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Applied and Environmental Microbiology, December 2006, p. 7912-7915, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01148-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Molecular Detection of Epiphytic Acaryochloris spp. on Marine Macroalgae{triangledown}

Satoshi Ohkubo,1 Hideaki Miyashita,1,2* Akio Murakami,3 Haruko Takeyama,4 Tohru Tsuchiya,1,2 and Mamoru Mimuro1,2

Graduate School of Human and Environmental Studies, Kyoto University, Kyoto 606-8501, Japan,1 Hall of Global Environmental Research, Kyoto University, Kyoto 606-8501, Japan,2 Kobe University Research Center for Inland Sea, Iwaya, Awaji, Hyogo 656-2401, Japan,3 Faculty of Engineering, Tokyo University of Technology and Agriculture, Koganei, Tokyo 184-8588, Japan4

Received 18 May 2006/ Accepted 26 September 2006


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ABSTRACT
 
A molecular method for detecting the epiphyte community on marine macroalgae was developed by using PCR-denaturing gradient gel electrophoresis. Selective amplification of 16S rRNA gene fragments from either cyanobacteria or algal plastids improved the detection of minor epiphytes. Two phylotypes of Acaryochloris, a chlorophyll d-containing cyanobacterium, were found not only on red macroalgae but also on green and brown macroalgae.


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INTRODUCTION
 
Acaryochloris species are unicellular cyanobacteria that contain chlorophyll (Chl) d as the predominant pigment (10-14). The first isolate, Acaryochloris marina MBIC11017, was obtained as a symbiont in colonial ascidians from the tropical coast (10). Recently, we discovered the epiphytic Acaryochloris sp. strain Awaji on the red macroalga Ahnfeltiopsis flabelliformis from the temperate coast (13). This indicates that Chl d, which had been assigned to a product of red macroalgae (5, 7, 8), was produced by the epiphytic Acaryochloris cells rather than the red algae themselves (13-14). Because of their small cell size and the simple morphology of the cyanobacterial epiphytes, a molecular method that enables detection and identification was required to analyze the distribution and diversity of epiphytic Acaryochloris spp. PCR-denaturing gradient gel electrophoresis (DGGE) has been used for the detection of environmental microorganisms in the last decade (1, 4, 6, 9, 16, 17). We developed a molecular method for analyzing the epiphyte community on macroalgae.

Nine species of macroalgae, including seven red, one green, and one brown algae, were collected in June 2003 from the rocky seashore of Awaji Island, Japan, where Acaryochloris sp. strain Awaji was discovered on A. flabelliformis (13). Individual samples were ground to fine powders in liquid nitrogen and transferred into a microtube containing 500 µl of lysis buffer (100 mM Tris-HCl [pH 8.0], 50 mM EDTA, 500 mM NaCl, 1% CTAB [cetyltrimethylammonium bromide]) and 50 µl of 10% sodium dodecyl sulfate. After incubation at 80°C for 5 min, 300 µl of 10 M potassium acetate and 6 µl of proteinase K (4 mg/ml) were added; the microtube was then incubated at 50°C for 60 min, put on ice for 20 min, and centrifuged at 4°C for 15 min at 16,000 x g. The upper aqueous phase was transferred into a new microtube, and DNA was then extracted by general phenol-chloroform extraction, isopropanol precipitation, and ethanol precipitation. Amplification of partial 16S rRNA gene fragments was conducted with the primers shown in Table 1. PCR was performed in a reaction mixture containing extracted DNA, primers, ExTaq polymerase (TaKaRa, Ohtsu, Japan), a deoxynucleoside triphosphate mixture, and 5x Ampdirect-D and and 5x AmpAddition-3 (Shimadzu Biotech, Kyoto, Japan). After incubation for 5 min at 95°C, 30 incubation cycles were conducted; each consisted of 1 min at 94°C, 1 min at 65 to 55°C (decreasing by 1°C during alternate cycles for the first 20 cycles), and 1 min at 72°C. PCR products (approximately 200 ng) were loaded onto 7% (wt/vol) polyacrylamide gels with a 20 to 40% denaturant gradient in 0.5x TAE buffer. Electrophoresis was carried out at 60°C with a constant voltage of 200 V for 6 h using the DCode system (Bio-Rad, Hercules, CA). All individual DGGE bands were excised from the gels, and the gene fragments were then used as templates for sequencing reactions after PCR reamplifications as described above. The sequence similarities of individual bands to known sequences were compared by using NCBI BLAST (2). The sequences determined in the present study were deposited in the DDBJ database under accession numbers AB232058 to AB232080.


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  		TABLE 1. Primer sequences and target sites

We adopted the oligonucleotide primer set comprised of the forward primer CYA359F and the reverse primer CYA781R (Table 1) (18). This primer set has been effectively applied for the community analysis of environmental cyanobacteria using PCR-DGGE (1, 3, 19, 20). However, it could amplify only the 16S rRNA gene fragments from the plastids of host macroalgae (bands 2 and 7 in Fig. 1A, lanes CC) when the template DNAs from red macroalgae Ahnfeltiopsis flabelliformis and Caulacanthus ustulatus was used. The bands from epiphytes were below detection limits, although epiphytic diatoms and cyanobacteria were identified microscopically on the thalli of these macroalgae. The abundance of plastid DNA may have impeded amplification of partial 16S rRNA gene fragments from the epiphytes.


Figure 1
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  		FIG. 1. DGGE profiles obtained using different primer combinations. (A) DGGE profiles of 16S rRNA gene fragments obtained after PCR amplifications with four different primer sets. Template DNAs were extracted from the two red macroalgae, Ahnfeltiopsis flabelliformis and Caulacanthus ustulatus, that were confirmed microscopically to possess chlorophyll d-containing cyanobacteria. PCR amplifications were performed with CYA353F-CYA781R (lanes CC), BAC341F-CYA781R (lanes BC), BAC341F-CYA781R(a) (lanes BCa), and BAC341F-CYA781R(b) (lanes BCb) independently. Band X was an artifact. No sequence was obtained from these bands; they migrated independently of both the concentration of the denaturant and the voltage. (B) DGGE profiles of 16S rRNA gene fragments obtained from genomic DNA of Acaryochloris sp. strain Awaji using the primer set BAC341F-CYA781R(b). The consecutive number of each band corresponds to that in Fig. 2 and Table 2.

We rearranged the combination of primers to avoid the PCR bias caused by the predominant plastid DNA and found that the primer set of BAC341F, a bacterium-universal forward primer (15), along with with CYA781R, could amplify 16S rRNA gene fragments of epiphytes together with those from the plastids of host macroalgae (Fig. 1A, lanes BC). Furthermore, when the reverse primers CYA781R(a) and CYA781R(b) were used separately in combination with BAC341F, all bands that were observed in lanes BC came out more clearly in either lanes BCa or BCb (Fig. 1A). In addition, two bands (bands 8 and 10) newly appeared, showing the improvement of the detection efficiency for minor epiphytes. Sequence analysis showed that all bands in lanes BCa (bands 1, 2, 6, 7, and 9) were originated from the plastids, and most of the bands in lanes BCb (bands 3, 4, 5, and 10) were from cyanobacteria. Band 4 was of Acaryochloris sp. strain Awaji (Fig. 1B and Table 2). Band 5 had 99.2% sequence similarity to the strain Awaji (Table 2), suggesting the existence of an additional phylotype of epiphytic Acaryochloris species on these macroalgae. Band X was an artifact, giving no sequence. These results showed that CYA781R(a) exhibited a biased affinity to plastid. In contrast, CYA781R(b) had affinity especially to unicellular cyanobacteria, which was consistent with the results of Boutte et al. (3). Therefore, parallel PCR amplifications with the BAC341F-CYA781R(a) and BAC341F-CYA781R(b) primer sets were useful for the detection of epiphytic cyanobacteria including Acaryochloris spp., as well as for the analysis of epiphyte diversity on macroalgae.


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  		TABLE 2. Sequence similarities of the excised DGGE bands

We analyzed the epiphyte communities on the nine macroalgae by using the method described above (Fig. 2). The BLAST search results for individual bands are listed in Table 2. The epiphyte community on each macroalga differed even though these algae were collected from the same site, indicating the presence of some selection mechanism between those macroalgae and epiphytes. In contrast, the two phylotypes of Acaryochloris (bands 4 and 5) were detected in all BCb lanes except that of Chondria crassicaulis (Fig. 2). This showed that Acaryochloris spp. existed widely not only on red macroalgae but also on green and brown macroalgae. The reason Chl d has been detected in red macroalgae might be that Acaryochloris cells tend to adhere to red macroalgae rather than the others.


Figure 2
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  		FIG. 2. Diversity of epiphytes on nine marine macroalgae as revealed by DGGE. PCR amplifications were performed with BAC341F-CYA781R(a) (lanes BCa) and BAC341F-CYA781R(b) (lanes BCb), together with DNAs extracted from nine marine macroalgae: Ahnfeltiopsis flabelliformis (R1), Carpopeltis prolifera (R2), Chondrus ocellatus (R3), Caulacanthus ustulatus (R4), Grateloupia lanceolata (R5), Gloiopeltis furcata (R6), Chondria crassicaulis (R7), Ulva pertusa (G1), and Undaria pinatifida (B1). Band X was an artifact as shown in Fig. 1.

In conclusion, we established a method for the detection of epiphytes on macroalgae using PCR-DGGE. This method and the results obtained in the present study are expected to facilitate a better understanding of the distribution of Acaryochloris spp. in the marine environment and of the epiphyte community on macroalgae.


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ACKNOWLEDGMENTS
 
We thank Tetsuro Ajisaka, Kyoto University, for the identification of macroalgae.

This study was partly supported by the NEDO program Construction of a Genetic Resource Library of Unidentified Microbes Based on Genome Information to H.M. and by the grants-in-aid for scientific research from the Ministry of Education, Sports, Culture, Science, and Technology of Japan to H.M. (grant 17370009) and to M.M. and A.M. (grant 17GS0314). We also acknowledged financial support from the Kansai Research Foundation for Technology Promotion to M.M. and from the Salt Science Research Foundation to A.M.


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FOOTNOTES
 
* Corresponding author. Mailing address: Graduate School of Human and Environmental Studies, Kyoto University, Kyoto 606-8501, Japan. Phone: 81-75-753-7928. Fax: 81-75-753-7928. E-mail: miyashita{at}hm1.mbox.media.kyoto-u.ac.jp. Back

{triangledown} Published ahead of print on 6 October 2006. Back


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Applied and Environmental Microbiology, December 2006, p. 7912-7915, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01148-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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