**Abstract**

Previous studies have suggested that a specific community of bacteria coexists within the phycosphere of marine dinoflagellates. In order to better understand the dinoflagellate-bacteria relationships, a fosmid clone library was constructed from the metagenome DNA and analyzed. Some of the fosmid clones were end-sequenced. A total of 1501 fosmid clones with insert sizes of 30–40 Kbp were produced. End sequencing of 238 clones showed that 55% of the genes had known functions, 11% were of putative function and 34% were genes of unknown function or had no match in Genbank. There were approximately 14% sequences with no classification and could potentially represent novel genes. Analysis of these partial sequences also revealed some promising enzymes that possess various potential industrial applications such as chitinases, kinases, agarases and oxygenases. The results also showed that the bacterial flora of the Alexandrium tamiyavanichii culture was dominated by the Alpha-proteobacteria, followed by Bacteroidetes and Gamma-proteobacteria. The findings in this study suggested that the bacterial community may play various role in the association with dinoflagellate. This study had also shown that dinoflagellate-associated bacterial community is a valuable source for discovery of novel genes and gene products.

**Keywords:** metagenomic, fosmid library, bacterial community, toxic dinoflagellate, genes discovery

## **1. Introduction**

Microalgae are the major producers of biomass and organic compounds in the ocean. More than 5000 species of marine microalgae are known to date and are separated into six major divisions: *Chlorophyta* (green algae), *Ochrophyta* (yellow algae, golden brown and diatoms), *Haptophyta* (coccolithophorids), *Pyrrhophyta* (dinoflagellates), *Euglenophyta* and *Cyanophyta* (blue-green algae) [1]. Among the 5000 species about 300 can proliferate in high numbers to form the so-called red tide and brown tide phenomena [1, 2].

Many planktonic organisms can form mass occurrences in the water column. When the cell densities reach values considerably higher than their general background distribution, they are called blooms [3]. Blooms can be almost mono specific, others are formed by a combination of species [4, 5]. Many prominent blooms can be traced back to high nutrient loads [6–8], but they can occur whenever a species is able to outgrow its competitors while partially reducing grazer pressure [9].

Microscopic marine algae can be vectors of microbial communities because they are universally associated in the ocean. In nature, most microbial communities are found adhered to microalgae, organism and inanimate surfaces. These interactions are dynamic and are important factors in microbial proliferation and survival. Aquatic algae in situ as well as in laboratory culture condition are often found to be associated with a variety of bacterial strains [10]. Bacteria community can be defined as multi – species of bacteria assemblages in which organisms live together in a contiguous environment (host) and interact with other [11]. Bacteria reproduce asexually, are sized between 0.1 to 20 μm, and can be rod, cocci or comma shaped.

For marine microorganisms (bacterioplankton), there are approximately 106 bacterial cells per ml of surface seawater throughout the world's oceans [12]. While this number has been known for at least 30 years, how many bacterial species are actually present in the bacterioplankton are still unknown. Bacterioplankton commonly found in marine environment are mainly from bacteria group of Proteobacteria, Cytophaga-Flexibactar-Bacteroides (CFB), marine Archaea and other groups of bacteria, where bacteria group from Proteobacteria is the largest. Proteobacteria group are divided to some class, which are Alpha (α-), Beta (β-), Delta (δ-), Epsilon (ε-) and Gamma (γ-) Proteobacteria [13]*.* Up until now, the estimated abundance and genetic diversity of bacterioplankton are based on the data in the GenBank database. Hagström et al. [14] had analyzed on all of the 16S rDNA sequences sent to GenBank to get the estimation of marine bacterioplankton species that were available in the GenBank database. Their studies show that the richness of marine bacterioplankton species in the GenBank database was low relatively.

The ecology of bacterioplankton and phytoplankton is widely recognized to be tightly coupled. Interactions between bacteria and phytoplankton such as dinoflagellates may play an important role in regulating dinoflagellate toxin production. Previous studies on the interactions between bacteria and dinoflagellates have been shown to be highly variable and are sometimes specific. Effects of bacteria on toxic dinoflagellates include negative effects such as cell lysis and death [15] and positive effects such as growth enhancement with an addition of bacteria to cultures [16]. Examples of factors which may cause stimulation or inhibition by bacteria include production of co-factors and secretion of signaling molecules controlling cellular processes of dinoflagellates [17]. In addition, bacterial influences on nutrient availability may result in stimulation or inhibition of the growth or toxin production of dinoflagellates. Both toxin production by dinoflagellates and bacteria associated with toxic or non-toxic dinoflagellates have been documented. For example, Gallacher et al. [18] described evidence of paralytic shellfish toxin (PST) production by bacteria associated with dinoflagellates cultures.

Cultures of dinoflagellates contain a considerable amount of bacteria which probably accompanied the dinoflagellates in the original sample. Bacterial assemblage found in the phycosphere of dinoflagellates may play an important role in regulating dinoflagellate toxin production. While several studies have suggested that bacteria-phytoplankton interactions have the potential to dramatically influence harmful algal bloom dynamics, little is known about how bacteria and phytoplankton communities interact at the species composition level. Other studies have indicated that inside a phytoplankton bloom, α-Proteobacteria overwhelm

**41**

*Construction and Analysis of Metagenome Library from Bacterial Community Associated…*

the free-living bacterioplankton, while microorganisms connected to phytoplankton are basically distinguished as fitting in with (CFB), γ-Proteobacteria, and

At present the precise association of bacteria with cultured dinoflagellates is still not well understood. Moreover, current estimates indicate that more than 99% of the microorganisms present in many natural environments are not readily culturable and therefore not accessible for biotechnology or basic research [21]. Technology to access the genomic DNA or RNA of microorganisms, directly from environmental samples without prior cultivation, has opened new ways of understanding microbial diversity and functions. Thus, this present study is an important

Metagenomics has become a powerful tool to investigate the biodiversity of complex microbial communities and for studying its metabolic pathways. This technique can be considered as a revolutionary approach to study the microbial community that is unapproachable by available conventional methods and this approach also can capture the total genomes that present in a community of interest. According to Schloss and Handelsman [22], metagenomics was builds on advances in microbial genomics and in the polymerase chain reaction (PCR) amplification and cloning of genes. The field of metagenomics has played a pivotal role for significant progress in microbial ecology, evolution, and diversity over the past years. This approach has allowed researchers to elucidate some possible mechanisms

**Dinoflagellate Culture.** Clonal culture of *Alexandrium tamiyavanichii* were obtained from UKM Microalgae Culture Collections and maintained in ES-DK medium [23] and growth in a light–dark cycle (14:10 hour) incubator at 26°C

**DNA Extraction.** Bulk genomic DNA were directly extracted from a 2.0 L of mid exponential growth phase dinoflagellate [24]. Firstly, culture medium was filtered through 0.2 μm nitrate cellulose membrane (Whatmann, England). Cell pellets were then concentrated and resuspended in buffer (100 mM EDTA, 10 mM Tris–HCl [pH 8.0]) and treated with proteinase K (0.5 mg/mL)-1% sodium dodecyl sulfate (SDS) for 1 h at 37°C. Lysates were further treated by CTAB extraction (0.5 M NaCl, 1% CTAB) for 10 min at 65°C. Then, DNA was extracted once with equal volume of chloroform-isoamyl alcohol (24:1) and phenol-chloroformisoamyl alcohol (25:4:1) and spin at 21000 x g for 5 min at 4°C. After that, DNA was precipitated with 0.6 volume of isopropanol by centrifugation at 21000 x g for 15 min. The DNA pellet was then washed with one volume of 70% ethanol and spin at 21000 x g for 10 min. Finally, DNA was resuspended in 50 μL of ddH2O and

**Metagenomic Library Construction and End-sequences Analysis.** Sheared DNA with sizes ranged from 30 kilo bases to 40 kilo bases were used to construct the metagenomic library. Metagenomic library was constructed using CopyControl pCC1FOS library construction kit (Epicenter, USA) following the manufacturer's protocol and fosmids were then purified using Millipore 96-well prep BAC purification kit (Millipore, USA) following the manufacturer's protocol and end-sequenced using T7 universal primer (5′- TAATACGACTCACTATAGGG-3′). End-sequences were edited by using Staden Package software [25]. Low quality DNA sequences were identified and trimmed using Pregap4. The resulting high-quality sequences were assembled into

step towards understanding bacteria-dinoflagellate interactions.

*DOI: http://dx.doi.org/10.5772/intechopen.88751*

governing ecosystem function and diversity.

**2. Methodology**

stored at −20°C.

(model 2015 Shelab, USA).

Planctomycetes groups [19, 20].

*Construction and Analysis of Metagenome Library from Bacterial Community Associated… DOI: http://dx.doi.org/10.5772/intechopen.88751*

the free-living bacterioplankton, while microorganisms connected to phytoplankton are basically distinguished as fitting in with (CFB), γ-Proteobacteria, and Planctomycetes groups [19, 20].

At present the precise association of bacteria with cultured dinoflagellates is still not well understood. Moreover, current estimates indicate that more than 99% of the microorganisms present in many natural environments are not readily culturable and therefore not accessible for biotechnology or basic research [21]. Technology to access the genomic DNA or RNA of microorganisms, directly from environmental samples without prior cultivation, has opened new ways of understanding microbial diversity and functions. Thus, this present study is an important step towards understanding bacteria-dinoflagellate interactions.

Metagenomics has become a powerful tool to investigate the biodiversity of complex microbial communities and for studying its metabolic pathways. This technique can be considered as a revolutionary approach to study the microbial community that is unapproachable by available conventional methods and this approach also can capture the total genomes that present in a community of interest. According to Schloss and Handelsman [22], metagenomics was builds on advances in microbial genomics and in the polymerase chain reaction (PCR) amplification and cloning of genes. The field of metagenomics has played a pivotal role for significant progress in microbial ecology, evolution, and diversity over the past years. This approach has allowed researchers to elucidate some possible mechanisms governing ecosystem function and diversity.
