Study of the Toxin Production Mechanism and the Algae-bacteria Association in Gambierdiscus balechii

關於岡比甲藻產毒機制以及藻菌共生的研究

Student thesis: Doctoral Thesis

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Award date4 Sep 2019

Abstract

Many benthic dinoflagellates of the genus Gambierdiscus are capable of producing ciguatoxins (CTXs) with polyether structures, which can be potentially bio-accumulated, bio-transformed, and bio-magnified in marine food chains. Humans consuming coral reef fishes that have accumulated CTXs from ingested algal food sources might suffer from ciguatera fish poisoning. Polyketide synthases (PKSs) are reported to be responsible for the synthesis of polyketide compounds. Understanding the molecular mechanism that regulates the toxin production is crucial for understanding the environmental triggers of the toxin and for informed fisheries management. The past few decades have witnessed the fast development of transcriptome technique in dinoflagellates, revealing the single domain PKS in dinoflagellates of sequence similarity with type I PKSs, while many multi-domain type I PKS are absent.

Nutrient availability is always an important indicator for the growth, photosynthesis, and toxin production of phytoplankton. Nitrogen (N), as the fifth most important element on earth, is involved in the storage, replication, and transcription of genetic information (major component of DNA, RNA, and proteins), and is also crucial in the cellular chemical energy production and photosynthetic process of autotrophic phytoplankton. Gambierdiscus spp. are usually found in tropical and subtropical oligotrophic waters, where the nutrient concentrations are low in the water column and can be limiting. N limitation might have impacts not only on the survival of Gambierdiscus spp. but also their capability to produce toxins.

Phytoplankton and heterotrophic bacteria, as primary producers and decomposers, are the dominant microorganisms in the aquatic environment and their interactions play a major role in important processes such as carbon fluxes and nutrient regeneration. Algae-bacteria associations have increasingly been recognized as important in shaping the growth of both algae and bacteria, as algae can provide carbon sources as well as nutrients for bacteria whereas bacteria can be a potential food source or provide vitamins for algae. Among the diverse types of algae-associated bacteria documented so far, Roseobacteria, Flavobacteria, and Proteobacteria are the most common. Rhizobiales bacteria that can fix N2 are important symbionts of legumes often developing nodules on plant roots. Rhizobiales bacteria have not been widely documented in association with algae, so far largely limited to green algae.

Firstly, we used transcriptome sequencing (RNA-Seq) and suppression subtractive hybridization (SSH) to compare a ciguatoxin-producing strain with a non-toxin-producing strain of Gambierdiscus balechii. RNA-Seq and bioinformatics yielded 84,642 unigenes. Among them, 19.61% of genes showed differential expression between the two strains. Using the SSH method, we obtained 3,134 unigenes in the toxin-producing strain and 3,508 unigenes in the non-toxin-producing strain. Using both methods, a total of 52 type I PKS genes were identified to be up-regulated in the toxin-producing G. balechii, including transcripts encoding single-domain proteins as well as transcripts encoding multi-domain proteins. Transit peptides targeting chloroplasts were detected in four of these type I PKS transcripts. Differential expression patterns of these genes were also verified using reverse transcription quantitative PCR (RT-qPCR).

Secondly, to investigate how N limitation can modify the growth, cell size, photosynthetic capacity, and toxin production of G. balechii, we exposed algal cells to the N-replete condition as well as N-limited condition. Under N limitation, the population growth rate was repressed; cell size was enlarged; photosynthetic capacity was decreased; and toxin production was decreased. To further identify the molecular response of G. balechii to N limitation, we compared the transcript level of 1,5-bisphosphate carboxylase oxygenase and PKSs between N-replete and N-limited conditions during the early exponential phase and late exponential phase using RT-qPCR. Results of these gene expression showed consistency with physiological variations.

Moreover, from the toxic G. balechii culture, we detected, isolated, and characterized a Rhizobium species. The sequence of the 16S rRNA gene (16S rDNA) exhibited 99% identity with that of Rhizobium rosettiformans. To characterize the function of the bacterium, we PCR-amplified and sequenced a cell wall hydrolase (CWH) gene, and phylogenetic analysis indicated that the sequence was also affiliated with homologs of Rhizobium and Hoeflea species in the order of Rhizobiales. Furthermore, we obtained a nifH homolog, bchX, which is known to exist in the bacteria that can conduct photosynthesis, from this bacterium. Using qPCR, we found that this bacterium increased in abundance during the late exponential growth phase of the Gambierdiscus culture relative to the early exponential phase. When the dinoflagellate culture was subjected to N limitation, the abundance of the bacterium represented by both 16S rDNA and CWH increased as well.

In conclusion, our results from RNA-seq and SSH suggest that in the CTX synthesis process, single domain type I PKSs as well as multi-domain type I PKSs may work together in chloroplasts of G. balechii. In addition, N availability is important in determining the growth, toxin production, photosynthesis, and cell size of G. balechii. Furthermore, the Rhizobium bacterium, which was isolated from G. balechii, may benefit from the association with G. balechii by obtaining carbon sources from hydrolyzing extracellular organic matter exudates released from the dinoflagellate. On the other hand, G. balechii might be able to benefit from the bacterium as food or as source of nutritional substances for survival under low N-nutrient conditions.

Clearly, more experiments should be conducted in the future. Firstly, how PKSs are regulated at the post-transcriptional level in the toxic G. balechii should be taken into consideration, in order to provide more comprehensive data on the toxin production pathway in this genus or dinoflagellates. Next, more physiological combined with molecular experiments should be carried out on G. balechii in order to get a whole picture of how this dinoflagellate might respond to the changing environment both at the physiological and molecular level. In addition, more studies on the evaluation of the N2-fixing and photosynthetic capability of the associated Rhizobium bacterium are needed to get a better understanding of its role in the alga-bacterium interactions.

    Research areas

  • Gambierdiscus, Ciguatoxins, Polyketide synthase, Transcriptome, Nitrogen limitation, Rhizobium, Cell wall hydrolase