Abstract
The relative abundance of bacteria in the mucus and crushed tissue of the Mediterranean coral Oculina patagonica was determined by analyses of the 16S rRNA genes of isolated colonies and from a 16S rRNA clone library of extracted DNA. By SYBR gold staining, the numbers of bacteria in mucus and tissue samples were 6.2 × 107 and 8.3 × 108/cm2 of coral surface, respectively, 99.8% of which failed to produce colonies on Marine Agar. From analysis of mucus DNA, the most-abundant bacterium was Vibrio splendidus, representing 68% and 50% of the clones from the winter and summer, respectively. After removal of mucus from coral by centrifugation, analyses of DNA from the crushed tissue revealed a large diversity of bacteria, with Vibrio species representing less than 5% of the clones. The most-abundant culturable bacteria were a Pseudomonas sp. (8 to 14%) and two different α-proteobacteria (6 to 18%). Out of a total 1,088 16S rRNA genes sequenced, 400 different operational taxonomic units were identified (>99.5% identity). Of these, 295 were novel (<99% identical to any sequences in the GenBank database). This study provides a comprehensive database for future examinations of changes in the bacterial community during bleaching events.
Corals consist of symbioses among the coral animal, endosymbiotic algae commonly referred to as zooxanthellae, and a large and diverse community of associated bacteria. In recent years, there have been numerous investigations of the different clones of zooxanthellae present in corals and their possible role in the sensitivity of corals to temperature-induced bleaching (3). Early studies using culturing methods demonstrated the importance of coral-associated bacteria in coral nutrition (24) and in response to stress (8). It was also shown that the bacterial community changes when corals are bleached (19) or exhibit white-band disease (17).
The first culture-independent studies of coral-associated bacteria demonstrated very high diversity, including a majority of novel species, and host specificity; i.e., similar bacterial populations were found on the same coral species that were geographically separated, and different populations were found on different coral species (20, 21). Coral-associated archaea have also been demonstrated by culture-independent methods (28). Recently, Bourne and Munn (7) used both culture-based and culture-independent techniques to investigate the microbial community of the reef-building coral Pocillopora damicornis. They found that the majority of clones obtained from coral tissue slurry libraries were γ-proteobacteria, whereas the coral mucus was dominated by α-proteobacteria. Many of the retrieved clone sequences were conserved between coral colonies, further supporting the hypothesis of specific bacterium-coral associations.
We report here a study of the bacterial community of the Mediterranean coral Oculina patagonica during the summer and winter. One of the unusual characteristics of O. patagonica is its ability to grow under highly variable conditions of temperature and salinity. Off the coast of Israel, seawater temperatures vary from 31°C (summer maximum) to 16°C (winter minimum). In shallow pools, where coral colonies are found, temperatures can reach 41°C and salinity can fluctuate between 2.8 and 5%. The ease of obtaining genetically identical coral fragments from the same colony and maintaining them in aquaria makes O. patagonica an excellent experimental coral model system (10).
The Vibrio shilonii-O. patagonica model system of bacterial bleaching of corals has been studied extensively (22). Each summer, at least for the last 12 years, approximately 70% of the coral colonies have shown bleaching. The causative agent of this bleaching is V. shilonii (13, 14). This pathogen is chemotactic to the coral mucus (4), binds to a β-galactoside-containing receptor in the coral mucus (27), and then penetrates the epidermal layer of the coral, where it multiplies, reaching >108 bacteria per cm3 of tissue (6). V. shilonii produces a proline-rich toxin (PYPVYAPPPVVP) which inhibits photosynthesis of the intracellular zooxanthellae (5). In the winter, when seawater temperatures drop below 20°C, V. shilonii cannot survive in the coral and the coral recovers (11). Information on the bacterial community of O. patagonica should be useful in understanding its sensitivity to infection and its ability to survive bleaching during the summer months and recover during the winter.
MATERIALS AND METHODS
Sample collection and mucus extraction.
The site sampled in this study was Sedot Yam, in the Mediterranean off the coast of Israel. Three healthy O. patagonica fragments were removed from separate colonies during September 2004 (summer, 27°C) and March 2005 (winter, 17°C) with a hammer and chisel. The colonies were located at 1- to 3-m depths and were separated by distances of at least 3 m. The samples were immediately placed in plastic bags underwater and were transported back to the laboratory in a cooler (in <2 h). The corals were then broken into pieces measuring ca. 2 by 2 cm and placed in 50-ml centrifuge tubes. The coral samples were centrifuged for 3 min at 2,675 × g to remove the mucus. After centrifugation, the coral samples were removed and placed in new 50-ml centrifuge tubes, which were allowed to stay for 60 min at 4°C. The coral samples were then centrifuged again to remove the newly secreted mucus, at 2,675 × g for 3 min. After the second extraction, the coral pieces were crushed in seawater with a mortar and pestle. After allowing the CaCO3 skeleton to settle, the supernatant was removed and is referred to as crushed tissue.
Isolation and enumeration of bacteria.
Tenfold serial dilutions of coral mucus and crushed coral pieces in filtered seawater were prepared and plated on Marine Agar 2216 (Difco, Detroit, MI). All plates were incubated at 30°C for a week. Colonies that appeared at the highest dilutions were restreaked and subsequently used for DNA isolation. Total bacterial counts were performed on coral mucus and crushed coral pieces with SYBR gold (Molecular Probes Inc.) according to the SYBR green protocol (16).
DNA extraction and PCR amplification of 16S rRNA genes.
Coral mucus, crushed tissue, and seawater samples were centrifuged at 9,300 × g for 15 min, and the pellets were used for DNA extraction. DNA was extracted from the pellets with the UltraClean Soil DNA kit (MoBio, Carlsbad, CA). For culturable bacteria, colony DNA was extracted with the GenElute bacterial genomic DNA kit (Sigma). Primers 8F and 1492R (15) were used for amplification of the 16S rRNA genes from isolated bacterial genomic DNA and environmentally extracted DNA from seawater, coral mucus, and crushed tissue samples. 16S rRNA genes were amplified in a 50-μl reaction mixture consisting of 5 μl of 10× buffer, 1 μl of a 2.5 mM total deoxynucleoside triphosphate mixture, each primer at 5 μM, 10 ng of template DNA, and 2.5 U of Ex Taq DNA polymerase (TaKaRa Bio Inc., Shiga, Japan). Amplification conditions for the PCR included an initial denaturation step of 95°C for 3 min, followed by 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min and a final extension step of 72°C for 10 min. Reaction products were checked for size and purity on 1% agarose gel. PCR products were cleaned with ExoSAP-IT (USB Corp.). DNA sequencing was performed by the chain termination method in an ABI Prism (model 377, version 2.1.1) automated sequencer. Primers used for the sequencing reaction were complementary to the conserved regions of the 16S rRNA genes.
Clone library construction.
Amplified DNA from water, mucus, and crushed coral samples was ligated into the pGEM-T Easy vector by the protocol of the manufacturer (Promega Corp., Madison, WI). The ligated vector and insert were transformed into competent Escherichia coli 12S cells. Each clone was amplified by colony PCR with M13 forward and reverse primers. Amplification conditions for the colony PCR included an initial denaturation step of 95°C for 4.5 min, followed by 30 cycles of 95°C for 0.5 min, 59.5°C for 0.5 min, and 72°C for 1 min and a final extension step of 72°C for 10 min. Reaction products were checked for size and purity on 1% agarose gel. Purification and sequencing were done according to the protocol described above.
Sequence analysis.
Sequences were aligned with ClustalX (26), and a DNA distance matrix was created with BioEdit. Sequences that had >99.5% identity were clustered together with DOTUR (23). Sequences shorter than 350 bp were removed from the alignment. BLASTN (2) (http://www.ncbi.nlm.nih.gov/BLAST/BLAST.cgi) was then used to characterize each sequence cluster.
Nucleotide sequence accession numbers.
The nucleotide sequence data for all of the clones and colonies reported in this paper will appear in the GenBank nucleotide sequence database under accession numbers DQ416209 to DQ416683.
RESULTS
Bacteria associated with O. patagonica during the winter.
Three coral fragments were obtained from different O. patagonica colonies in the winter of 2005. Before isolation of DNA, the coral mucus from each fragment was separated from the remainder of the coral by centrifugation. Microscopic observations indicated that the coral tissue was not damaged by the centrifugation procedure. The average volume of mucus ± the standard error was 0.21 ± 0.02 ml/cm2 of coral surface. Thus, the average thickness of the mucus was 1 mm. Following SYBR gold staining, the average numbers of bacteria in the coral mucus and tissue (coral minus mucus) were (6.2 ± 4.2) × 107 and (8.3 ± 1.6) × 108/cm2, respectively. Thus, the concentration of bacteria in the mucus was 3.0 × 108/ml.
Table 1 summarizes the data on the most-abundant 16S rRNA gene sequences obtained from the clone libraries of O. patagonica winter DNA. Clones which matched known Vibrio species dominated the mucus, representing more than 68% of the clones sequenced. Clusters 1, 2, and 4, which closely matched Vibrio splendidus strains, made up 63% of the mucus clones. Despite their higher abundance in the mucus, Vibrio species made up only a small percentage of the tissue bacteria, less than 5%. In general, the tissue bacteria showed much greater diversity than the mucus bacteria. Of the 253 sequences analyzed, 112 or 44% appeared in 1 of the 10 clusters shown in Table 1. The remaining sequences appeared only once (127 times) or twice (7 times). Thus, the number of different bacterial operational taxonomic units (OTUs) sequenced was 144 (by using >99.5% identity to define an OTU). Of these 144, 129 were novel (by using <99% identity to sequences in the GenBank database to define a novel OTU).
TABLE 1.
Most-abundant bacterial clusters associated with O. patagonica in winter 2005a
| Clusterb or parameter | Class | Closest match in Blast (% identity)c | No. of clones | Abundance (% of)
|
||
|---|---|---|---|---|---|---|
| Total | Mucusd | Tissued | ||||
| 1 | γ-Proteobacteria | V. splendidus AJ874360 (100) | 72 | 28.5 | 51.1 | 4.1 |
| 2 | γ-Proteobacteria | V. splendidus biovar II AB038030 (99) | 11 | 4.3 | 8.4 | 0 |
| 3 | γ-Proteobacteria | Sulfur-oxidizing bacterium AF170424 (97) | 5 | 2.0 | 0 | 4.1 |
| 4 | γ-Proteobacteria | V. splendidus strain B17 AY046955 (100) | 5 | 2.0 | 3.8 | 0 |
| 5 | γ-Proteobacteria | Uncultured γ-proteobacterium AF424056 (98) | 3 | 1.2 | 1.5 | 0.8 |
| 6 | γ-Proteobacteria | V. litoralis DQ097524 (97) | 3 | 1.2 | 2.3 | 0 |
| 7 | γ-Proteobacteria | V. superstes AF519806 (100) | 3 | 1.2 | 2.3 | 0 |
| 8 | α-Proteobacteria | α-Proteobacterium AJ890099 (97) | 3 | 1.2 | 0 | 2.5 |
| 9 | Nitrospira | Uncultured isolate A93 AY373422 (96) | 4 | 1.6 | 1.5 | 1.6 |
| 10 | Unclassified proteobacteria | Uncultured isolate T26-22 AF332308 (90) | 3 | 1.2 | 0.0 | 2.5 |
| Total in the 10 clusters | 112 | 44 | 71 | 16 | ||
Based on analysis of 253 16S rRNA clones.
A cluster is defined here as 16S rRNA clones that have at least 99.5% sequence identity.
The consensus sequence of the cluster was used in searching the GenBank database.
Mucus bacteria were separated from tissue bacteria by centrifugation (see Materials and Methods).
The same winter coral mucus and tissue fractions that were used to prepare the clone libraries were also used to determine culturable bacteria (Table 2). The viable counts of the mucus and tissue fractions were (1.6 ± 0.8) × 105 and (9.6 ± 4.2) × 105 CFU/cm2 of coral surface, respectively. These values are ca. 0.2% of the total count. The 20 most-abundant clusters made up 76% of the total culturable bacteria (Table 2). The most-abundant culturable bacteria were very different from the nonculturable bacteria from the same source. For example, none of the eight most-abundant clusters shown in Table 2 were found even once in the 253 clones sequenced from the 16S rRNA gene clone library. Interestingly, two clusters (Table 2, clusters 2 and 5) matching V. splendidus were found in the culturable bacteria, although at much lower frequency than in the nonculturable bacteria. The colonies not shown in Table 2 consisted of 39 sequences that appeared only once and 12 sequences that appeared twice. Thus, the total number of different culturable clusters was 71 out of the 263 colonies analyzed. Of the 71, 39 were novel species (<99% identity to bacterial 16S rRNA gene sequences in the GenBank database).
TABLE 2.
Most-abundant cultured bacterial species associated with O. patagonica in winter 2005a
| Clusterb or parameter | Class | Closest match in Blast (% identity)c | No. of colonies | Abundance (% of)
|
||
|---|---|---|---|---|---|---|
| Total | Mucusd | Tissued | ||||
| 1 | γ-Proteobacteria | Pseudomonas sp. AF500211 (99) | 21 | 8.0 | 4 | 11.7 |
| 2 | γ-Proteobacteria | V. splendidus AY046955 (99) | 4 | 1.5 | 2.4 | 0.7 |
| 3 | γ-Proteobacteria | V. litoralis DQ097524 (99) | 3 | 1.1 | 2.4 | 0 |
| 4 | γ-Proteobacteria | V. superstes AF519806 (99) | 3 | 1.1 | 4.8 | 0 |
| 5 | γ-Proteobacteria | V. splendidus biovar II AB038030 (99) | 3 | 1.1 | 1.6 | 0.7 |
| 6 | γ-Proteobacteria | Vibrio sp. AF456335 (100) | 3 | 1.1 | 2.4 | 0 |
| 7 | γ-Proteobacteria | Uncultured isolate PDA-OTU4 AY700602 (99) | 3 | 1.1 | 2.4 | 0 |
| 8 | γ-Proteobacteria | Photobacterium sp. AY576761 (99) | 3 | 1.1 | 2.4 | 0 |
| 9 | α-Proteobacteria | α-Proteobacterium AB026194 (99) | 48 | 18.3 | 20.6 | 16.1 |
| 10 | α-Proteobacteria | α-Proteobacterium AY584527 (99) | 33 | 12.5 | 6.3 | 18.3 |
| 11 | α-Proteobacteria | α-Proteobacterium AY562560 (100) | 21 | 8.0 | 5.6 | 10.2 |
| 12 | α-Proteobacteria | Vibrio sp. DQ146982 (99) | 19 | 7.2 | 10.3 | 4.4 |
| 13 | α-Proteobacteria | Pseudovibrio sp. AB112827 (99) | 7 | 2.7 | 1.6 | 3.7 |
| 14 | α-Proteobacteria | Uncultured isolate 20BSU27 AJ863180 (99) | 6 | 2.3 | 4.8 | 0 |
| 15 | α-Proteobacteria | Roseobacter sp. AY349460 (99) | 5 | 1.9 | 0.8 | 2.9 |
| 16 | α-Proteobacteria | α-Proteobacterium AY576758 (98) | 4 | 1.5 | 3.2 | 0 |
| 17 | α-Proteobacteria | Uncultured isolate 4%N-26d-5 AF432337 (99) | 3 | 1.1 | 1.6 | 0.7 |
| 18 | α-Proteobacteria | α-Proteobacterium AB015896 (99) | 3 | 1.1 | 2.4 | 0 |
| 19 | Flavobacteria | Muricauda aquimarina AY445076 (97) | 4 | 1.5 | 0 | 2.9 |
| 20 | Sphingobacteria | Bacterial isolate DG1129 AY258133 (97) | 4 | 1.5 | 0 | 2.9 |
| Total in 20 clusters | 200 | 76 | 80 | 75 | ||
Based on analysis of 263 colonies.
A cluster is defined here as colonies that have at least 99.5% sequence identity.
The consensus sequence of the cluster was used in searching the GenBank database.
Mucus bacteria were separated from tissue bacteria by centrifugation (see Materials and Methods).
Bacteria associated with O. patagonica during the summer.
Three coral fragments were obtained from different O. patagonica colonies in the summer of 2004. As described above, the coral mucus was separated from the coral tissue before analyses. The volume of mucus was 0.19 ± 0.01 ml/cm2. Thus, the average thickness of the mucus was 0.95 mm. The total number of bacteria in the mucus was (4.9 ± 2.1) × 107/cm2, corresponding to a concentration of bacteria in the mucus of 2.5 × 108/ml.
Table 3 summarizes the data on the bacterial clusters obtained from summer coral mucus and tissue DNAs. Cluster 1, which yielded a consensus sequence identical to that of cluster 1 in the winter analysis (Table 1) and the V. splendidus sequence with accession no. AJ874360 in the GenBank database, represented 35% of the 432 clones sequenced. Again, the Vibrio bacteria were located primarily in the mucus (54% of the clones) and not in the tissue (2.9% of the clones). The 10 most-abundant clusters shown in Table 3 represented 64% of the total number of clones sequenced, 81% of the clones obtained from mucus, and 30% of the clones obtained from tissue. The sequenced clones that do not appear in Table 3 consist of 5 clusters of 4 clones each, 5 clusters of 3 clones, 16 clusters of 2 clones, and 87 clones which appear only once. Thus, the number of different bacterial clusters found in the summer coral samples was 123 out of the 425 clones sequenced. Ninety-two of these 123 were novel OTUs (<99% identity to bacterial 16S rRNA gene sequences in the GenBank database).
TABLE 3.
Most-abundant bacterial clusters associated with O. patagonica in summer 2004a
| Clusterb or parameter | Class | Closest match in Blast (% identity)c | No. of clones | Abundance (% of)
|
||
|---|---|---|---|---|---|---|
| Total | Mucusd | Tissued | ||||
| 1 | γ-Proteobacteria | V. splendidus AJ874360 (100) | 147 | 34.6 | 49.8 | 2.9 |
| 2 | γ-Proteobacteria | V. harveyi strain LB5 DQ146936 (100) | 12 | 2.8 | 4.2 | 0 |
| 3 | γ-Proteobacteria | Alcanivorax sp. AB167044 (99) | 6 | 1.4 | 2.1 | 0 |
| 4 | δ-Proteobacteria | δ-Proteobacterium AY750148 (100) | 19 | 4.5 | 6 | 2.2 |
| 5 | Chlorobia | Prosthecochloris sp. AJ888467 (99) | 34 | 8.0 | 7.0 | 10.1 |
| 6 | Flavobacteria | Muricauda aquimarina strain SW-72 AY445076 (97) | 9 | 2.1 | 3.1 | 0 |
| 7 | Nitrospira | Uncultured isolate A93 AY373422 (96) | 5 | 1.2 | 0 | 3.6 |
| 8 | Unclassified bacteria | Uncultured Cytophaga sp. AB189358 (96) | 25 | 5.9 | 4.9 | 8.0 |
| 9 | Unclassified bacteria | Uncultured Cytophaga sp. AB189358 (93) | 9 | 2.1 | 3.1 | 0 |
| 10 | Unclassified bacteria | Uncultured isolate C12-9 AF332263 (89) | 5 | 1.2 | 0.3 | 2.9 |
| Total in the 10 clusters | 271 | 64 | 81 | 30 | ||
Based on analysis of 425 16S rRNA clones.
A cluster is defined here as 16S rRNA clones that have at least 99.5% sequence identity.
The consensus sequence of the cluster was used in searching the GenBank database.
Mucus bacteria were separated from tissue bacteria by centrifugation (see Materials and Methods).
The viable counts of the mucus and tissue samples from summer coral samples were (7.6 ± 2.1) × 104 and (2.7 ± 1.0) × 106 CFU/cm2 of coral surface, respectively. Bacteria isolated from summer coral samples (Table 4) were similar to those isolated in the winter (Table 2). Abundant summer clusters 1, 6, 7, 9, and 10 correspond to abundant winter clusters 1, 9, 11, 12, and 13, respectively. As in the winter, the most-abundant culturable bacteria were very different from the nonculturable bacteria from the same source. Only 1 (cluster 9, corresponding to V. splendidus) of the 18 most-abundant culture species from summer coral samples was present in the 10 most-abundant clusters obtained by the DNA cloning technique. In addition to the 18 clusters (88 colonies) shown in Table 4, 59 colonies appeared only once. Thus, the total number of different culturable species was 77 out of the 147 colonies analyzed. Of these 77, 35 were novel species (<99% identity to bacterial 16S rRNA gene sequences in the GenBank database).
TABLE 4.
Most-abundant cultured clusters associated with O. patagonica in summer 2004a
| Clusterb or parameter | Class | Closest match in Blast (% identity)c | No. of colonies | Abundance (% of)
|
||
|---|---|---|---|---|---|---|
| Total | Mucusd | Tissued | ||||
| 1 | γ-Proteobacteria | Pseudomonas sp. AF500211 (99) | 21 | 14.3 | 7.8 | 19.3 |
| 2 | γ-Proteobacteria | Agarivorans albus AB076560 (99) | 3 | 2.0 | 4.7 | 0 |
| 3 | γ-Proteobacteria | V. splendidus AY620972 (100) | 2 | 1.4 | 3.1 | 0 |
| 4 | γ-Proteobacteria | Thalassomonas viridans AJ294747 (99) | 2 | 1.4 | 0 | 2.4 |
| 5 | γ-Proteobacteria | Photobacterium ganghwense AY960847 (97) | 2 | 1.4 | 0 | 2.4 |
| 6 | α-Proteobacteria | α-Proteobacterium AB026194 (99) | 18 | 12.2 | 4.7 | 18.1 |
| 7 | α-Proteobacteria | α-Proteobacterium AY562560 (100) | 8 | 5.4 | 1.6 | 8.4 |
| 8 | α-Proteobacteria | Uncultured isolate AY711168 (93) | 7 | 4.8 | 9.4 | 1.2 |
| 9 | α-Proteobacteria | Vibrio sp. DQ146982 (99) | 4 | 2.7 | 3.1 | 2.4 |
| 10 | α-Proteobacteria | Pseudovibrio sp. AB112827 (99) | 4 | 2.7 | 3.1 | 2.4 |
| 11 | α-Proteobacteria | Rhodobacteraceae sp. AY442178 (95) | 3 | 2.0 | 4.7 | 0 |
| 12 | α-Proteobacteria | Uncultured isolate AJ633968 (100) | 2 | 1.4 | 0 | 2.4 |
| 13 | α-Proteobacteria | Uncultured isolate AM162572 (90) | 2 | 1.4 | 3.1 | 0 |
| 14 | α-Proteobacteria | Sphingomonadaceae family member AY676115 (96) | 2 | 1.4 | 3.1 | 0 |
| 15 | α-Proteobacteria | Marine bacterium Y4I AF388307 (98) | 2 | 1.4 | 3.1 | 0 |
| 16 | α-Proteobacteria | α-Proteobacterium AJ244796 (97) | 2 | 1.4 | 3.1 | 0 |
| 17 | α-Proteobacteria | α-Proteobacterium AY584527 (99) | 2 | 1.4 | 1.6 | 1.2 |
| 18 | Flavobacteria | Cytophaga sp. AB073566 (91) | 2 | 1.4 | 3.1 | 0 |
| Total in 18 clusters | 88 | 60 | 59 | 60 | ||
Based on analysis of 147 colonies.
A cluster is defined here as colonies that have at least 99.5% sequence identity.
The consensus sequence of the cluster was used in searching the GenBank database.
Mucus bacteria were separated from tissue bacteria by centrifugation (see Materials and Methods).
Seawater controls.
Seawater surrounding the coral samples yielded a total count of (5.2 ± 2.1) × 106/ml and a viable count of (2.2 ± 0.9) × 103/ml. These values are approximately 1% of the concentration of bacteria in the mucus samples. The most-abundant and culturable species belonged to the genera Vibrio (30.3%) and Alteromonas (10.9%). The eight most-abundant culturable seawater bacteria represented less than 1% of the bacteria in any of the coral samples. The most-abundant bacteria in seawater determined by DNA analysis belonged to the genus Synechococcus (20.0%). The nine most-abundant bacterial clusters from clone libraries of seawater represented less than 1% of the bacteria in any of the coral samples.
Distribution of the most-abundant clusters between coral fragments.
The data summarized in Tables 1 and 3 combine values from six different O. patagonica fragments, three from the winter and three from the summer. Table 5 presents the results for the most-abundant clusters obtained from the separate DNA libraries of each coral fragment. The most-abundant cluster, which has 16S rRNA gene sequences very close to the V. splendidus sequence with accession no. AJ874360, was present in higher abundance in all summer and winter fragments. The other abundant clusters showed large differences from summer to winter and from one fragment to another. For example, the cluster corresponding to the V. splendidus sequence with accession no. AB038030 represented 2.3 to 6.8% of the clones from the winter coral samples but was not detected in the summer coral samples, and the cluster corresponding to the δ-proteobacterium sequence with accession no. AY750148 represented 12.9% of the clones of summer coral 3 but was not detected in any of the other coral fragments.
TABLE 5.
Distribution of most-abundant species between different coral fragments
| Season | Closest match in Blast | Abundance (% of)
|
||
|---|---|---|---|---|
| Coral 1 | Coral 2 | Coral 3 | ||
| Wintera | V. splendidus AJ874360 | 35.4 | 39.1 | 11.4 |
| V. splendidus biovar II AB038030 | 3.8 | 2.3 | 6.8 | |
| Summerb | V. splendidus AJ874360 | 35.7 | 34.1 | 32.7 |
| Prosthecochloris sp. AJ888467 | 13.3 | 8.1 | 2.7 | |
| Uncultured Cytophaga sp. AB189358 | 14.0 | 0.7 | 2.7 | |
| δ-Proteobacterium AY750148 | 0 | 0 | 12.9 | |
DISCUSSION
As has been shown with several other corals (18, 20), the mucus layer of O. patagonica contains a high concentration of bacteria, 3 × 108/ml, 99.8% of which failed to produce colonies on MB agar under aerobic conditions. It should be pointed out that during the day, coral mucus and tissue are supersaturated with oxygen because of endosymbiotic algal photosynthesis (12), so that the presence of strict anaerobes is unlikely. In addition to the mucus bacteria, an even larger amount of bacteria was associated with the coral tissue, even after removal of the mucus by two centrifugation steps. It is not known if these latter bacteria are bound firmly to the outside of the coral or are present in coral tissues. What is clear is that the tissue bacteria are qualitatively very different from the mucus bacteria. No previous study of coral bacteria (e.g., reference 21) has examined the culturable and nonculturable bacteria, the mucus- and tissue-associated bacteria, and the bacteria present in different seasons. Since we now know the 16S rRNA gene sequences of the most-abundant tissue and mucus bacteria, it should be possible to use fluorescence in situ hybridization technology to locate the position of these bacteria on the coral surface or tissues.
By molecular methods, the most-abundant bacterium in O. patagonica mucus was V. splendidus, representing 68% and 50% of the clones from the winter and summer, respectively. At present, the species V. splendidus contains a rather broad group of bacteria (25). In this study, two clusters of V. splendidus were found. Each cluster consisted of sequences that showed more than 99.5% identity to the consensus sequence. However, the identity of the two consensus sequences to each other was only 98.7%. Since a few representatives of each of the V. splendidus clusters were present in the culturable bacteria, we are now able to study the physiological and biochemical properties of these bacteria in order to classify them more precisely and to attempt to understand the reason for their high concentration in mucus. Little is known about the role of coral mucus bacteria in coral health and disease (18).
Most studies of the bacterial populations of environmental samples, including the present one, have found very different data by culture and molecular methods. Since the number of colonies formed from the O. patagonica mucus and tissue samples represented only 0.2% of the total number of bacteria estimated from SYBR gold staining of the same samples, it is clear that the culturable bacteria are not representative of the total community. However, there is also no proof that the molecular methods, which employed DNA isolation, PCR with one set of primers, and cloning, did not introduce bias into the results. Furthermore, it has been reported (9) that O. patagonica contains a large number of filamentous, autofluorescent prokaryotes (probably cyanobacteria) tightly bound to the CaCO3 skeleton. The techniques used in this study would not reveal these bacteria because the skeleton was removed before analysis of the tissue.
The difference between the seawater temperatures at the times of sampling in the winter and summer was 10°C. By the molecular method, the only cluster that was present among the 10 most-abundant clusters in both seasons was the major V. splendidus cluster. By the culture method, the three most-abundant clusters were present in both seasons. We suggest that by changing the resident bacterial population, a coral holobiont may adapt more easily to different environmental conditions. The data presented in this report provide a comprehensive baseline for examining changes that occur when coral is stressed, e.g., during bleaching.
In considering the diversity of bacteria associated with O. patagonica, out of a total of 1,088 16S rRNA genes sequenced, ca. 400 were different OTUs. This is based on the arbitrary assumption that a bacterium or a group of bacteria that show less than 99.5% identity in their 16S rRNA gene sequence to any other bacterium represent a separate OTU. It has been reported that bacteria have multiple 16S rRNA operons and that the sequence divergence of these operons can exceed 0.5% (1). If this were the case with bacteria associated with O. patagonica, then our arbitrary assumption that a 16S rRNA gene sequence that differs from all other sequences by more than 0.5% is a separate OTU would lead to an overestimation of diversity. On the other hand, since most of the 400 OTUs appeared only once, it is clear that many more clones would have to be sequenced to estimate the full diversity of the coral bacteria. By using less than 99% identity to any 16S rRNA gene sequence in the GenBank database to define a novel species, 295 out of the 1,088 were novel OTUs. High bacterial diversity and novel species have been previously been reported for Caribbean corals (20). The bacterial communities associated with bleached O. patagonica and those found in dark caves (azooxanthellates) are under investigation.
Acknowledgments
We thank Uri Gophna for helpful discussions.
This study was supported by the Israel Center for the Study of Emerging Diseases and the Coral Disease Group of the World Bank.
REFERENCES
- 1.Acinas, S. G., L. Marcelino, V. Klepac-Ceraj, and M. F. Polz. 2004. Divergence and redundancy of 16S rRNA sequences in genomes with multiple rrn operons. J. Bacteriol. 186:2629-2635. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Altschul, S. F., W. Gish, W. Miller, E. W. Meyers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410. [DOI] [PubMed] [Google Scholar]
- 3.Baker, A. 2004. Symbiont diversity on coral reefs and its relationship to bleaching resistance and resilience, p. 177-194. In E. Rosenberg and Y. Loya (ed.), Coral health and disease. Springer-Verlag, Berlin, Germany.
- 4.Banin, E., T. Israely, M. Fine, Y. Loya, and E. Rosenberg. 2001. Role of endosymbiotic zooxanthellae and coral mucus in the adhesion of the coral-bleaching pathogen Vibrio shiloi to its host. FEMS Microbiol. Lett. 199:33-37. [DOI] [PubMed] [Google Scholar]
- 5.Banin, E., T. Israely, A. Kushmaro, Y. Loya, E. Orr, and E. Rosenberg. 2000. Penetration of the coral-bleaching bacterium Vibrio shiloi into Oculina patagonica. Appl. Environ. Microbiol. 66:3031-3036. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Banin, E., K. H. Sanjay, F. Naider, and E. Rosenberg. 2001. A proline-rich peptide from the coral pathogen Vibrio shiloi that inhibits photosynthesis of zooxanthellae. Appl. Environ. Microbiol. 67:1536-1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Bourne, D. G., and C. B. Munn. 2005. Diversity of bacteria associated with the coral Pocillopora damicornis from the Great Barrier Reef. Environ. Microbiol. 7:1162-1174. [DOI] [PubMed] [Google Scholar]
- 8.Ducklow, H. W., and R. Mitchell. 1979. Bacterial populations and adaptations in the mucus layers of living corals. Limnol. Oceanogr. 24:715-725. [Google Scholar]
- 9.Fine, M., and Y. Loya. 2002. Endolithic algae: an alternative source of photoassimilates during coral bleaching. Proc. Biol. Sci. R. Soc. 269:1205-1210. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Fine, M., and Y. Loya. 2004. Coral bleaching in a temperate sea: from colony physiology to population ecology, p. 143-156. In E. Rosenberg and Y. Loya (ed.), Coral health and disease. Springer-Verlag, Berlin, Germany.
- 11.Israely, T., E. Banin, and E. Rosenberg. 2001. Growth, differentiation and death of Vibrio shiloi in coral tissue as a function of seawater temperature. Aquat. Microb. Ecol. 24:1-8. [Google Scholar]
- 12.Kuhl, M., Y. Cohen, T. Dalsgard, B. B. Jorgensen, and N. P. Revsbech. 1995. Microenvironment and photosynthesis of zooxanththellae in scleractinian corals studied with microsensors for O2, pH and light. Mar. Ecol. Prog. Ser. 117:159-172. [Google Scholar]
- 13.Kushmaro, A., Y. Loya, M. Fine, and E. Rosenberg. 1996. Bacterial infection and coral bleaching. Nature 380:396. [Google Scholar]
- 14.Kushmaro, A., E. Rosenberg, M. Fine, and Y. Loya. 1997. Bleaching of the coral Oculina patagonica by Vibrio AK-1. Mar. Ecol. Prog. Ser. 147:159-165. [Google Scholar]
- 15.Lane, D. J. 1991. 16S/23S rRNA sequencing. John Wiley & Sons, Chichester, United Kingdom.
- 16.Noble, R. T., and J. A. Fuhrman. 1998. Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria. Aquat. Microb. Ecol. 14:113-118. [Google Scholar]
- 17.Ritchie, K. B., and G. W. Smith. 1995. Preferential carbon utilization by surface bacterial communities from water mass, normal and white-band diseased Acropora cervicornis. Mol. Mar. Biol. Biotechnol. 4:345-352. [Google Scholar]
- 18.Ritchie, K. B., and G. W. Smith. 2004. Microbial communities of coral surface mucopolysaccharide layers, p. 259-264. In E. Rosenberg and Y. Loya (ed.), Coral health and disease. Springer-Verlag, Berlin, Germany.
- 19.Ritchie, K. B., J. H. Dennis, T. McGrath, and G. W. Smith. 1994. Bacteria associated with bleached and nonbleached areas of Monastrea annularis. Proc. Symp. Nat. Hist. Bahamas 5:75-80. [Google Scholar]
- 20.Rohwer, F., M. Breitbart, J. Jara, F. Azam, and K. Knowlton. 2001. Diversity of bacteria associated with the Caribbean coral Monastrea franksi. Coral Reefs 20:85-91. [Google Scholar]
- 21.Rohwer, F., V. Seguritan, F. Azam, and K. Knowlton. 2002. Diversity and distribution of coral-associated bacteria. Mar. Ecol. Prog. Ser. 243:1-10. [Google Scholar]
- 22.Rosenberg, E., and L. Falkowitz. 2004. The Vibrio shiloi/Oculina patagonica model system of coral bleaching. Annu. Rev. Microbiol. 8:143-159. [DOI] [PubMed] [Google Scholar]
- 23.Schloss, P. D., and J. Handelsman. 2005. Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl. Environ. Microbiol. 71:1501-1506. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Sorokin, Y. I. 1973. Trophical role of bacteria in the ecosystem of the coral reef. Nature 242:415-417. [Google Scholar]
- 25.Thompson, J. R., S. Pacocha, C. Pharino, V. Klepac-Ceraj, D. E. Hunt, J. Benoit, R. Sarma-Rupavtarm, D. L. Distel, and M. F. Polz. 2005. Genotypic diversity within a natural coastal bacterioplankton population. Science 307:1311-1313. [DOI] [PubMed] [Google Scholar]
- 26.Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The ClustalX Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24:4876-4882. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Toren, A., L. Landau, A. Kushmaro, Y. Loya, and E. Rosenberg. 1998. Effect of temperature on adhesion of Vibrio strain AK-1 to Oculina patagonica and on coral bleaching. Appl. Environ. Microbiol. 64:1379-1384. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Wegley, L., Y. Yu, M. Breitbart, V. Casas, D. I. Kline, and F. Rohwer. 2004. Coral-associated Archaea. Mar. Ecol. Prog. Ser. 273:89-96. [Google Scholar]