Abstract
The coral reef microbiome is central to reef health and resilience. Competitive interactions between opportunistic coral pathogens and other commensal microbes affect the health of coral. Despite great advances over the years in sequencing-based microbial profiling of healthy and diseased coral, the molecular mechanism underlying colonization competition has been much less explored. In this study, by examining the culturable bacteria inhabiting the gastric cavity of healthy Galaxea fascicularis, a scleractinian coral, we found that temperate phages played a major role in mediating colonization competition in the coral microbiota. Specifically, the non-toxigenic Vibrio sp. inhabiting the healthy coral had a much higher colonization capacity than the coral pathogen Vibrio coralliilyticus, yet this advantage was diminished by the latter killing the former. Pathogen-encoded LodAB, which produces hydrogen peroxide, triggers the lytic cycle of prophage in the non-toxicogenic Vibrio sp. Importantly, V. coralliilyticus could outcompete other coral symbiotic bacteria (for example, Endozoicomonas sp.) through LodAB-dependent prophage induction. Overall, we reveal that LodAB can be used by pathogens as an important weapon to gain a competitive advantage over lysogenic competitors when colonizing corals.
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Data availability
All data generated or analysed during this study are included in this manuscript and its Supplementary Information files. The complete genome sequences of the strains used in this study and 16S rRNA gene amplicon sequencing reads have been deposited in GenBank under BioProject PRJNA416452 and PRJNA668462. The accession numbers of bacterial genomes are listed in the Supplementary Table 2. The accession numbers of sequencing reads of 16S rRNA gene amplicon sequencing are listed in Supplementary Table 7. Source data are provided with this paper.
Code availability
The software and codes used in this study are publicly available and customized parameters are indicated in Methods.
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Acknowledgements
X.W. was supported by the National Natural Science Foundation of China (grant nos. 42188102, 91951203 and 31625001), the National Key R&D Programme of China (grant no. 2018YFC1406500), Local Innovative and Research Teams Project of Guangdong Pearl River Talents Programme (grant no. 2019BT02Y262), Guangdong Major Project of Basic and Applied Basic Research (grant no. 2019B030302004) and the Key Special Project for Introduced Talents Team of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou) (grant no. GML2019ZD0407). T.L. was supported by K.C. Wong Education Foundation (grant no. GJTD-2020-12). P.W. was supported by the National Natural Science Foundation of China (grant no. 32070175) and Youth Innovation Promotion Association CAS (grant no. 2021345).
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X.W. and K.T. designed the study. W.W., K.T., P.W., Z.Z., T.X., W.Z., T.L., Y.W. and X.W. performed the experiments. W.W., K.T. and P.W. analysed the data. X.W., K.T. and Y.W. contributed materials/analysis tools. X.W., P.W., K.T. and W.W. wrote the manuscript. All the authors read and approved the final manuscript.
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Extended data
Extended Data Fig. 1 The culture-dependent and culture-independent analysis of G. fascicularis microbiota.
(a) Bacterial isolates were identified by 16S rRNA gene sequencing. Data from our cultivable 405 isolates were reanalyed. The number after each genus indicates the number of isolates obtained. Vibrio and Pseudoalteromonas species were assigned according to the top hit using NCBI 16S_ribosomal_RNA database. During isolation from different coral colonies, three gammaproteobacterial genera, Vibrio, Pseudoalteromonas and Halomonas, were consistently isolated. Vibrio and Pseudoalteromonas accounted for ~50% of the total 405 isolates from either healthy or heat-stressed corals sampled from different locations around Hainan Island. Among 121 Vibrio isolates, 81 isolates were classified as V. alginolyticus and two isolates were classified as V. coralliilyticus. Among 73 Pseudoalteromonas isolates, 29 isolates were classified as P. flavipulchra. (b) Bacteria with different colony morphologies isolated from gastric fluid samples on 2216E plates (the geographical locations of these corals are shown below each plate). The red arrow indicates transparent spots formed on Vibrio colonies. (c) Distribution and relative abundance of major microbial family in G. fascicularis polyps (n = 2) at 26 °C and 32 °C based on 16S rRNA gene V4 amplicon sequencing. The relative abundance of Vibrio and Pseudoalteromonas accounted for ~5% of total reads of the 16S rRNA gene amplicon in G. fascicularis polyps and up to ~20% after heating for 7 days with the occurrence of tissue necrosis.
Extended Data Fig. 2 Va43009 was less pathogenic than Vc43001.
(a) The percentage of tissue loss was measured as the area of tissue lost relative the total area of the coral fragment of the immerse-based infection assay in Fig. 1. (b) G. fascicularis fragments before 32 °C treatment (upper panel) and under 32 °C treatment for 1 day (middle panel) and 2 days (lower panel). Three coral fragments were prepared in the same way as those used in the immersion infection assay. For each coral fragment, only the right side was injected. Red arrows indicate the side (two sides were separated by elastic bands on each fragment) that was injected with Va43009 and Vc43001 or seawater (control). A total of 0.2–1 ×108 cells were injected into the gastric cavity of one corallite, and 5-8 corallites in the center of the right region received an injection. The coral fragments were acclimatized separately at 26 °C for 2 days before temperate was increased to 32 °C. After 1 day, the right side of the coral fragment that had been injected with Vc43001 showed stronger signs of necrosis than the left side. After 2 days, the whole fragments lost tissue on the side injected with Vc43001, suggesting that Vc43001 could achieve a successful infection in the context of the native microbiota in the coral gastric cavity. There was no obvious difference between the right side injected with Va43009 and the left side after 1 day, and sporadic tissue loss of the coenenchyma appeared similar on both sides (~10%) after 2 days. (c) Quantitation of the bleaching rate of the corals in panel b. The percentage of tissue loss was calculated as the area of lost tissue divided by the area of the coral fragment.
Extended Data Fig. 3 Construction and verification of mutant strains.
(a) The GfP2 prophage deletion mutant ΔGfP2 in Va43009. (b) The gfp gene fused with the mcp gene of GfP2 in Va43009; integration of the gfp gene with the gentamicin promoter (PGm) in the chromosome of strain Va43009 (c) and strain ΔGfP2 (d). (e) In-frame deletion lodAB in strain Vc43001. (f) Integration of the gfp gene with the gentamicin promoter (PGm) in the chromosome of strain Vc43001.
Extended Data Fig. 4 GfP2 excision alters the promoter sequence but not dsbA expression.
(a) A schematic diagram illustrating the excision of the prophage GfP2 from the Va43009 chromosome and the formation of GfP2 replicative form (RF) molecules in the cytoplasm of Va43009. The PattR promoter flanking the attR site drives the expression of dsbA when the GfP2 prophage is integrated, and the PattB promoter flanking the attB site drives the expression of dsbA when GfP2 is excised. (b) Sequence analysis of the PattR and PattB promoters. The -35 and -10 boxes predicted from BPROM in Softberry and the Shine–Dalgarno (SD) sequences are marked. The same sequence in the two promoters is highlighted in red, the coding region of dsbA is italicized, and the start codon of dsbA is shown in bold letters. The core phage attachment sites (45 bp) are in red letters and underlined. (c) A schematic of lacZ reporter constructs containing the PattR and PattB promoters shown in (b), with a qualitative lacZ assay using X-gal plates shown. 1–4 are four independent colonies containing PattR in ER2738, and 5-8 are four independent colonies containing PattB in ER2738. (d) Quantitative results showing PattR and PattB promoter activity in both Va43009ΔGfP2 and E. coli ER2738 hosts according to β-gal activity. Data are shown as the mean ± SD. Each pair of groups was compared using an unpaired t test with 95% confidence intervals (n = 3; two-tailed p value).
Extended Data Fig. 5 The addition of NAC reduced the ability of Vc43001 to activate GfP2 in Va43009.
The excision rate of GfP2 and the number of GfP2 RFs when coculturing Va43009 and Vc43001 with or without N-acetyl L-cysteine (NAC) (an inhibitor of reactive oxygen species). Data are shown as the mean ± SD. Each pair of groups was compared using an unpaired t test with 95% confidence intervals (n = 3; two-tailed p value).
Extended Data Fig. 6 Construction of chromosomally tagged antibiotic resistance gene strains and growth measurements in monoculture or coculture.
PCR verification of the mutant strains Vc43001 Gmr, Vc43001ΔlodAB Gm (a) and Va43009 Cmr (b). (c-f) Growth of different strains. Chromosome tagging of antibiotic resistance genes did not affect the growth of these strains. (g) Comparison of monoculture growth of strains. Data are the same as in panel c-f. (h-j) Comparison of the growth of cocultured strains. Growths of strains in coculture are highlighted in red. Growth of strains in monoculture is shown black, the data for which are the same as those in panel g. Data are shown as the mean ± SD. n = 3 for c-j.
Extended Data Fig. 7 Analysis of colonized bacteria using agar plates with corresponding antibiotics.
(a) Boxplots of CFUs of Va43009 and Vc40001 from polyps in each group were determined in 2216E plates containing chloramphenicol and gentamicin, respectively. n = 5 (Va43009 in Group 3; Va43009 and Vc43001ΔlodAB in Group 4); n = 6 (Va43009 in Group 1; Vc43001 in Group 2; and Vc43001 in Group 3). Statistically significant differences were detected by Kruskal–Wallis test with Dunn’s correction. (b) Ratio of Va43009:Vc43001 in Group 3 and Va43009:Vc43001ΔlodAB in Group 4. Statistically significant differences were detected by for multiple comparisons and Mann–Whitney test with 95% confidence intervals. n = 6, two-tailed p value. In boxplots, centre lines indicate median values and the whiskers extend from the 25th or 75th percentiles to the minimum or maximum values.
Extended Data Fig. 8 Vc43001 outcompetes other coral symbiotic bacteria through LodAB-dependent prophage induction.
(a) Competition between Vc43001/Vc43001ΔlodAB and other coral symbiotic bacteria. Data are shown as the mean ± SD. Each pair of groups was compared using an unpaired t test with 95% confidence intervals (n = 5; two-tailed p value). (b) The induction of prophage by Vc43001 but not by Vc43001ΔlodAB was evaluated by qPCR to quantify the phage structural gene and gyrB gene. Data are shown as the mean ± SD. Each pair of groups was compared using an unpaired t test with 95% confidence intervals (n = 3; two-tailed p value).
Extended Data Fig. 9 Schematic diagram of interplay between coral pathogen and commensal bacteria.
LodAB-mediated prophage induction plays an important role in colonization competition of gastric bacteria, affecting coral health.
Supplementary information
Supplementary Information
Supplementary Text (Results, Materials and Methods), Figs. 1–11, Discussion and Tables 1–7.
Supplementary Data 1 and 2
Supplementary Data 1, List of P2 major capsid proteins retrieved from IMG/M database. Supplementary Data 2, List of LodA homologues and domain annotation.
Supplementary Data 3
Source data for Supplementary Information.
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Wang, W., Tang, K., Wang, P. et al. The coral pathogen Vibrio coralliilyticus kills non-pathogenic holobiont competitors by triggering prophage induction. Nat Ecol Evol 6, 1132–1144 (2022). https://doi.org/10.1038/s41559-022-01795-y
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DOI: https://doi.org/10.1038/s41559-022-01795-y
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