprehensive summary of differentially expressed genes (DEGs) in Pseudomonas sp. J380 under iron-limited and iron-enriched conditions as identified by RNA-Seq, Table S6. Comprehensive summary of enriched gene ontology (GO) terms in Pseudomonas sp. J380 under iron-limited and iron-enriched conditions, Table S7. Comprehensive summary of enriched KEGG pathways in Pseudomonas sp. J380 under iron-limited and iron-enriched conditions, Table S8. Additional genes related to iron homeostasis, File S1. Comprehensive details of GO cluster groups and individual GO terms for iron homeostasis-related DEGs.