Arch Microbiol DOI 10.1007/s00203-015-1133-0
SHORT COMMUNICATION
Potential DMSP‑degrading Roseobacter clade dominates endosymbiotic microflora of Pyrodinium bahamense var. compressum (Dinophyceae) in vitro Deo Florence L. Onda1,2 · Rhodora V. Azanza1 · Arturo O. Lluisma1
Received: 5 March 2015 / Revised: 18 June 2015 / Accepted: 28 June 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Many aspects of the biology and ecology of the toxic dinoflagellate Pyrodinium bahamense var. compressum are still poorly understood. In this brief note, we present identification of its associated intracellular bacteria or endosymbionts via PCR cloning and 16s rRNA gene sequencing and their localization by confocal microscopy, a first for Pyrodinium. The most frequently observed species in the endosymbiotic microflora were from Roseobacter clade (Alphaproteobacteria, 68 %) and Gilvibacter sediminis (Flavobacteriaceae, 20 %). Roseobacter lineage, the most abundant taxa in this study, is known to be involved in dimethylsulfoniopropionate metabolism which is highly produced in dinoflagellates—a possible strong factor shaping the structure of the associated bacterial community.
Communicated by Erko Stackebrandt. Electronic supplementary material The online version of this article (doi:10.1007/s00203-015-1133-0) contains supplementary material, which is available to authorized users. * Arturo O. Lluisma
[email protected] 1
The Marine Science Institute, University of the Philippines, 1101 Diliman, Quezon City, Philippines
2
Present Address: Takuvik/Quebec‑Ocean/IBIS, Pavillon Eugene Marchand, Université Laval, Québec City, QC G1V0A6, Canada
Keywords Endosymbiotic bacteria · Dimethylsulfoniopropionate (DMSP) catabolism · Symbiosis · Pyrodinium bahemense var. compressum
Introduction Pyrodinium bahamense var. compressum (Bӧhm) Steidinger, Tester and J.F.R. Taylor is one of the most studied toxic dinoflagellates. However, many aspects of its biology and ecology still remain to be understood including its association with its intracellular bacterial community or endosymbionts (Azanza et al. 2006). Identifying these intracellular bacteria is a crucial step toward the elucidation of their roles in the biology and ecology of dinoflagellates. For example, recent evidence showed that the removal of culturable and possibly most bacteria by antibiotic treatment significantly affected the growth or caused death of Pyrodinium cultures (Santos and Azanza 2012). They have also been implicated in the initiation, maintenance and decline of algal blooms (Doucette 1995). Studies showed that dinoflagellates have specifically structured associated bacterial communities beneficial to the host organism. Previous investigations that identified potential symbionts of P. bahamense var. compressum were limited to culture-dependent methods (Azanza et al. 2006; Chin et al. 2013). Hence, the uncultivable members of the endosymbiotic community and factors shaping community structures have remained to be identified and characterized. In this study, we investigated the genetic diversity of the endocytic bacterial community in Pyrodinium. The localization and spatial distribution of these bacteria were also investigated via confocal laser scanning microscopy (CLSM) using a fast and reproducible sample preparation
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method. We then inferred potential interactions based on the characteristics of most similar cultured bacterial isolates.
Materials and methods Sample preparation for CLSM Pyrodinium bahamense var. compressum PbcMZRVA042595 isolated from Masinloc Bay, Zambales, Philippines, was used in this study. Samples for CLSM imaging were prepared by fixing 1 mL algal sample with 1 % glutaraldehyde (Sigma-Aldrich) and pelleting at 2500×g for 15 min. Cells were resuspended in 100 μL of 1X phosphate buffer saline (PBS) and transferred to a polycarbonate membrane with a hole in the center mounted in a glass slide. Five microliters (5 μL) of each of 5000× SybrGreen (Invitrogen) and 50 % calcofluor white (Sigma-Aldrich) was added. Observation was done in an inverted CLSM (Carl Zeiss LSM 710, Germany), and images were taken by excitation at 385 nm (cell wall), 488 nm (algal nucleus and bacteria) and 580–720 nm (chloroplast autofluorescence). Cell cleaning and bacterial DNA extraction Fifteen milliliters (15 mL) of cell culture samples was fast-fixed (0.1 % glutaraldehyde), harvested by centrifugation at 2500×g for 15 min, resuspended in 1 mL 1 % lysozyme solution and then incubated at room temperature for another 15 min. Subsequent centrifugation was done at 2300×g (13 min) and 2200×g (11 min) with resuspension in 500 µL 1X PBS in between. Extracellular bacterial contamination was assessed by CLSM and PCR (i.e., resuspending medium) before proceeding. Upon confirmation of efficiency of cleaning, cells were pelleted at 2200×g for 10 min and finally resuspended in 1 mL sterile deionized water (Nanopure). For DNA extraction, 100 μL of the resuspended cells was transferred to a 1.5-mL microfuge tube, and four sterile silica beads were added. The sample was then bead-beaten using a vortex mixer (Disruptor Genie) for 3–4 min. Enzymatic purification was done by adding 3 μL of proteinase K (30 mg mL−1, Macherey– Nagel) with incubation at 55 °C for 30 min and stopped at 85 °C for 40 min. Cells were harvested on the 7th, 12th and 21st day of the batch cultures and were then pooled together after extraction. PCR, cloning and 16s rRNA gene library construction The 16s rRNA genes of the endosymbionts were amplified using the universal primers 27F and 1492R following conditions described by Isenbarger et al. (2008) with
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annealing temperature at 40 °C. Amplification was done in 25 µL reaction mixtures as described by Onda et al. (2014) but with 5 μL DNA template (suspended in water). After PCR, target amplicons (~1.4 kb) were gel purified using Qiaquick Gel Purification kit (Qiagen) and then stored at −20 °C. High-efficiency transformation Electromax DH10BT1 phage resistant cells (Invitrogen) were used for cloning by electroporation using the GenePulser electroporator (BioRad) at 2.0 kV pulse. Cells were plated on LB medium with 50 μg mL−1 kanamycin at 37 °C overnight. A loopful of the colonies was individually resuspended in 40 μL sterile water and lysed at 95 °C for 15 min. The flanking M13 regions of the vector were amplified to check for correct inserts. Amplicons with expected band size (~1.6 kb amplicons) were gel purified and sequenced using the M13F primer (1st Base Gene, Malaysia). Sequence data processing was carried out in MEGA 5.0 (Tamura et al. 2011), and chimeras were screened in DECIPHER (Wright et al. 2011). Most similar sequences were determined by batch BLASTn search against the NCBI GenBank 16s rRNA reference sequence database, then downloaded and aligned using Muscle (Edgar 2004). Phylogenetic trees were generated via maximum likelihood method (using the Kimura-2 parameter model as determined by model test) with bootstrap analysis using 1000 replicates. Since not all sequences were sequenced in the same orientation, separate trees were built using representative sequences from targeted taxa together with the closest related reference sequences. Sequences generated in this study were deposited in NCBI GenBank database under accession numbers KP453874–KP453973.
Results and discussion Taxonomic diversity of associated bacteria In this study, we removed the extracellular/attached bacteria prior to extraction of DNA from the Pyrodinium cells to focus on the endosymbiotic communities by applying washing protocols (Fig. 1a, b). Results show that despite cleaning, we still obtained higher bacterial diversity than previously reported (Azanza et al. 2006; Chin et al. 2013). A total of 100 clones of screened transformants were analyzed and mostly with 98–99 % sequence similarity to the reference sequences (Table 1, Supplementary Fig. 1a, b). However, some sequences did not return hits with significant scores, consistent with previous studies done on environmental/natural samples (Alavi et al. 2001). The most frequent clones are from class α-proteobacteria, clade Roseobacter. Highly abundant putative species observed include Ruegeria sp. and Roseovarius
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Fig. 1 CLSM images of Pyrodinium bahamense var. compressum showing a the extracellular/attached bacteria (green dots); b a cleaned algal cell after the washing protocol; c a cross section of the algal cell
showing the localization of the endosymbiotic (end) bacteria along with the NR nuclear material, OM outer membrane, Th theca, Chl chloroplasts
sp. which are also reported in other toxic dinoflagellates and generally in marine environment (Green et al. 2010; Buchan et al. 2005). The closest matches to our sequences were also species from seawater, sand and marine sediments supporting that our results are not artifacts or contamination. Other sequences are similar to putative biofilmassociated bacteria such as Phaeobacter sp. and Leisingera sp., and a phytoplankton phycosphere-associated Marinovum algicola (formerly Roseobacter algicola) (Green et al. 2010). Also, the abundance of single-genus sequences from Flavobacteriaceae of the species G. sediminis (20 %) in the library is surprising since most of its representative cultures are associated with sediments. Recently, it was observed that breaks in the cell wall are created during the regeneration of temporary cysts in sediments which could possibly provide entry of bacteria from the environment (Onda et al. 2014). There are also some representatives from other Bacteroidetes such as Cyclobacterium sp. Inconsistency in the observed diversity with previous reports was also observed (Azanza et al. 2006; Chin et al. 2013) which may be attributed to the biases in the methods used, the differences in conditions used in maintaining the culture and other unknown factors. Biological or even statistical explanations, resulting in disparity in the datasets, are also possible.
As much as 10 % of fixed carbon is directed to DMSP production by marine phytoplankton (Howard et al. 2008). In this study, one interesting common characteristic of the most abundant bacterial groups is their possible involvement in DMSP decomposition (Table 1, Supplementary Fig. 1a, b). Dimethylsulfoniopropionate is a relatively stable molecule, but also a labile bacterial substrate that can supply up to 15 and 100 % of bacterial carbon (C) and sulfur (S) demand, respectively (Simó et al. 2002). Evidence showed that alphaproteobacteria lineage, which makes up to 68 % of the clones in our library (Table 1), is involved in the metabolism and recycling of DMSP (González et al. 1999). DMSP turnover rate in phytoplankton blooms is thus hastened by bacterial uptake (Pinhassi et al. 2005). Most of the DMSP in seawater is degraded through the demethylation/demethiolation pathway which produces intermediates (e.g., methanethiol, methyl mercaptopropionate) that are significant for the assimilation of C and S. When DMSP-S is not assimilated (i.e., low demand for S), the molecule is cleaved by either the algal or bacterial enzyme DMSP lyase which produces the climate active gas dimethylsulfide (DMS) and acrylate. Although different copies of DMSP lyase genes have been found in several phytoplankton, it has not yet been reported in Pyrodinium. The DMS then diffuses into the atmosphere, or oxidized by reactive oxygen species (ROS) or DMS-oxidizing bacteria in seawater producing the end product dimethylsulfoxide [DMSO, (Sunda et al. 2002)] or tetrathionate (Boden et al. 2010), respectively. These two pathways (demethylation/demethiolation and DMSP cleavage), however, are estimated to only account for 5–10 % of the fate of DMSP. Incidentally, as much as 58 % of cells surveyed via metagenomics from ocean surface were found to possibly participate in the
Bacteria–algae association Phytoplankton species produce organic compounds and metabolites that attract the neighboring bacterial community. Dinoflagellates in particular are significant producers of dimethylsulfoniopropionate (DMSP) that serves as osmolyte and osmoprotectant during salinity fluctuations.
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13 99 96 92 96
NR029273.1, NR112651.1 NR116682.1 NR044547.1 NR043611.1
Marinovum algicola (now Roseobacter algicola) Marivita byunsanensis Oceanibaculum indicum
426
368, 373
97
95
NR113209.1
99 97
NR108232.1 NR109594.1
Roseovarius indicus
95
98 96 92
NR108509.1 NR036802.1 NR074599.1
Ruegeria pomeroyi Ruegeria sp. Tropicibacter multivorans Methylophaga thalassica Pseudomonas protegens Thioalkalivibrio sulfidophilus
66, 73, 84, 125, 244, 246, 353, 370
114, 132
211, 232, 240
Gammaproteobacteria 88
113, 134
103, 356, 449
Bacteroidetes Flavobacteriacea 62, 69, 71, 77, 108, 110, 116, 124, 126, 133, 141, 213, 380, 381, 404, 422, 427, 432, 434, 435 Flexibacteriaceae 91, 119
93
NR074151.1
Ruegeria arenilitoris
99
91
NR114011.1
NR040920.1
Marinoscillum furvescens
88
98
98
Gilvibacter sediminis
NR074692.1
NR074150.1
NR109635.1
99
70, 92, 204, 236, 362, 371, 376, 377, 379, 445
NR043564.1
Roseovarius litoreus Roseovarius pacificus
65, 120, 131, 358
127, 378, 430, 431
94
231, 237
NR043932.1 NR121734.1
Pseudoruegeria aquimaris Roseibacterium elongatum
95
450
NR113210.1
118
Poseidonocella pacifica Poseidonocella sedimentorum
50
428
97
NR074144.1
Phaeobacter gallaciensis
98
364
NR118542.1
Pelagibaca bermudensis Phaeobacter caeuruleus
369, 420
17, 49, 80, 89, 111, 352, 410, 410b
90
36, 52, 83, 86, 93, 112, 117, 212, 242, 363, 412
NR042670.1 NR074160.1
Leisingera aquimarina
96
Maricaulis maris
NR102914.1
Marine organism
Marine sediment
Highly saline bioreactor
Plant symbiont
Marine microbial mat
Seawater
Type strain
Atlantic coastal water
Seashore sand
Deep-sea sediment
Seawater
Deep seawater
Sand
Seawater, Korea
Shallow sandy sediment
Shallow sandy sediment
Coastal environment
Marine environment, Palau and Japan
Sargasso sea
Deep seawater, Indian Ocean
Tidal flat sediment
Marine environment, Spain; phycosphere of Prorocentrum lima
Type strain
Marine electroactive biofilm
Industrial strain
% similarityb (%) Source
101, 115, 249, 419
Ketogulonicigenium vulgare
Alphaproteobacteria 415
Reference sequence NCBI accession number
441
Taxonomic groupa
Clones
Table 1 Summary of taxonomic assignments based on sequence similarity search using BLASTn against the NCBI GenBank
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Type strain
Marine oilfield sediment, SCS
85
97
Maximum percentage of identical nucleotides in the noted alignment length
107
Full name or description of the closest matched sequence
b
a
Planctomyces maris
NR025327.1
NR043903.1 Cyclobacterium lianum Cyclobacteriaceae 122, 135, 360
Planctomycetes
Taxonomic groupa Clones
Table 1 continued
Reference sequence NCBI accession number
% similarityb (%) Source
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marine sulfur cycle via the DMSP catabolic pathway (Howard et al. 2008). Several isolates of Roseobacter in particular were observed to demethylate DMSP in culture. The corresponding genes for DMSP catabolism were also found in some of the reference type strains in this study such as Ruegeria pomeroyi DSS-3 and Phaeobacter gallaeciensis DSM 17395. Although species belonging to Roseobacter clade are cosmopolitan in nature, they have been frequently and abundantly found to be associated with DMSP-producing dinoflagellates such as Pfiesteria sp. (Alavi et al. 2001), Gymnodinium catenatum (Lafay and Ruimy 1995, Green et al. 2004), Alexandrium sp., Scripsiella sp. (Hold et al. 2001), Prorocentrum lima (Lafay and Ruimy 1995), A. tamarense (Jasti et al. 2005) and Pyrodinium bahamense var. compressum (this study) among others. These studies suggest how conditions inside the cell could possibly shape the structure of the associated bacterial communities which could have beneficial role to the host organism. For example, a symbiont Ruegeria sp. TM1040 which is capable of DMSP demethylation was isolated from a culture of the heterotrophic dinoflagellate Pfisteria piscicida (Miller and Bellas 2004). Further studies later suggested that the association is obligate and is affected by changing conditions. Other derivatives from DMSP decomposition may also be reabsorbed by the alga or used up by other bacteria. Methylophaga thalassica, for example (Supplementary Fig. 1b), an obligate methylotrophic bacteria found in environments with high DMS (Boden et al. 2010), was also observed to be present in Pyrodinium. The dominance of potential DMSP catabolic clades inside the Pyrodinium cell raises a lot of other questions that may help further understand DMSP regulation and DMS production in dinoflagellates. Further investigations using more laboratory-based approaches, however, are still necessary to provide evidence for such hypothesis. Confocal laser scanning microscopy images also showed that the associated bacteria seem to be more abundant and interspersed together with the chloroplasts in the cytoplasm in very close proximity with the theca (Fig. 1c). It has been shown that Roseobacter clade uniquely produces bacteriochlorophyll a with some species capable of aerobic anoxygenic photosynthesis (Allgaier et al. 2003). Recent reports demonstrated that DMSP uptake of Dinoroseobacter shibae associated with Prorocentrum minimum could be regulated by the absence or presence of light. Depending on necessity, light availability and aerobic anoxygenic photosynthetic activity could also influence shift from mutualistic to parasitic relationships between the host and the symbionts (Wang et al. 2014). Further studies are needed, however, to show that the physical closeness of chloroplasts to the bacteria in Pyrodinium implies possible functional relationships. Other taxa such as Bacteroidetes could also supply iron and vitamins, recycle organic compounds and
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metabolize toxins (Kodama et al. 2006). Gilvibacter sediminis which consists 20 % of the clones in our library also singly dominated the Flavobacteriaceae group. It has been previously reported in the benthic dinoflagellate Coolia monotis (Ruh et al. 2009), in sediments (Khan et al. 2007) and as free-living bacterioplankton (Brettar et al. 2012). Interestingly, although most Flavobacteria have been generally implicated in the conversion of high molecular weight substrates to smaller components (Buchan et al. 2014), the particular role potentially played by G. sediminis in associations has not yet been fully explored. The contribution of the associated bacterial species to algal toxicity, specifically saxitoxin (STX), has also been suggested (Azanza et al. 2006; Hold et al. 2001). Gene homologs most similar to bacteria that could possibly be involved in the STX biosynthetic pathway in Pyrodinium have also been observed (Onda et al., unpublished data). Although phylogenetic data do not directly suggest functionality in the bacterial community, it could still provide insights into the functional roles of the endosymbionts and how groups correlate to certain variables significant to the host organism. The high diversity of intracellular bacteria found in this study, for example, suggests strong environmental conditions that favored such associations to exist which in turn could have ecological implications. These associations and their still unknown interactions may be truly significant that their removal from the host and/or disruption of the association may be detrimental to the host alga. For example, loss of viability of cultured Pyrodinium was observed when associated cultivable bacteria were attempted to be removed by antibiotic treatment (Santos and Azanza 2012). These results are the foundations of our ongoing investigations to further understand the significance of the “microalgal microbiome” in such ecological relationships using high-throughput sequencing approach. Pyrodinium’s relationship with its associated bacterial community is complex and still enigmatic. This model system, however, provides a wealth of information for further understanding coevolution and host–symbiont interactions. These symbionts are also known producers of secondary metabolites—a mine for potential novel genes with biotechnological applications. Acknowledgments This study was part of the research program “Ecology and Oceanography of Harmful Algal Blooms in the Philippines (PhilHABs), Project 1: Biodiversity/Genetic Diversity of selected HAB-forming species in the Philippines and their associated bacterial communities”, funded and supported by the Department of Science and Technology (DOST) through the Philippine Council for Aquatic and Marine Research and Development (PCAMRD, now part of PCAARRD). We would also like to thank the Biotechnology and Molecular Genetics Research Laboratory at the UP-NIMBB headed by Dr. Ameurfina D. Santos for the use of the electroporator, Dr. Neda Barghi for her assistance in cloning, Emelita Eugenio for her help in
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Arch Microbiol maintaining the algal cultures, Dr. Mary Anne Santos for her comments on Pyrodinium biology, and Margaux Goudal for her valuable insights on DMSP regulation and metabolism. Compliance with Ethical Standards Conflict of interest The authors express no conflict of interest in publishing this article.
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