- Email: [email protected]
Sequence analysis of 16S rRNA gene of cyanobacteria associated with the marine sponge Mycale (Carmia) hentscheli
FEMS Microbiology Letters 207 (2002) 43^47
www.fems-microbiology.org
Sequence analysis of 16S rRNA gene of cyanobacteria associated with the marine sponge Mycale (Carmia) hentscheli Victoria L. Webb a
a;
*, Elizabeth W. Maas
b
National Institute of Water and Atmospheric Research Ltd, P.O. Box 14-901, Kilbirnie, Wellington, New Zealand b Institute of Environmental and Science and Research Ltd., P.O. Box 50-348, Porirua, New Zealand Received 29 August 2001 ; received in revised form 22 November 2001; accepted 27 November 2001 First published online 7 January 2002
Abstract Marine sponges frequently contain a complex mixture of bacteria, fungi, unicellular algae and cyanobacteria. Epifluorescent microscopy showed that Mycale (Carmia) hentscheli contained coccoid cyanobacteria. The 16S rRNA gene was amplified, fragments cloned and analysed using amplified rRNA gene restriction analysis. The nearly complete 16S rRNA gene of distinct clones was sequenced and aligned using ARB. The phylogenetic analysis indicated the presence of four closely related clones which have a high (8%) sequence divergence from known cyanobacteria, Cyanobacterium stanieri being the closest, followed by Prochloron sp. and Synechocystis sp. All belong to the order Chroococcales. The lack of non-molecular evidence prevents us from proposing a new genus. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Sponge; Cyanobacteria; 16S rDNA ; Ampli¢ed rRNA gene restriction analysis
1. Introduction Marine sponges contain a complex mixture of bacteria (both symbiotic and incidental), fungi, unicellular algae and cyanobacteria (also both symbiotic and incidental). In this situation, where a large number of species occur and many species are unculturable, phylogenetic analysis of sequence data from the 16S ribosomal RNA (rRNA) gene provides a powerful tool for the classi¢cation of microbes including cyanobacteria [1,2]. For a number of years cyanobacteria have been the focus of intense molecular studies in the hopes of determining the origins of the higher plant plastid [3^5]. These studies have provided abundant sequence data to the 16S rRNA gene together with phylogenetic analysis, thus providing good material for future comparisons. Sponges provide an ideal habitat for microorganisms including cyanobacteria. They have two distinct layers, the outer ectosome and the inner endosome. It is in the endosome that some sponges also harbour vast numbers of other organisms, particularly bacteria, including cyano-
* Corresponding author. Tel. : +64 (4) 386 0300; Fax: +64 (4) 386 0574. E-mail address : [email protected] (V.L. Webb).
bacteria, and microalgae. The role these organisms play in the life history of the sponge is unclear although some bacteria are certainly symbiotic [6]. Some cyanobacteria have also been classi¢ed as symbiotic with various marine sponges [7,8]. This study aims to determine the phylogenetic a¤liation of the cyanobacteria associated with the marine sponge Mycale (Carmia) hentscheli using sequence data generated to the 16S rRNA with comparisons made to databases through the BLAST programme [1] and ARB (available at www.biol.chemie.tu-muenchen.de/pub/ARB/). 2. Materials and methods 2.1. Preparation of sponge for microscopy and DNA extraction Sponges were collected by scuba diving from New Zealand waters. A 1-cm-cube sample of the sponge M. (C.) hentscheli [9,10] (Porifera, order Poecilosclerida, family Mycalidae) was thoroughly rinsed with sterile ¢ltered seawater to remove surface debris. The sample was aseptically cut into 2-mm-thick slices and incubated in calcium^magnesium-free sea water (CMF-SW) for 25 min on ice. This produced free sponge cells as well as freeing any bacteria
0378-1097 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 5 5 5 - 9
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V.L. Webb, E.W. Maas / FEMS Microbiology Letters 207 (2002) 43^47
and cyanobacteria that were resident within the sponge endosome. The cells were collected by centrifugation at 600Ug for 8 min. They were washed twice with CMFSW, each time being collected by centrifugation as above. Cells were re-suspended in CMF-SW to a ¢nal volume of 650 Wl. The dissociated sponge sample, before centrifugation, was examined for the presence of cyanobacteria using both epi£uorescent and light microscopy. The criteria for con¢rmation of the presence of cyanobacteria follows those described in [11]. Total DNA was extracted from 600 Wl of the cell suspension using BIO 101 FastDNA spin kit (Qbiogene, Carlsbad, CA, USA). The DNA was quanti¢ed against a low-mass DNA ladder (Life Technologies, Rockville, MD, USA) run in a 6% polyacrylamide gel. 2.2. Ampli¢cation of the 16S rRNA gene A 50-ng aliquot of DNA was ampli¢ed using 10 WM of each universal primer to the 16S rRNA of bacteria, cyanobacteria and chloroplast 16S rRNA, PB36: AGR GTT TGA TCM TGG CTC AG (nucleotide positions 8^27 Escherichia coli bp numbering) and PB38 GKT ACC TTG TTA CGA CTT (nucleotide positions R1492^1508 E. coli bp numbering); MgCl2 ¢nal concentration 2.0 mM; 10U bu¡er; 1 mM each dNTP and 1 U Taq Pol (Boehringer Mannheim, Germany). PCR conducted on a Perkin Elmer Cetus had the following pro¢le: 94³C for 3 min; 25 cycles of 94³C for 55 s; 55³C for 45 s; 72³C for 90 s. The expected 1500-bp product was visualised using 6% PAGE and ethidium bromide staining. 2.3. Cloning of the 16S rRNA genes pGEMT vector was used with a vector:insert molar ratio of 3:1. Rapid Ligation Bu¡er (Promega, Madison, WI, USA) was used according to manufacturer's instructions with 1 U Ligase (Promega). Tubes containing samples and suitable controls were incubated overnight at 4³C. Plasmids were transformed into Max e¤ciency competent cells (Life Technologies, Rockville, MD, USA) following manufacturer's protocol and plated onto LB Amp (60 Wg ml31 ) agar. Plates were incubated overnight at 37³C. 2.4. Ampli¢ed rRNA gene restriction analysis (ARDRA) screening of 16S rDNA clones Twenty-four white colonies were randomly picked from the plates and transformants cultured in LB Amp (100 Wg ml31 ) and incubated overnight at 37³C. A 50-Wl aliquot of the overnight culture was added to 100 Wl dH2 O, vortexed and heat-denatured for 20 min at 94³C. A 5-Wl aliquot was used in a 25-ml PCR reaction using 10 mM of each pGEMF and pGEMR primers; 25 mM MgCl2 ; 10U
PCR bu¡er ; 10 mM each dNTP and 1 U Taq Polymerase (Boehringer Mannheim, Germany). The PCR pro¢le was the same as above. Products were run in 1% agarose gels to establish the size of the clones. For ARDRA, the PCR products were digested with the endonuclease HaeIII (Life Technologies, Rockville, MD, USA) at 37³C for 3 h. The restriction fragments were resolved in 1.5% agarose gels and analysed for heterogeneity. 2.5. Sequencing of the 16S rDNA Three clones that had identical HaeIII digestion patterns and ¢ve clones that had unique digestion patterns were re-ampli¢ed in 75 Wl PCR reactions using the pGEMF and pGEMR primers and PCR pro¢le as above. The PCR products were puri¢ed using the PCR Product Puri¢cation Kit (Qiagen, Germany) according to manufacturer's instructions. Puri¢ed products were quanti¢ed using the DyNA Quant 200 system (Hoefer, Pharmacia Biotech, San Francisco, CA, USA). Eight clones were sequenced directly, using a protocol for `Taq Cycle sequencing', using £uorescently labelled dideoxynucleotide `terminators', as per the manufacturer's instructions (PE Biosystems, Foster City, CA, USA), using pGEMF primer and conserved 16S rDNA sequencing primers [12]. 2.6. Phylogeny analysis of cloned 16S rDNA sequence Sequences were initially aligned using BioEdit (available at www.mbio.ncsu.edu.RnaseP/info/programs/BIOEDIT/ bioedit.html) and Clustal W programme (within BioEdit) and determined to be identical or unique. Sequences were compared to the compilation of 16S rRNA gene sequences available in databases using NCIB/BLAST [1] to determine highest similarity to GenBank and EMBL database sequences. The 16S rRNA gene sequence data was aligned with rRNA gene sequences from EMBL/GenBank using evolutionary conserved primary sequence and secondary structure [12]. Evolutionary distances were calculated from sequence pair dissimilarities and phylogenetic relationships were estimated using neighbour-joining with Jukes^Cantor correction [13] using programmes contained in ARB (available at www.biol.chemie.tu-muenchen.de/ pub/arb/). The 16S rDNA sequences determined in this study have been deposited in the EMBL database under accession numbers: AJ292192 (MY2) ; AJ292193 (MY3) ; AJ292194 (MY4) ; AJ292195 (MY17). 3. Results 3.1. Microscopic examination Examination of the dissociated sponge sample, as described in Section 2, using epi£uorescent microscopy revealed the presence of unicellular coccoid organisms. The
FEMSLE 10297 26-2-02
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45
organisms were approximately 4 Wm in diameter and £uoresced in the orange spectrum.
The distance matrix (Table 1 and Fig. 2) shows that the clones are closely related with the di¡erence between the four clones only being in the order of 0.2^0.8% divergence. There are many explanations for microvariations in sequence data and not all indicate the presence of separate species. Analyses were performed using sequences of seven other cyanobacteria. In general, the results obtained by using neighbour-joining showed similar relationships between the positions of our clones in relation to the clusters of known taxa (Fig. 2). The results obtained supported placing the clones in a separate cluster, possibly constituting a new genus within the cyanobacteria (Fig. 2).
3.2. ARDRA, 16S rDNA sequence and phylogenetic analysis
4. Discussion
PCR ampli¢cation, cloning and subsequent sequencing were performed as described in Section 2. For rapid screening, the clones were analysed by HaeIII digestion in agarose gels. Of the 24 clones analysed by ARDRA, ¢ve had unique restriction patterns, while the remaining 19 showed an identical pattern (Fig. 1). A subset (three) of the 19 clones were sequenced as well as the ¢ve unique clones. For sequence analysis, the nearly complete 16S rRNA gene covering variable regions between bases 31 and 1407 (E. coli numbering of 16S rRNA gene) was selected. BLAST search showed that all inserts analysed coded for 16S rRNA segments either from cyanobacteria, eubacteria or chloroplasts. In addition to the three clones (MY2, 3, 4) with identical restriction digest patterns, sequence analysis showed that one other clone (MY17) also had high similarity to the original three clones. BLAST search showed that these four displayed the highest similarity to Cyanobacterium stanieri with approximately 91% similarity followed by approximately 89% similarity to Prochloron sp. and approximately 88% similarity to Synechocystis sp. PCC6803 (Table 1). Of the remaining four unique clones sequenced, one showed sequence similarity to a Chlorophyte. The remaining sequences were to incidental bacteria (not shown).
While the presence of cyanobacteria in sponges has been documented (see Section 1), most have not been identi¢ed. The development of 16S rRNA sequencing techniques has played an important part in unraveling the phylogenetic relationships of microbes when they are found in close association with another organism. To date, the cyanobacteria under study have not been able to be cultured, thus, much of the data so important for classifying cyanobacteria is unavailable. Sequence data to the 16S rRNA has presented the opportunity to place these organisms within the cyanobacterial group. In this study, whole DNA was extracted from the marine sponge M. (C.) hentscheli, which epi£uorescent microscopy showed contained coccoid organisms. The orange £uorescence under UV excitation is consistent with the cells being a cyanobacterium, based on pigment content [11]. Also, the size of approximately 4 Wm in diameter, while putting it above the range for picoplanktic cyanobacteria, is still within the size range of many cyanobacteria [11]. Grouping of the 16S rDNA clones by ARDRA provided an e¤cient way to examine the diversity of microbes present in the sponge endosome. It also provided a useful way to reduce the number of clones committed for further analyses [14]. Thus, of the original 24 clones analysed by
Fig. 1. Image of ARDRA patterns observed on an ethidium bromidestained 1.5% agarose gel. Lanes 2^6, 10 and 14 all show the dominant pattern that was further analysed by sequencing.
Table 1 Percent sequence similarity between a 800-nucleotide-long stretch of the 16S rDNA sequences of clones from M. (C.) hentscheli and the closest related species Clone MY2 Clone MY2 Clone MY3 Clone MY4 Clone MY17
100 99.4
Clone MY3
Clone MY4
Clone MY17
99.4
99.8 99.5
100
99.8
99.5
99.2
99.5
100 99.3
C. stanieri
Cyanothece sp. Prochloron PCC7424 sp.
Symbiont of Climacodium (diatom)
Symploca hydnoides
Synechococcus elongatus
Synechocystis sp. PCC6803
99.2
91.0
87.6
89.4
87.6
87.3
85.0
88.3
99.5
91.3
87.7
89.7
87.9
87.5
85.2
88.6
99.3
91.1
87.7
89.5
87.8
87.3
85.1
88.4
91.1
87.5
89.6
87.7
87.5
85.1
88.6
100
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V.L. Webb, E.W. Maas / FEMS Microbiology Letters 207 (2002) 43^47
Fig. 2. Unrooted phylogenetic tree depicting the phylogenetic position of the clones of M. (C.) hentscheli within the cyanobacteria (error bar equals 1% sequence divergence).
ARDRA, only eight were submitted for sequencing with four of these ¢nally being con¢rmed as belonging to the cyanobacteria. Following the alignment of the sequence data, the clone MY17 was included with the original three as all being highly similar. The sequence divergence between the four clones is small (0.8^0.2%, Table 1 and Fig. 2) and may be due to a number of factors. A number of studies [15,16] have reported the occurrence of closely related sequences in phylogenetic studies of bacterial communities, while others [17] suggest that the di¡erences may stem from clonal variation within microbial populations, gene families or may represent true subspecies. Another study [18] discusses the possibility of small sequence heterogeneity being contained within multiple rRNA operons. Other data [19] suggests small variations can be introduced by PCR and cloning. However, radiations of closely related bacterial populations that co-exist in relatively closed microenvironments have been observed in other studies [15,18,20], and a similar state may also occur for cyanobacteria populations. Phylogenetic analysis indicates the three species closest to the clones are C. stanieri, Prochloron sp. and Synechocystis sp. PCC6803 (Fig. 2). These cyanobacteria are all members of the order Chroococcales [21]. Members of this order can be free living or endosymbiotic as in Prochloron sp. [22] and inhabit a range of environments including freshwater and marine [21]. In addition, most of the order are considered [11] to be picoplanktic, although one group, the Cyanothece, have individuals larger than 3 Wm in diameter. Thus, while cytomorphological and ecophysiological data are not available for the cluster of clones, it appears likely that they belong in the order Chroococcales of the cyanobacteria. The high degree of sequence divergence from other know cyanobacteria suggests the organisms under study should be assigned to a new genus. However, the lack of non-molecular evidence prevents the proposal of a new genus for this organism at this time.
Acknowledgements We thank Susan Turner for technical help and the use of her laboratory and Peter Berquist for information on primers. The authors would like to thank Michelle KellyShanks for the taxonomic identi¢cation of the sponge. This work was supported by funding from the Foundation of Research, Science and Technology (CO1X0001).
References [1] Altschul, S., Madden, T.L., Scha¡er, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI-Blast: a new generation of protein database search programms. Nucleic Acids Res. 25, 3389^3402. [2] Woese, C. (1987) Bacterial evolution. Microbiol. Rev. 51, 221^271. [3] Palenik, B. and Haselkorn, R. (1992) Multiple evolutionary origins of prochlorophytes, the chlorophyll b-containing prokaryotes. Nature 355, 265^267. [4] Urbach, E., Robertson, D.L. and Chisholm, S.W. (1992) Multiple evolutionary origins of prochlorophytes within the cyanobacterial radiation. Nature 355, 267^270. [5] Turner, S., Pryer, K.M. and Miao, V.P.W. (1999) Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis. J. Eukaryot. Microbiol. 46, 327^ 338. [6] Altho¡, K., Schutt, C. and Batal, R. (1998) Evidence for a symbiosis between bacteria of the genus Rhodobacter and the marine sponge Halichondria panicea: harbor also for putatively toxic bacteria ? Mar. Biol. 130, 529^536. [7] Unson, M. and Faulkner, D. (1993) Cyanobacterial symbiont biosynthesis of chlorinated metabolites from Dysidea herbacea (Porifera). Experientia 49, 349^353. [8] Bewley, C., Holland, N. and Faulkner, D. (1996) Two classes of metabolites from Theonella swinhoei are localised in bacterial symbionts. Experientia 52, 716^722. [9] Gray, J.E. (1867) Notes on the arrangement of sponges, with the description of some new genera. Proc. Zool. Soc. Lond., 492^558. [10] Bergquist, P.R. and Fromont, P.J. (1988) The marine fauna of New Zealand : Porifera, Demospongiae, Part 4 (Poecilosclerida), New Zealand Oceanographic Institute Memoir 96. [11] Komarek, J. (1996) Towards a combined approach for the taxonomy
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[12]
[13]
[14]
[15]
[16]
[17]
and species delimitation of picoplanktic cyanoprokaryotes. Arch. Hydrobiol. Suppl. Algol. Stud. 83, 377^401. Lane, D.J. (1991) 16S/23S rRNA Sequencing. In: Nucleic Acid Techniques in Bacterial Systematics (Stackebrandt, E. and Goodfellow, M. Eds.), pp. 115^175. John Wiley and Sons Ltd., Chichester. Jukes, T. and Cantor, C.R. (1969) Evolution of protein molecules. In: Mammalian Protein Metabolism (Munro, H., Ed.), pp. 21^132. Academic Press, New York. Vaneechoutte, M., Rossau, R., De Vos, P., Gillis, M., Janssens, D., Paepe, N., De Rouck, A., Fiers, T., Claeys, G. and Fersters, K. (1992) Rapid identi¢cation of bacteria of the Comamonadaceae with ampli¢ed ribosomal DNA-restriction analysis (ARDRA). FEMS Microbiol. Lett. 39, 227^234. Giovannoni, S., Britschgi, T.B., Moyer, C.L. and Field, K.G. (1990) Genetic diversity in Sargasso Sea bacterioplankton. Nature 345, 60^ 63. Liesack, W. and Stackebrandt, E. (1992) Occurrence of novel groups of the domain Bacteria as revealed by analysis of genetic material isolated from an Australian terrestrial environment. J. Bacteriol. 174, 5072^5078. Mullins, T., Britschgi, T.B., Krest, R.L. and Giovannoni, S.J. (1995)
[18]
[19]
[20]
[21]
[22]
47
Genetic comparisons reveal the same unknown bacterial lineages in Atlantic and Paci¢c bacterioplankton communities. Limnol. Oceanogr. 40, 148^158. Fuhrman, J., McCallum, K. and Davis, A.A. (1993) Phylogenetic diversity of subsurface marine microbial communities from the Atlantic and Paci¢c oceans. Appl. Environ. Microbiol. 59, 1294^1302. Speksnijder, A., Kowalchuk, G.A., De Long, S., Kline, E., Stephen, J.R. and Laanbroek, H.J. (2001) Microvariation artifacts introduced by PCR and cloning of closely related 16S rRNA gene sequences. Appl. Environ. Microbiol. 67, 469^472. Acinas, S., Anton, J. and Rodriguez-Valera, F. (1999) Diversity of free-living and attached bacteria in o¡shore western Mediterranean waters as depicted by analysis of genes encoding 16S rRNA. Appl. Environ. Microbiol. 65, 514^522. Waterbury, J., and Rippka, R (1989) Subsection I, order Chroococcales Wettstein 1924, emend. Rippka et al., 1979. In: Bergey's Manual of Systematic Bacteriology (Stanley, J., Bryant, J.T., Pfennig, N. and Holt, J.G, Eds.), pp. 1746^1770. Williams and Wilkins, Baltimore, MD. Lewin, R. (1975) A marine Synechocystis (Cyanophyta, Chroococcales) epizoic on ascidians. Phycologia 14, 153^160.
FEMSLE 10297 26-2-02
www.fems-microbiology.org
Sequence analysis of 16S rRNA gene of cyanobacteria associated with the marine sponge Mycale (Carmia) hentscheli Victoria L. Webb a
a;
*, Elizabeth W. Maas
b
National Institute of Water and Atmospheric Research Ltd, P.O. Box 14-901, Kilbirnie, Wellington, New Zealand b Institute of Environmental and Science and Research Ltd., P.O. Box 50-348, Porirua, New Zealand Received 29 August 2001 ; received in revised form 22 November 2001; accepted 27 November 2001 First published online 7 January 2002
Abstract Marine sponges frequently contain a complex mixture of bacteria, fungi, unicellular algae and cyanobacteria. Epifluorescent microscopy showed that Mycale (Carmia) hentscheli contained coccoid cyanobacteria. The 16S rRNA gene was amplified, fragments cloned and analysed using amplified rRNA gene restriction analysis. The nearly complete 16S rRNA gene of distinct clones was sequenced and aligned using ARB. The phylogenetic analysis indicated the presence of four closely related clones which have a high (8%) sequence divergence from known cyanobacteria, Cyanobacterium stanieri being the closest, followed by Prochloron sp. and Synechocystis sp. All belong to the order Chroococcales. The lack of non-molecular evidence prevents us from proposing a new genus. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Sponge; Cyanobacteria; 16S rDNA ; Ampli¢ed rRNA gene restriction analysis
1. Introduction Marine sponges contain a complex mixture of bacteria (both symbiotic and incidental), fungi, unicellular algae and cyanobacteria (also both symbiotic and incidental). In this situation, where a large number of species occur and many species are unculturable, phylogenetic analysis of sequence data from the 16S ribosomal RNA (rRNA) gene provides a powerful tool for the classi¢cation of microbes including cyanobacteria [1,2]. For a number of years cyanobacteria have been the focus of intense molecular studies in the hopes of determining the origins of the higher plant plastid [3^5]. These studies have provided abundant sequence data to the 16S rRNA gene together with phylogenetic analysis, thus providing good material for future comparisons. Sponges provide an ideal habitat for microorganisms including cyanobacteria. They have two distinct layers, the outer ectosome and the inner endosome. It is in the endosome that some sponges also harbour vast numbers of other organisms, particularly bacteria, including cyano-
* Corresponding author. Tel. : +64 (4) 386 0300; Fax: +64 (4) 386 0574. E-mail address : [email protected] (V.L. Webb).
bacteria, and microalgae. The role these organisms play in the life history of the sponge is unclear although some bacteria are certainly symbiotic [6]. Some cyanobacteria have also been classi¢ed as symbiotic with various marine sponges [7,8]. This study aims to determine the phylogenetic a¤liation of the cyanobacteria associated with the marine sponge Mycale (Carmia) hentscheli using sequence data generated to the 16S rRNA with comparisons made to databases through the BLAST programme [1] and ARB (available at www.biol.chemie.tu-muenchen.de/pub/ARB/). 2. Materials and methods 2.1. Preparation of sponge for microscopy and DNA extraction Sponges were collected by scuba diving from New Zealand waters. A 1-cm-cube sample of the sponge M. (C.) hentscheli [9,10] (Porifera, order Poecilosclerida, family Mycalidae) was thoroughly rinsed with sterile ¢ltered seawater to remove surface debris. The sample was aseptically cut into 2-mm-thick slices and incubated in calcium^magnesium-free sea water (CMF-SW) for 25 min on ice. This produced free sponge cells as well as freeing any bacteria
0378-1097 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 5 5 5 - 9
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V.L. Webb, E.W. Maas / FEMS Microbiology Letters 207 (2002) 43^47
and cyanobacteria that were resident within the sponge endosome. The cells were collected by centrifugation at 600Ug for 8 min. They were washed twice with CMFSW, each time being collected by centrifugation as above. Cells were re-suspended in CMF-SW to a ¢nal volume of 650 Wl. The dissociated sponge sample, before centrifugation, was examined for the presence of cyanobacteria using both epi£uorescent and light microscopy. The criteria for con¢rmation of the presence of cyanobacteria follows those described in [11]. Total DNA was extracted from 600 Wl of the cell suspension using BIO 101 FastDNA spin kit (Qbiogene, Carlsbad, CA, USA). The DNA was quanti¢ed against a low-mass DNA ladder (Life Technologies, Rockville, MD, USA) run in a 6% polyacrylamide gel. 2.2. Ampli¢cation of the 16S rRNA gene A 50-ng aliquot of DNA was ampli¢ed using 10 WM of each universal primer to the 16S rRNA of bacteria, cyanobacteria and chloroplast 16S rRNA, PB36: AGR GTT TGA TCM TGG CTC AG (nucleotide positions 8^27 Escherichia coli bp numbering) and PB38 GKT ACC TTG TTA CGA CTT (nucleotide positions R1492^1508 E. coli bp numbering); MgCl2 ¢nal concentration 2.0 mM; 10U bu¡er; 1 mM each dNTP and 1 U Taq Pol (Boehringer Mannheim, Germany). PCR conducted on a Perkin Elmer Cetus had the following pro¢le: 94³C for 3 min; 25 cycles of 94³C for 55 s; 55³C for 45 s; 72³C for 90 s. The expected 1500-bp product was visualised using 6% PAGE and ethidium bromide staining. 2.3. Cloning of the 16S rRNA genes pGEMT vector was used with a vector:insert molar ratio of 3:1. Rapid Ligation Bu¡er (Promega, Madison, WI, USA) was used according to manufacturer's instructions with 1 U Ligase (Promega). Tubes containing samples and suitable controls were incubated overnight at 4³C. Plasmids were transformed into Max e¤ciency competent cells (Life Technologies, Rockville, MD, USA) following manufacturer's protocol and plated onto LB Amp (60 Wg ml31 ) agar. Plates were incubated overnight at 37³C. 2.4. Ampli¢ed rRNA gene restriction analysis (ARDRA) screening of 16S rDNA clones Twenty-four white colonies were randomly picked from the plates and transformants cultured in LB Amp (100 Wg ml31 ) and incubated overnight at 37³C. A 50-Wl aliquot of the overnight culture was added to 100 Wl dH2 O, vortexed and heat-denatured for 20 min at 94³C. A 5-Wl aliquot was used in a 25-ml PCR reaction using 10 mM of each pGEMF and pGEMR primers; 25 mM MgCl2 ; 10U
PCR bu¡er ; 10 mM each dNTP and 1 U Taq Polymerase (Boehringer Mannheim, Germany). The PCR pro¢le was the same as above. Products were run in 1% agarose gels to establish the size of the clones. For ARDRA, the PCR products were digested with the endonuclease HaeIII (Life Technologies, Rockville, MD, USA) at 37³C for 3 h. The restriction fragments were resolved in 1.5% agarose gels and analysed for heterogeneity. 2.5. Sequencing of the 16S rDNA Three clones that had identical HaeIII digestion patterns and ¢ve clones that had unique digestion patterns were re-ampli¢ed in 75 Wl PCR reactions using the pGEMF and pGEMR primers and PCR pro¢le as above. The PCR products were puri¢ed using the PCR Product Puri¢cation Kit (Qiagen, Germany) according to manufacturer's instructions. Puri¢ed products were quanti¢ed using the DyNA Quant 200 system (Hoefer, Pharmacia Biotech, San Francisco, CA, USA). Eight clones were sequenced directly, using a protocol for `Taq Cycle sequencing', using £uorescently labelled dideoxynucleotide `terminators', as per the manufacturer's instructions (PE Biosystems, Foster City, CA, USA), using pGEMF primer and conserved 16S rDNA sequencing primers [12]. 2.6. Phylogeny analysis of cloned 16S rDNA sequence Sequences were initially aligned using BioEdit (available at www.mbio.ncsu.edu.RnaseP/info/programs/BIOEDIT/ bioedit.html) and Clustal W programme (within BioEdit) and determined to be identical or unique. Sequences were compared to the compilation of 16S rRNA gene sequences available in databases using NCIB/BLAST [1] to determine highest similarity to GenBank and EMBL database sequences. The 16S rRNA gene sequence data was aligned with rRNA gene sequences from EMBL/GenBank using evolutionary conserved primary sequence and secondary structure [12]. Evolutionary distances were calculated from sequence pair dissimilarities and phylogenetic relationships were estimated using neighbour-joining with Jukes^Cantor correction [13] using programmes contained in ARB (available at www.biol.chemie.tu-muenchen.de/ pub/arb/). The 16S rDNA sequences determined in this study have been deposited in the EMBL database under accession numbers: AJ292192 (MY2) ; AJ292193 (MY3) ; AJ292194 (MY4) ; AJ292195 (MY17). 3. Results 3.1. Microscopic examination Examination of the dissociated sponge sample, as described in Section 2, using epi£uorescent microscopy revealed the presence of unicellular coccoid organisms. The
FEMSLE 10297 26-2-02
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45
organisms were approximately 4 Wm in diameter and £uoresced in the orange spectrum.
The distance matrix (Table 1 and Fig. 2) shows that the clones are closely related with the di¡erence between the four clones only being in the order of 0.2^0.8% divergence. There are many explanations for microvariations in sequence data and not all indicate the presence of separate species. Analyses were performed using sequences of seven other cyanobacteria. In general, the results obtained by using neighbour-joining showed similar relationships between the positions of our clones in relation to the clusters of known taxa (Fig. 2). The results obtained supported placing the clones in a separate cluster, possibly constituting a new genus within the cyanobacteria (Fig. 2).
3.2. ARDRA, 16S rDNA sequence and phylogenetic analysis
4. Discussion
PCR ampli¢cation, cloning and subsequent sequencing were performed as described in Section 2. For rapid screening, the clones were analysed by HaeIII digestion in agarose gels. Of the 24 clones analysed by ARDRA, ¢ve had unique restriction patterns, while the remaining 19 showed an identical pattern (Fig. 1). A subset (three) of the 19 clones were sequenced as well as the ¢ve unique clones. For sequence analysis, the nearly complete 16S rRNA gene covering variable regions between bases 31 and 1407 (E. coli numbering of 16S rRNA gene) was selected. BLAST search showed that all inserts analysed coded for 16S rRNA segments either from cyanobacteria, eubacteria or chloroplasts. In addition to the three clones (MY2, 3, 4) with identical restriction digest patterns, sequence analysis showed that one other clone (MY17) also had high similarity to the original three clones. BLAST search showed that these four displayed the highest similarity to Cyanobacterium stanieri with approximately 91% similarity followed by approximately 89% similarity to Prochloron sp. and approximately 88% similarity to Synechocystis sp. PCC6803 (Table 1). Of the remaining four unique clones sequenced, one showed sequence similarity to a Chlorophyte. The remaining sequences were to incidental bacteria (not shown).
While the presence of cyanobacteria in sponges has been documented (see Section 1), most have not been identi¢ed. The development of 16S rRNA sequencing techniques has played an important part in unraveling the phylogenetic relationships of microbes when they are found in close association with another organism. To date, the cyanobacteria under study have not been able to be cultured, thus, much of the data so important for classifying cyanobacteria is unavailable. Sequence data to the 16S rRNA has presented the opportunity to place these organisms within the cyanobacterial group. In this study, whole DNA was extracted from the marine sponge M. (C.) hentscheli, which epi£uorescent microscopy showed contained coccoid organisms. The orange £uorescence under UV excitation is consistent with the cells being a cyanobacterium, based on pigment content [11]. Also, the size of approximately 4 Wm in diameter, while putting it above the range for picoplanktic cyanobacteria, is still within the size range of many cyanobacteria [11]. Grouping of the 16S rDNA clones by ARDRA provided an e¤cient way to examine the diversity of microbes present in the sponge endosome. It also provided a useful way to reduce the number of clones committed for further analyses [14]. Thus, of the original 24 clones analysed by
Fig. 1. Image of ARDRA patterns observed on an ethidium bromidestained 1.5% agarose gel. Lanes 2^6, 10 and 14 all show the dominant pattern that was further analysed by sequencing.
Table 1 Percent sequence similarity between a 800-nucleotide-long stretch of the 16S rDNA sequences of clones from M. (C.) hentscheli and the closest related species Clone MY2 Clone MY2 Clone MY3 Clone MY4 Clone MY17
100 99.4
Clone MY3
Clone MY4
Clone MY17
99.4
99.8 99.5
100
99.8
99.5
99.2
99.5
100 99.3
C. stanieri
Cyanothece sp. Prochloron PCC7424 sp.
Symbiont of Climacodium (diatom)
Symploca hydnoides
Synechococcus elongatus
Synechocystis sp. PCC6803
99.2
91.0
87.6
89.4
87.6
87.3
85.0
88.3
99.5
91.3
87.7
89.7
87.9
87.5
85.2
88.6
99.3
91.1
87.7
89.5
87.8
87.3
85.1
88.4
91.1
87.5
89.6
87.7
87.5
85.1
88.6
100
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V.L. Webb, E.W. Maas / FEMS Microbiology Letters 207 (2002) 43^47
Fig. 2. Unrooted phylogenetic tree depicting the phylogenetic position of the clones of M. (C.) hentscheli within the cyanobacteria (error bar equals 1% sequence divergence).
ARDRA, only eight were submitted for sequencing with four of these ¢nally being con¢rmed as belonging to the cyanobacteria. Following the alignment of the sequence data, the clone MY17 was included with the original three as all being highly similar. The sequence divergence between the four clones is small (0.8^0.2%, Table 1 and Fig. 2) and may be due to a number of factors. A number of studies [15,16] have reported the occurrence of closely related sequences in phylogenetic studies of bacterial communities, while others [17] suggest that the di¡erences may stem from clonal variation within microbial populations, gene families or may represent true subspecies. Another study [18] discusses the possibility of small sequence heterogeneity being contained within multiple rRNA operons. Other data [19] suggests small variations can be introduced by PCR and cloning. However, radiations of closely related bacterial populations that co-exist in relatively closed microenvironments have been observed in other studies [15,18,20], and a similar state may also occur for cyanobacteria populations. Phylogenetic analysis indicates the three species closest to the clones are C. stanieri, Prochloron sp. and Synechocystis sp. PCC6803 (Fig. 2). These cyanobacteria are all members of the order Chroococcales [21]. Members of this order can be free living or endosymbiotic as in Prochloron sp. [22] and inhabit a range of environments including freshwater and marine [21]. In addition, most of the order are considered [11] to be picoplanktic, although one group, the Cyanothece, have individuals larger than 3 Wm in diameter. Thus, while cytomorphological and ecophysiological data are not available for the cluster of clones, it appears likely that they belong in the order Chroococcales of the cyanobacteria. The high degree of sequence divergence from other know cyanobacteria suggests the organisms under study should be assigned to a new genus. However, the lack of non-molecular evidence prevents the proposal of a new genus for this organism at this time.
Acknowledgements We thank Susan Turner for technical help and the use of her laboratory and Peter Berquist for information on primers. The authors would like to thank Michelle KellyShanks for the taxonomic identi¢cation of the sponge. This work was supported by funding from the Foundation of Research, Science and Technology (CO1X0001).
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