Fig 1.
The influence of temperature cultivation in growth dynamics of Pdd.
Growth of RM-71 strain was assayed at 15°C and 25°C in TSB-1 for 48 h. Vertical error bars represent standard deviation of biological triplicates.
Fig 2.
Smear plot of differentially expressed genes (DEGs) in Pdd RM-71 exposed to two temperatures, 15°C and 25°C.
The smear plot shows the relationship between the log FC and mean of normalized counts. Grey points represent genes with non-significant changes in expression, whereas red points represent genes that are significantly differentially expressed.
Fig 3.
Graphical representation of DEGs distribution among the two Pdd chromosomes and pPHDD1 virulence plasmid.
Numbers denote DEGs upregulated at 25°C (red), downregulated at 25°C (blue), and genes not differentially regulated (grey).
Table 1.
List of selected DEGs with enhanced expression at 25°C including the 50 top upregulated genes at 25°C.
Enhanced expression at 25°C is denoted by positive FC values. Genes with VDA codes correspond to the annotation in the CIP102761 genome, and genes with A0J47 codes correspond to the annotation in the RM-71 genome.
Fig 4.
Virulence factors encoded within pPHDD1 plasmid are upregulated at 25°C.
(A) pPHDD1 encodes a series of membrane-associated proteins and toxins with demonstrated and potential roles for virulence in fish. (B) plasmid genes upregulated at 25°C are mainly distributed along 5 plasmid regions. Numbers denote the VDA gene codes of the type strain CIP102761 (GenBank Acc. No. NZ_ADBS00000000.1). Genes with A0J47 labels correspond to genes unique to RM-71. Colour codes of genes (represented as arrows) denote each of the three ranges of FC values, whereas white arrows denote genes that are not differentially expressed (NO DEG).
Table 2.
List of the top DEGs in Pdd with lower expression at 25°C than at 15°C.
Note that downregulated expression at 25°C is denoted by negative FC values.
Table 3.
List of most expressed genes at 15°C and their corresponding FPKM values.
Damselysin (Dly) toxin is highlighted in bold. FPKM values shown correspond to mean values of the three biological replicates.
Table 4.
List of most expressed genes at 25°C and their corresponding FPKM values.
FPKM values shown correspond to mean values of the three biological replicates.
Fig 5.
Damselysin toxin gene dly is one of the most highly expressed genes in Pdd.
FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values at the two assayed temperatures (S6 Table) were obtained for a selection of virulence and regulatory genes, secretion system genes and housekeeping genes including the top-expressed gene encoding ribosomal protein L24p, and compared using a logarithmic scale. Vertical error bars represent standard deviation of biological triplicates.
Fig 6.
Diagrammatic summary of genes upregulated (right side) and downregulated (left side) at 25°, relative to 15°C, in Pdd. Growth at 25°C upregulated motility and chemotaxis-related functions, as well as nutrient uptake and utilization genes. Potential virulence factors encoded within pPHDD1 plasmid were also upregulated at 25°C. Genes with lower expression at 25°C include a number of gene operons of yet unknown function, functions related to cell envelope, rpoS, and specific amino acid biosynthesis routes, among others. This diagram has been constructed based on transcriptomic data, and further studies are needed to establish the proposed model.