TEXT
Reef-building corals are “metaorganisms”; i.e., the coral animal lives in a mutualistic relationship with photosynthetic, endosymbiotic dinoflagellates of the genus
Symbiodinium along with microorganisms including bacteria, archaea, fungi, and viruses. The significance of the bacterial assemblage to the coral animal is not well understood, although coral bacteria have been characterized as species specific (
1) and may have roles in nitrogen fixation, carbon fixation, antibiotic production, and other features that enable their health and functioning (
1–5). A substantial component of the coral bacterial community resides within the mucus layer (
6,
7), and there is little understanding of which microbial partners are actually in residence within the coral tissues and potentially interacting with the coral. Here, we address this lack of knowledge by examining if and how a dominant group of bacteria frequently recovered in sequencing-based studies is located internally within the coral tissues of the Red Sea coral
Stylophora pistillata. This study used Sanger and 454 pyrosequencing of the bacterial 16S rRNA gene to document the bacterial community of
S. pistillata and a fluorescence
in situ hybridization (FISH) approach to examine if the dominant bacteria (
Endozoicomonas) associated with
S. pistillata reside within the coral. Additional analyses of other Red Sea corals, as well as an
in silico analysis of worldwide corals, were used to examine the prominence of
Endozoicomonas in other coral species.
Five
S. pistillata samples (Sp1 to Sp5) were collected in the southern Red Sea in June 2009 by scuba diving at depths between 2 and 5 m at five sites (see Fig. S1 in the supplemental material). DNA was extracted from airbrushed tissue with the PowerPlant DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA), with some modifications (
8). Several primer pairs were tested prior to 454 pyrosequencing to ensure specificity to bacterial DNA (see the supplemental material), and primers 784F and 1061R (
9), which include variable regions 5 and 6 of the 16S rRNA gene, were used for this analysis. Libraries were generated with the GS FLX Titanium emPCR kit (Lib-A; Roche, Branford, CT) and sequenced by Titanium FLX chemistry. Data analysis was conducted with the mothur software v.1.24.1 (
10). Sequencing resulted in 287,488 reads, of which 131,421 remained (median length, 250 bp) after cleaning (
Table 1). The sequences were clustered into operational taxonomic units (OTUs) at 97% similarity and classified against the 2011 version of the GreenGenes database (
11) as described by Wang et al. (
12), with a bootstrap cutoff of 60%. Rarefaction curves demonstrate that for samples Sp4 and Sp5, most of the diversity has been sampled but curves did not plateau for the other samples (see Fig. S2 in the supplemental material). The evenness was very low, indicating that few bacterial OTUs make up the majority of the microbiome (
Table 1). In fact, two OTUs dominated the bacterial community in
S. pistillata, and they were classified in the genus
Endozoicomonas of the order
Oceanospirillales and the genus
Burkholderia of the order
Burkholderiales (
Fig. 1A).
Full-length sequences were also obtained from each sample (Sp1 to Sp5, 86 to 94 clones per sample after chimera removal; see the supplemental material) and yielded bacterial community composition results similar to those of the 454 data (
Fig. 1A). Phylogenetic analyses indicated that the
Endozoicomonas OTUs obtained in this study all clustered with cultivated species (
Endozoicomonas numazuensis,
E. montiporae, and
E. elysicola), as well as other
Endozoicomonas sequences from a diverse range of marine invertebrates, including many species of reef-building and gorgonian corals (see Fig. S3 in the supplemental material). The closest relative of the
Burkholderia sequences was isolated from a white-rot fungus (see Fig. S4 in the supplemental material), suggesting that the
Burkholderia bacteria found on
S. pistillata were associated with a fungus on the coral.
In order to examine the ubiquity of
Endozoicomonas bacteria in other coral species from the Red Sea, healthy specimens of
Acropora humilis and
Pocillopora damicornis (three of each species) were obtained from the same area as the
S. pistillata samples (see Fig. S1 in the supplemental material). Bacterial small-subunit (SSU) rRNA genes were examined by cloning and sequencing (
n = 20 to 102 per sample after chimera check and contaminant removal; see the supplemental material).
Endozoicomonas bacteria accounted for 70 to 95% of the bacterial abundance in
P. damicornis and
A. humilis (
Fig. 1B). Each coral species was associated with one to three
Endozoicomonas OTUs (see Fig. S3 in the supplemental material).
To determine how prevalent
Endozoicomonas bacteria are in other corals, we performed an
in silico analysis of full- and partial-length
Endozoicomonas SSU rRNA gene sequences, which revealed that these bacteria associate with 14 species of scleractinian corals (
Table 2) (
8,
13–19). The fact that sequences were recovered from corals over vast geographic regions suggests that
Endozoicomonas bacteria probably have an important relationship with corals. Interestingly, none of the
Endozoicomonas OTUs were detected in more than one species, suggesting that each coral harbors its own unique
Endozoicomonas strain. Additionally, closely related OTUs may associate with the same coral species in different geographic regions. For example,
Endozoicomonas sequences recovered from
P. damicornis in the Red Sea (this study) formed a clade with sequences from
P. damicornis from the Great Barrier Reef, Australia (
13) (see Fig. S3 in the supplemental material). One of the
Endozoicomonas OTUs from
A. humilis (this study) also clustered with an
Endozoicomonas isolate from an acroporid in the Caribbean (
8) (see Fig. S3). These results suggest coevolution of
Endozoicomonas bacteria and coral, an important strategy to maintain their association over time and space.
To further investigate the relationship of
Endozoicomonas bacteria and
S. pistillata, FISH experiments were conducted with novel
Endozoicomonas-specific oligonucleotide probes (see the supplemental material). Five or six specimens from colonies Sp1 to Sp5 were examined by imaging in three or four areas of each specimen and compared with control specimens (no probe, NON338 nonsense probe) of each respective colony. In all of the colonies examined, the
Endozoicomonas probes were found to hybridize to cells located in close proximity to
Symbiodinium cells (
Fig. 2A and
C) and within the autofluorescent coral tissues, suggesting that they reside within the coral endoderm.
Endozoicomonas cells were arranged in multiple dense aggregates generally containing 10 to 50 cells and represented the majority of the cells that hybridized to the general bacterial probe (
Fig. 2B and
D). These are the first observations of
Endozoicomonas in association with corals, but the aggregations do resemble the “ovoid bacterial clusters” that have been previously identified with corals (
20–22). Hybridization of the
S. pistillata samples with the NON338 probe resulted in the probe hybridizing to aggregates similar to the
Endozoicomonas probed cells (
Fig. 2E), but the intensity and abundance of the apparent nonspecific probe binding were much lower than those of the specific probes (
Fig. 2A to
D). The nonspecific binding to portions of the aggregates, as well as to coral nematocysts (
Fig. 2F), suggests that an adhesive-type substance may surround the aggregates. Aggregates were also dimly visible in the no-probe controls at the same intensity as the autofluorescent coral tissues (not shown), and this suggests that the cells are embedded within the host endoderm.
Endozoicomonas bacteria appear to have an intimate and established relationship with many Red Sea corals and other corals worldwide. One feature of
Endozoicomonas bacteria associated with
S. pistillata is that they may produce quorum-sensing molecules (reviewed in reference
23). The dense cell aggregations found suggest that reaching a critical mass may provide an advantage to the cells. Despite our limited knowledge of
Endozoicomonas bacteria, they appear to be an important group of bacteria that require further investigation of their potential role in the functional system of the coral holobiont, as well as their interactions with other invertebrate associates.
Nucleotide sequence accession numbers.
The sequences determined in this study have been deposited in the NCBI Sequence Read Archive under accession number PRJNA189184 and in the GenBank database under accession numbers KC668414 to KC669277.