The Study on the Biological Characteristics of Photobacterium damselae subsp. damselae Affected by the Deletion of the Iron Uptake System Genes tonB1 and tonB2

Abstract

Photobacterium damselae subsp. damselae (PDD), a marine fish pathogen, has an unclear contribution of tonB genes to iron acquisition and pathogenicity. ΔtonB1-PDD and ΔtonB2-PDD mutants as well as their complemented strains were generated via homologous recombination from the virulent strain PDD1605. Growth was evaluated by monitoring OD600 with viable-count calibration, intracellular iron was measured using a colorimetric assay, biofilm was detected by crystal violet staining, transcription was analyzed through qRT-PCR, and pathogenicity was assessed via an 8-day intramuscular challenge in black rockfish (Sebastes schlegelii). Under iron limitation induced by 100 μM 2,2′-dipyridyl, the maximum density decreased from 4.51 × 108 CFU/mL in wild-type (WT-PDD) to 4.00 × 108 and 3.64 × 108 CFU/mL in ΔtonB1-PDD and ΔtonB2-PDD respectively, and was largely restored after complementation. The intracellular iron content declined from 3.75 × 10−4 ± 4.00 × 10−6 nmol/106 cells in WT-PDD to 3.13 × 10−4 ± 1.05 × 10−5 nmol/106 cells in ΔtonB1-PDD and 2.55 × 10−4 ± 2.00 × 10−5 nmol/106 cells in ΔtonB2-PDD. Compared with WT-PDD, biofilm formation was reduced by 22.1% in ΔtonB1-PDD and by 24.8% in ΔtonB2-PDD. Deletion of tonB2 induced mild yet statistically significant transcriptional alterations in multiple iron acquisition and virulence-related genes, while colony morphology, swarming motility, hemolysis, phospholipase activity, biochemical traits, and antimicrobial susceptibility remained unchanged. In the high-dose challenge assay, WT-PDD caused 100% mortality within 2 days, whereas ΔtonB1-PDD and ΔtonB2-PDD caused 17/20 and 18/20 deaths respectively; all infected groups reached 100% mortality by day 3. In the low-dose challenge assay, WT-PDD, ΔtonB1-PDD, and ΔtonB2-PDD caused 12/20, 12/20, and 13/20 deaths by day 2, with cumulative mortalities reaching 80%, 70%, and 80% by day 8, respectively. Kaplan–Meier analysis detected no significant differences among the survival curves at either challenge dose. These findings suggest that tonB1 and tonB2 play roles in iron acquisition, low-iron adaptation, and biofilm formation, with tonB2 deletion exerting greater effects on growth and intracellular iron accumulation under iron-limited conditions.

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