Presentation Information
[P02-247]Opposing effects of Dermacoccus nishinomiyaensis strains on pyrene degradation by Mycolicibacterium parafortuitum PO1 through interspecies interactions
○Satoko Suzuki1, Chiho Suzuki-Minakuchi1,2, Onruthai Pinyakong3, Hideaki Nojiri1,2 (1. Grad. Sch. Agric. Life Sci., UTokyo (Japan), 2. CRIIM, UTokyo (Japan), 3. Chulalongkorn Univ. (Thailand))
Keywords:
Bacterial interspecies interactions,Biodegradation,Polycyclic aromatic hydrocarbons,Pyrene,Mycolicibacterium
[Purpose]
The degradation ability of xenobiotic-degrading bacteria is modulated through interspecies interactions. However, traditional enrichment and agar-based isolation methods reduce the likelihood of isolating interacting strains, thereby limiting our understanding of microbial community behavior. In this study, we used fluorescence-based, single-cell approach to screen environmental bacteria that modulate pyrene degradation by Mycolicibacterium parafortuitum PO1 and investigated the underlying mechanisms.
[Method]
Pyrene was crystallized at the bottom of 96-well plates, and a minimal mineral medium was added to prepare pyrene micro-crystal plates. PO1 and an environmental bacteria extracted from wheat cultivation soil were single-cell inoculated using flow cytometry to generate 756 two-strain consortia. Residual pyrene was quantified by fluorescence microscopy during static incubation at 30ºC for up to 18 days.
[Results & Consideration]
We obtained 11 and 19 two-strain consortia containing environmental bacteria that enhanced or suppressed pyrene degradation by PO1, respectively. 16S rRNA gene sequencing revealed that Dermacoccus nishinomiyaensis was the most frequently identified species, including both enhancing and suppressing strains. Two D. nishinomiyaensis strains, DO1 (suppressing) and DO2 (enhancing), were isolated and co-cultured with PO1_mS, an mScarlet-tagged derivative of PO1, for further analysis.
When co-cultured with PO1_mS at 103 CFU/mL, DO1 suppressed pyrene degradation, while DO2 enhanced it, consistent with the screening results. Both strains formed aggregates composed of their own cells; however, DO2 formed larger aggregates that attracted more number of PO1_mS, potentially facilitating pyrene degradation. At a higher initial cell density (107 CFU/mL), both strains significantly inhibited pyrene degradation and formed mixed multi-species biofilms with PO1_mS. PO1_mS exhibited weak and dispersed fluorescence, suggesting impaired biofilm formation. Disk diffusion assay indicated that both DO1 and DO2 produced antimicrobial compounds, and the decreased abundance of PO1 under high cell density conditions likely contributed to the reduced pyrene degradation.
Whole-genome sequencing revealed that DO1 and DO2 possessed nearly identical genomes (average nucleotide identity, 100%; alignment percentage, 99%). Comparative genomic analysis identified nine SNPs and four insertions/deletions between the two strains. Notably, two transcriptional regulators in DO2 contained mutations, which may contribute to the differing effects on pyrene degradation by PO1.
[Conclusion]
These findings demonstrate that pyrene micro-crystal plate screening platform enables efficient identification of interacting bacteria which might be overlooked by conventional methods. Furthermore, we revealed that closely related strains can exert opposing effects on pyrene degradation, and that the interaction outcome was dependent on the initial cell density.
The degradation ability of xenobiotic-degrading bacteria is modulated through interspecies interactions. However, traditional enrichment and agar-based isolation methods reduce the likelihood of isolating interacting strains, thereby limiting our understanding of microbial community behavior. In this study, we used fluorescence-based, single-cell approach to screen environmental bacteria that modulate pyrene degradation by Mycolicibacterium parafortuitum PO1 and investigated the underlying mechanisms.
[Method]
Pyrene was crystallized at the bottom of 96-well plates, and a minimal mineral medium was added to prepare pyrene micro-crystal plates. PO1 and an environmental bacteria extracted from wheat cultivation soil were single-cell inoculated using flow cytometry to generate 756 two-strain consortia. Residual pyrene was quantified by fluorescence microscopy during static incubation at 30ºC for up to 18 days.
[Results & Consideration]
We obtained 11 and 19 two-strain consortia containing environmental bacteria that enhanced or suppressed pyrene degradation by PO1, respectively. 16S rRNA gene sequencing revealed that Dermacoccus nishinomiyaensis was the most frequently identified species, including both enhancing and suppressing strains. Two D. nishinomiyaensis strains, DO1 (suppressing) and DO2 (enhancing), were isolated and co-cultured with PO1_mS, an mScarlet-tagged derivative of PO1, for further analysis.
When co-cultured with PO1_mS at 103 CFU/mL, DO1 suppressed pyrene degradation, while DO2 enhanced it, consistent with the screening results. Both strains formed aggregates composed of their own cells; however, DO2 formed larger aggregates that attracted more number of PO1_mS, potentially facilitating pyrene degradation. At a higher initial cell density (107 CFU/mL), both strains significantly inhibited pyrene degradation and formed mixed multi-species biofilms with PO1_mS. PO1_mS exhibited weak and dispersed fluorescence, suggesting impaired biofilm formation. Disk diffusion assay indicated that both DO1 and DO2 produced antimicrobial compounds, and the decreased abundance of PO1 under high cell density conditions likely contributed to the reduced pyrene degradation.
Whole-genome sequencing revealed that DO1 and DO2 possessed nearly identical genomes (average nucleotide identity, 100%; alignment percentage, 99%). Comparative genomic analysis identified nine SNPs and four insertions/deletions between the two strains. Notably, two transcriptional regulators in DO2 contained mutations, which may contribute to the differing effects on pyrene degradation by PO1.
[Conclusion]
These findings demonstrate that pyrene micro-crystal plate screening platform enables efficient identification of interacting bacteria which might be overlooked by conventional methods. Furthermore, we revealed that closely related strains can exert opposing effects on pyrene degradation, and that the interaction outcome was dependent on the initial cell density.
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