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. 2020 Jan 21;11(1):e01019-19.
doi: 10.1128/mBio.01019-19.

A Zeaxanthin-Producing Bacterium Isolated from the Algal Phycosphere Protects Coral Endosymbionts from Environmental Stress

Keisuke Motone  1   2 , Toshiyuki Takagi  3 , Shunsuke Aburaya  1   2 , Natsuko Miura  4 , Wataru Aoki  1   5 , Mitsuyoshi Ueda  6   5
Affiliations

A Zeaxanthin-Producing Bacterium Isolated from the Algal Phycosphere Protects Coral Endosymbionts from Environmental Stress

Keisuke Motone et al. mBio. .

Abstract

Reef-building corals form a complex consortium with photosynthetic algae in the family Symbiodiniaceae and bacteria, collectively termed the coral holobiont. These bacteria are hypothesized to be involved in the stress resistance of the coral holobiont, but their functional roles remain largely elusive. Here, we show that cultured Symbiodiniaceae algae isolated from the reef-building coral Galaxea fascicularis are associated with novel bacteria affiliated with the family Flavobacteriaceae Antibiotic treatment eliminated the bacteria from cultured Symbiodiniaceae, resulting in a decreased maximum quantum yield of PSII (variable fluorescence divided by maximum fluorescence [Fv/Fm]) and an increased production of reactive oxygen species (ROS) under thermal and light stresses. We then isolated this bacterial strain, named GF1. GF1 inoculation in the antibiotic-treated Symbiodiniaceae cultures restored the Fv/Fm and reduced the ROS production. Furthermore, we found that GF1 produces the carotenoid zeaxanthin, which possesses potent antioxidant activity. Zeaxanthin supplementation to cultured Symbiodiniaceae ameliorated the Fv/Fm and ROS production, suggesting that GF1 mitigates thermal and light stresses in cultured Symbiodiniaceae via zeaxanthin production. These findings could advance our understanding of the roles of bacteria in Symbiodiniaceae and the coral holobiont, thereby contributing to the development of novel approaches toward coral protection through the use of symbiotic bacteria and their metabolites.IMPORTANCE Occupying less than 1% of the seas, coral reefs are estimated to harbor ∼25% of all marine species. However, the destruction of coral reefs has intensified in the face of global climate changes, such as rising seawater temperatures, which induce the overproduction of reactive oxygen species harmful to corals. Although reef-building corals form complex consortia with bacteria and photosynthetic endosymbiotic algae of the family Symbiodiniaceae, the functional roles of coral-associated bacteria remain largely elusive. By manipulating the Symbiodiniaceae bacterial community, we demonstrated that a bacterium that produces an antioxidant carotenoid could mitigate thermal and light stresses in cultured Symbiodiniaceae isolated from a reef-building coral. Therefore, this study illuminates the unexplored roles of coral-associated bacteria under stressful conditions.

Keywords: Symbiodiniaceae; antioxidant; coral; microbiome; stress tolerance; zeaxanthin.

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Figures

FIG 1
FIG 1
Bacterial community analysis of cultured Symbiodiniaceae. (a) Symbiodiniaceae were isolated from G. fascicularis and cultured in the absence (control) or presence of antibiotics. The antibiotic-treated Symbiodiniaceae were subsequently incubated in the absence of antibiotics without (Abx) or with (Abx+GF1) inoculation of the bacterial strain GF1 (see Fig. 3a and b). (b) Bacterial community composition based on 16S rRNA amplicon sequencing. Data were provided as the means of relative abundances from three biological replicates. OTU, operational taxonomic unit.
FIG 2
FIG 2
Effects of bacterial community on thermal and light tolerance of cultured Symbiodiniaceae. Maximum quantum yield of PSII (Fv/Fm) in cultured Symbiodiniaceae under nonstress (24°C, 50 μmol photons m−2 s−1) (a), thermal stress (31.5°C, 50 μmol photons m−2 s−1) (b), and light stress (24°C, 200 μmol photons m−2 s−1) (c) conditions. Relative ROS production in cultured Symbiodiniaceae after 5 days of exposure to thermal (31.5°C, 50 μmol photons m−2 s−1) (d) or light (24°C, 200 μmol photons m−2 s−1) (e) stress. Data ae provided as the relative fluorescence to that of control under nonstress conditions (24°C, 50 μmol photons m−2 s−1). Error bars indicate standard errors of the means (SEMs) from three biological replicates, and significant differences were determined by Tukey’s post hoc tests. *, P < 0.05; **, P < 0.01 (between control and Abx), #, P < 0.05, ##, P < 0.01 (between Abx and Abx+GF1). Control, cultured Symbiodiniaceae without manipulation of bacterial community; Abx, cultured Symbiodiniaceae treated with antibiotics; Abx+GF1, cultured Symbiodiniaceae treated with antibiotics followed by inoculation of the bacterial strain GF1.
FIG 3
FIG 3
Isolation and inoculation of GF1. (a) GF1 colonies grown on a marine agar plate. (b) Neighbor-joining tree based on 16S rRNA gene sequences showing the phylogenetic relationships of GF1 and related taxa in the family Flavobacteriaceae. Numbers on branches represent bootstrap values (1,000 replications). Bar, 0.01 substitutions per nucleotide position. (c) Principal-coordinate analysis of weighted UniFrac distances of bacterial community composition. Control, cultured Symbiodiniaceae without manipulation of bacterial community; Abx, cultured Symbiodiniaceae treated with antibiotics; Abx+GF1, cultured Symbiodiniaceae treated with antibiotics followed by inoculation of the bacterial strain GF1.
FIG 4
FIG 4
Zeaxanthin production by GF1. (a) Zeaxanthin biosynthesis pathway and corresponding genes of GF1. GGPP, geranylgeranyl diphosphate. (b) Zeaxanthin biosynthesis gene cluster. The annotation of each gene, which starts with “peg,” is listed in Table S4 in the supplemental material. (c) LC-MS/MS chromatograms of zeaxanthin in the methanol extract of GF1. Zeaxanthin was detected in positive ion mode by MRM from m/z 568.30 to 476.35, 119.15, and 145.15. A 1-μg/ml zeaxanthin standard is shown for retention time comparison. Zeaxanthin was not detected from the methanol used for extraction.
FIG 5
FIG 5
Effect of zeaxanthin supplementation on cultured Symbiodiniaceae. Fv/Fm and relative ROS production in cultured Symbiodiniaceae supplemented with zeaxanthin under thermal stress (31.5°C, 50 μmol photons m−2 s−1) (a and b) and light stress (24°C, 200 μmol photons m−2 s−1) (c and d) conditions. (b, d) Data are provided as the relative fluorescence to NC. Error bars indicate SEMs from three biological replicates, and significant differences were determined by Tukey’s post hoc tests. *, P < 0.05; **, P < 0.01; NC, negative control (no supplementation); DMSO, dimethyl sulfoxide.
FIG 6
FIG 6
Estimation of zeaxanthin production. CFU/ml of GF1 in Abx+GF1 algal culture under thermal stress (31.5°C, 50 μmol photons m−2 s−1) (a) and light stress (24°C, 200 μmol photons m−2 s−1) (c) conditions. Estimated zeaxanthin productions by GF1 under thermal stress (b) and light stress (d) conditions. Zeaxanthin productions were calculated based on CFU of each condition. Error bars indicate SEMs from three biological replicates.
FIG 7
FIG 7
Fluorescence in situ hybridization (FISH) analysis of bacteria in algal cultures. DAPI and FAM fluorescence were detected by 405 and 488 nm excitation, respectively. Symbiodiniaceae cells were autofluorescent at 488 nm excitation. Arrowheads indicate the presence of rod-shaped bacteria hybridized with the CF319a probe labeled with FAM. S, Symbiodiniaceae; control, cultured Symbiodiniaceae without manipulation of bacterial community; Abx, cultured Symbiodiniaceae treated with antibiotics; Abx+GF1, cultured Symbiodiniaceae treated with antibiotics followed by inoculation of the bacterial strain GF1.
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