By continuing to browse this site you agree to us using cookies as described in About Cookies
Notice: Wiley Online Library will be unavailable on Saturday 7th Oct from 03.00 EDT / 08:00 BST / 12:30 IST / 15.00 SGT to 08.00 EDT / 13.00 BST / 17:30 IST / 20.00 SGT and Sunday 8th Oct from 03.00 EDT / 08:00 BST / 12:30 IST / 15.00 SGT to 06.00 EDT / 11.00 BST / 15:30 IST / 18.00 SGT for essential maintenance. Apologies for the inconvenience.
Fabiano L. Thompson, Avenida Brigadeiro Trompowsky, s/nº. Centro de Ciências da Saúde, Departamento de Genética, Bloco A, Sala 105-A2, Ilha do Fundão, Rio de Janeiro, RJ, CEP 21941-590, Brazil. E-mail: email@example.com
Aims: A taxonomic survey of the vibrios associated with the Brazilian endemic coral Mussismilia hispida and the sympatric zoanthids (i.e. Palythoa caribaeorum, Palythoa variabilis and Zoanthus solanderi).
Methods and Results: Mucus of 54 cnidarian specimens collected in three different places at São Sebastião in two consecutive years (i.e. 2005 and 2006) was used for taxonomic characterization of the cnidarian microbiota. Ninety-eight of the 151 vibrio isolates fell within the vibrio core group according to partial 16S rDNA sequences. We performed the sequencing of recA and pyrH genes of all vibrio isolates. The most abundant taxa belonged to the vibrio core group (Vibrio harveyi, Vibrio rotiferianus, Vibrio campbellii and Vibrio alginolyticus), Vibrio mediterranei (=Vibrio shillonii) and Vibrio chagasii. With the exception of V. chagasii which was found only in the mucus of M. hispida, the other species appeared in different hosts with no evidence for the presence of host-specific clones or species. Using rep-PCR analysis, we observed a high genomic heterogeneity within the vibrios. Each vibrio isolate generated a different rep-PCR fingerprint pattern. There was a complete agreement between the grouping based on rep-PCR and concatenated sequences of pyrH, recA and 16S rDNA, but the pyrH gene has the highest discriminatory power for vibrio species identification.
Conclusion: The vibrio core group is dominant in the mucus of these cnidarians. There is a tremendous diversity of vibrio lineages within the coral mucus. pyrH gene sequences permit a clear-cut identification of vibrios.
Significance and Impact of the Study: The taxonomic resolution provided by pyrH (but not recA) appears to be enough for identifying species of vibrios and for disclosing putative new taxa. The vibrio core group appears to be dominant in the mucus of the Brazilian cnidarians. The overrepresentation of these vibrios may reflect as yet unknown ecological functions in the coral holobiont.
There has been a growing number of studies on the diversity of coral microbiota in the last 10 years (Rohwer et al. 2001; Rosenberg et al. 2007; Dinsdale et al. 2008). Corals have a key role in the construction of reefs and form the benthic fauna in a variety of rocky shores worldwide. The reef Biome has a key role for the health of the Oceans, but it is also extremely important for the economy of several countries via fishing, ecotourism and nursery place. It became evident that corals harbour an enormous diversity of bacteria, both well-known cultivable taxa (e.g. vibrios) and novel groups awaiting formal taxonomic characterization. Rohwer et al. (2001, 2002) studied the diversity of the coral microbiota by culture-independent methods using 16S rRNA libraries and observed that each of the sympatric coral species Montastraea franksi, Diploria strigosa and Porites astreoides had a different microbiota. In addition, they found that the microbiota of the same coral species was similar even in specimens collected in different years and >3000 km apart from each other. Based on these data, these authors argued that each coral species has its own microbiota, possibly acquired via host-microbe co-evolution. Apparently, the composition of the coral mucus has a considerable effect on the composition of the microbiota. On the other hand, it was shown more recently that some bacterial species (e.g. in the Rickettsiales) are widespread in multiple species of corals (Casas et al. 2004). So far, the host-specific hypothesis put forward by Rohwer et al. (2001, 2002) has not been evaluated for the microbiota of the Brazilian corals.
This is the first study aiming at the taxonomic characterization of the heterotrophic microbiota, particularly vibrios, associated with the Brazilian endemic coral Mussismilia hispida and the sympatric zoanthids (Palythoa caribaeorum, Palythoa variabilis and Zoanthus solanderi). These shallow water zooxanthellated species are sympatric on the Brazilian coast, sharing the same habitat with M. hispida. The microbiological examination of different sympatric species would allow us to evaluate if different vibrio species appear in different hosts, indicating a pattern of host-microbe evolution. Mussismilia hispida is one of the most important reef builders in the South Atlantic Ocean. It has the largest spatial distribution, occurring along the coast of Brazil, from Rio Grande do Norte to Santa Catarina State (c. 5000 km) (Leão et al. 2003; Leão and Kikuchi 2005). Recent studies suggest that the genus Mussismilia is in danger of extinction (Francini-Filho et al. 2008). On the other hand, the genus Palythoa is widespread around the world, most notably at rocky shores and coral reefs. Palythoa caribaeorum may cover up to 50% of the total area of rocky shores in some locations at the coast of Brazil (Oigman-Pszczol et al. 2004). This organism is well known for its ability to produce a very potent nonproteinaceous toxin (called palitoxin) and copious amounts of mucus. These features may allow it to occupy areas of periodic desiccation and intense sunlight stress. We screened the diversity of heterotrophic bacteria associated with the cnidarians species using 16S rDNA gene sequencing in three locations at the coast of São Sebastião city (São Paulo State). In each location, we obtained several specimens of different cnidarians species, normally distant 1 m from each other. The dominant groups of vibrios were analysed in more depth with recA and pyrH gene sequencing, two proven taxonomic markers, with complementary evolutionary stories (Thompson et al. 2005, 2007; Sawabe et al. 2007). According to previous studies, the recA marker unravels any fuzziness between sister-species (due to recombination and/or slower molecular clock), whereas the pyrH marker clearly discriminates sister-species of vibrios.
Materials and methods
In total 54 cnidarians, specimens of M. hispida, P. caribaeorum, P. variabilis and Z. solanderi were sampled at rocky shores. The specimens were collected on 03/02/2005 and 01/02/2006 at three sites: Grande (23°50′25″S; 045°24′59″W), Portinho (23°50′25″S; 045°24′22″W) and Preta (23°49′10″S; 045°24′37″W) beaches located near the Marine Biology Center of USP (São Sebastião Channel, São Paulo, Brazil) by SCUBA diving between depths of 3–7 m. The beaches Grande, Portinho and Preta are about 2 km apart from each other, the latter being on the continental side of the Channel and the first two in the opposite facing CEBIMAR-USP (Fig. S1). In the sampling performed in 2006, the mucus of each specimen was collected using individual sterile 50ml syringes and placed in 50ml sterile tubes in situ on board. The mucus samples were kept at c. 10°C for 6h prior microbiological examination. Whereas intact colonies of M. hispida and fragments of zoanthids were placed in sterile plastic bags and kept at c. 10°C for 6h prior microbiological examination in the 2005 sampling.
Isolation and preservation of strains
Bacterial isolation, purification and preservation were performed at University of Campinas. Aliquots of 1 ml of mucus samples were transferred to sterile tubes and diluted in 10 ml in sterile saline solution (3% NaCl, SSS). The mucus was resuspended by vigorous vortex for 3 min. Representative aliquots of 1 ml of diluted mucus samples were 10fold diluted in SSS and plated onto the culture medium Thiosulfate-citrate-bile salt-sucrose agar (Oxoid). Plates were incubated at 28°C for 48 h. Five representative colony morphotypes were picked in the highest dilution (105) for further purification. The pure cultures are maintained in vials with 20% glycerol at −80°C.
The preliminary characterization of all 151 vibrio isolates was obtained by partial 16S rDNA gene sequences (430 nt) as described previously with minor modifications (Thompson et al. 2001). Almost complete 16S rDNA sequences (>1300 nt) were performed for representative strains of putative new taxa. The total DNA from each culture was extracted with 50 μl sterile lysis buffer (10% SDS, 2·5 ml; 1 N NaOH, 5 ml and MilliQ water 92 ml), diluted in 150 μl of sterile MilliQ water and boiled for 15 min. The PCR reactions were composed of 37·5 μl sterile MilliQ water, 5·0 μl PCR buffer (10X), 1·5 μl Mg2Cl (1·5 mmol l−1), 0·4 μl dNTP’s (0·2 mmol l−1 each), 1 μl forward primer (p27f – 5′AGA GTT TGA TCM TGG CTC AG3′, 20 μmol l−1), 1 μl reverse primer (1401r – 5′CGG TGT GTA CAA GGC CCG GGA ACG3′, 20 μmol l−1), 0·4 μl AmpliTaq DNA Polymerase (2 U μl−1) and 1 μl template DNA (0·02 μg μl−1). The thermal programme consisted of (i) 2 min at 95°C, (ii) 30 cycles of 1 min at 94°C + 1 min at 55°C and 3 min at 72°C, and (iii) 3 min at 72°C. PCR was performed using an Eppendorf thermocycler. The PCR products were purified using a solution of PEG8000 (20%)/2 mol l−1 NaCl. Purified PCR products were eluted in 50 μl sterile MilliQ water. Subsequently, 5·0 μl of purified PCR product was mixed with 4·0 μl ET TerminatorTM Mix (GE Health Care), 0·2 μl sequencing primers (782r – 5′ACC AGG GTA TCT AAT CCT GT3′, 20 μmol l−1), (10f – 5′GAG TTT GAT CCT GGC TCA G3′), and 0·8 μl MilliQ water. The thermal program consisted of 30 cycles of 20 s at 95°C + 15 s at 50°C + 1 min at 60°C. The purification of the sequencing products was obtained by mixing 1 μl ammonium acetate (7·5 mol l−1) and 27·5 μl absolute ethanol, followed by incubation in dark for 30 min and subsequent centrifugation at 20 800 g for 75 min at 4°C, after this the supernates were removed and 100 μl of 70% ethanol was added, a new centrifugation was performed at 3700 rev min−1 for 45 min at 4°C. Separation of the DNA fragments was obtained in a MegaBace 1000 system (GE Health Care). Voltage and time of injection were 3 kV and 80 s. Running was performed at 9 kV for 100 min at 44°C.
Vibrios were further characterized by partial sequencing of pyrH (440 nt) and recA (600 nt) as described previously (Thompson et al. 2005, 2007). Briefly, PCR was composed of 38·2 μl sterile MilliQ water, 1·5 μl MgCl2 (1·5 mmol l−1), 5·0 μl PCR buffer (10X), 0·4 μl dNTP’s (0·2 mmol l−1 each), 1·2 μl forward primer (pyrH80F – 5′GAT CGT ATG GCT CAA GAA G3′, 20 μmol l−1), 1·2 μl reverse primer (pyrH530R – 5′TAG GCA TTT TGT GGT CAC G3′, 20 μmol l−1), 0·4 μl AmpliTaq DNA Polymerase (2 U μl−1) and 2·0 μl template DNA (0·05 μg μl−1). The thermal programme consisted of (i) 5 min at 95°C, (ii) three cycles of 1 min at 95°C + 2 min 15 s at 55°C and 1 min 15 s at 72°C, (iii) 30 cycles of 30 s at 95°C + 1 min 15 s at 55°C and 1 min 15 s 72°C, and (iv) a final 7 min at 72°C. pyrH PCR products were purified with the enzyme Exosap according to the instructions of the manufacturer (GE Health Care). Sequencing was performed as described above using 0·6 μl of each PCR primer (pyrH80F and pyrH530R, 20 μmol l−1) and an annealing temperature of 50°C. Sequencing of the recA gene was performed using the forward primers (recA1F – 5′TGA RAA RCA RTT YGG TAA AGG3′ and recA130F – 5′GTC TAC CAA TGG GTC GTA TC3′; 20 μmol l−1) and reverse primers (recA2R – 5′TCR CCN TTR TAG CTR TACC3′ and recA720R – 5′GCC ATT GTA GCT GTA CCA AG3′; 20 μmol l−1). Purification of the recA PCR products and sequencing was performed as described above with an annealing temperature of 55°C.
Raw sequence data were transferred to the Gene Builder module within Kodon package 2.03 (Applied Maths, Belgium) and chromaspro ver. 1.34 (Technelysium Pty. Ltd, Tewantin, Australia) where consensus sequences were determined. The sequences were aligned using ClustalW. Similarity matrices and phylogenetic trees were constructed using the software mega ver. 4.0 (Tamura et al. 2007). Trees were drawn using the Neighbour-Joining method (Saitou and Nei 1987). The robustness of each topology was checked by 1000 bootstrap replications. The gene sequence data obtained in this study are also available through our website TAXVIBRIO (http://www.taxvibrio.lncc.br/). The gene sequences are deposited in the GenBank under the accession nos EU716656–EU717075.
Representative vibrio isolates were characterized by rep-PCR analysis as described previously (Gomez-Gil et al. 2004, 2007). Briefly, genomic DNA was extracted with the Promega Wizard DNA extraction kit (Promega) according to the manufacture’s instructions. The DNA obtained was adjusted to 50 ng ml−1 spectrophotometrically. DNA fingerprinting of all the strains was performed with rep-PCR using the (GTG)5 primer. Rep-PCR products were amplified with the AmpliTaq® DNA polymerase enzyme (Applied-Biosystems, USA) and the products underwent electrophoresis in 2·25% 20 × 20 cm agarose gels for 18 h at 55 V and 4–8°C. The gels were stained with ethidium bromide and visualized after integration in a gel documentation system (UVP). TIFF files obtained were analysed with the gelcompar II software (ver. 4.5; Applied-Maths), a similarity matrix was calculated with the Jaccard coefficient and the dendrogram constructed with Ward (position tolerance of 0·59%). Type and references strains of all valid species of vibrios were incorporated into the analysis, as well as many strains from other origins, allowing clear species identification.
We analysed the diversity of 151 vibrio isolates originating in the mucus of M. hispida and the sympatric zoanthids (P. caribaeorum, P. variabilis and Z. solanderi). The species and the numbers of isolates obtained from different cnidarians are summarized in the Table 1. According to the preliminary screening based on partial 16S rDNA sequences, most of the isolates were allocated in to the vibrio core group (Fig. S2, Table S1). To identify unambiguously the species of vibrios, we sequenced segments of the taxonomic markers recA and pyrH. We defined groups on the basis of the monophyletic groupings using concatenated sequences of recA, pyrH and 16S rDNA (Fig. 1). Isolates within the same group had more than 97% concatenated sequence similarity. Most of the vibrio isolates fell into known species with an apparent dominance of certain taxa. The vibrio core group (Vibrio alginolyticus, Vibrio rotiferianus, Vibrio harveyi and Vibrio campbelli), Vibrio mediterranei (including its latter synonymous Vibrio shilonii), Vibrio tubiashii, Vibrio sinaloensis and Vibrio chagasii were found in 2005 and 2006. Vibrio campbellii, V. rotiferianus and V. harveyi isolates were associated with M. hispida and P. caribaeorum in 2 years (2005 and 2006) and in three different sites. Isolates identified into the species V. tubiashii were found mainly in 2006. Several isolates obtained in 2005 and 2006 were identified into the species V. mediterranei. The V. chagasii isolates were found only in association with M. hispida in 2005 and 2006. Most of the vibrio species were found in the three different places (Beach Grande, Portinho and Preta) (Fig. S4).
The concatenated sequences did not provide enough taxonomic resolution for the differentiation of the vibrio core group (Fig. S2). However, the analysis based only on pyrH and rep-PCR revealed that the vibrio core group isolates form distinct groups corresponding to the species V. harveyi, V. campbellii and V. rotiferianus (Fig. 1). The recA alone did not allow the clear-cut differentiation of isolates of the vibrio core group. We observed a rather low intraspecies sequence variation (<0·5%) in the concatenated segments of pyrH, recA and 16S rDNA in the various taxa detected in this study (Fig. S2). The recA gene appeared to show more intra-species variation, while the pyrH gene clearly discriminated closely related sister-species of vibrios (Fig. 1).
Fifty-seven isolates distributed in three groups (L1–L3) appeared to belong to new taxa as they formed separate groups on the basis of concatenated sequences and showed differences in the complete 16S rDNA gene sequences (1%) with their closest neighbours. The group L1 (N = 46) had <97% concatenated sequence similarity towards V. harveyi. Isolates of this group clustered with the strain LMG 20370. Group L2 comprised five isolates originating in M. hispida and Palythoa, represented for R-616, R-619 (Fig. S2) and R-77, R-78, and R-91 with the pyrH and 16S rDNA sequence obtained (Table S1). Group L3 comprised seven Photobacterium isolates of P. caribaeorum (R-15, R-89, R-13, R-16, R-17, R-34 and R90; Table S1, Fig. S2).
The rep-PCR analyses showed that the vibrio isolates formed cohesive species clusters clearly separated from each other on the basis of rep-PCR analysis. The rep-PCR analysis was reassuring and confirmed the grouping obtained by pyrH, recA and 16S rDNA gene sequences (Figs 2 and S2). For instance, the V. harveyi isolates R-647 and R-691 had c. 45% rep-PCR pattern similarity and 96% concatenated sequence similarity. Whereas the V. harveyi R-54 and R-601 had 55% rep-PCR pattern similarity, and 97% concatenated sequence similarity. Overall, the rep-PCR analysis provided a finer discrimination between isolates than the pyrH gene sequences. A negative correlation was found between the nucleotide substitutions in the concatenated sequences and the rep-PCR (Fig. S3). In general, the higher the rep-PCR similarity values, the lower the nucleotide substitutions (Spearman correlation = −0·324, P < 0·0001, n = 3486). The correlation became more evident when only pyrH sequences were compared with rep-PCR. It became evident that most of the isolates within each species generated a different rep-PCR pattern (<85% band pattern similarity).
The vibrio core group appears to be a dominant group among the vibrios of the microbiota of the four species Brazilian cnidarians. The overrepresentation of these vibrios may reflect as yet unknown ecological functions in the coral holobiont. Vibrio alginolyticus has been considered a water-column resident previously (Ritchie 2006), but we showed in this study that this species is present in the mucus of different cnidarians species. Vibrio alginolyticus is a coral pathogen (Cervino et al., 2004, 2008), but other studies suggest that it could belong to the normal coral microbiota (Lampert et al. 2006). In Nitrogen fixing, V. alginolyticus strains might have a beneficial effect on M. hispida. Vibrio mediterranei was frequently found in association with the coral M. hispida and the sympatric zoanthids (i.e. P. caribaeorum, P. variabilis and Z. solanderi). A recent study suggested that vibrios, including V. mediterranei, no longer can be detected in mucus of health and bleached Oculina patagonica. This fact may be related to shortcomings of the FISH analysis used in this study (Ainsworth et al. 2008).
The species V. chagassi was proposed to encompass isolates originated of rotifer and fish cultures (Thompson et al. 2003), but we now expand its occurrence. This species was found in 2005 and 2006 associated only with M. hispida. However, we have no evidence to suggest that different cnidarians species select different vibrio species as is the case of the squid Euprymna scolopes and Aliivibrio fischeri. If a host-microbe interaction exists involving vibrios and corals as argued by Rohwer et al. (2001, 2002), we hypothesize that it would involve the selection of strains rather than species. Additional work would be needed to prove that specific clones of vibrios are picked up and maintained by the cnidarian hosts, with the examination of large co-occurring populations of dominant vibrios, such as the vibrio core group.
The analysis of the concatenated sequences of the genes pyrH, recA and 16S rDNA did not provide enough taxonomic resolution for the differentiation of the vibrio core group (Fig. S2). Although recA was suggested to be an identification marker for vibrios (Thompson et al. 2003), our current results show that this locus alone does not allow the differentiation of fresh environmental isolates of the vibrio core group. However, the analysis based only on pyrH and rep-PCR revealed that the vibrio core group isolates form distinct groups corresponding to the species V. harveyi, V. campbellii and V. rotiferianus (Fig. 1). The taxonomic resolution provided by pyrH appears to be enough for identifying species of vibrios and for disclosing putative new taxa. Several isolates allocated in the clusters L1, L2 and L3 appear to belong to new taxa as they do not cluster with any known vibrio species. Isolates of L1 group clustered with the strain LMG 20370. This strain was originally allocated within V. harveyi because it had 71% DDH similarity with the type strain of V. harveyi, but its AFLP patterns were clearly different from the type and reference strains of V. harveyi (Thompson et al. 2001). The results presented in this study enforce that L1 represents a new species. A polyphasic taxonomic approach will be undertaken to describe formally these putative new taxa, including the sequencing of additional genetic loci (i.e. topA, ftsZ, mreB and atpA) and phenotypic characterization.
We performed rep-PCR analysis to check if there was a clonal spread of successful vibrio genotypes among different cnidarian hosts. This analysis would give us information on intraspecific genome-wide variation of the most abundant taxa. Our results clearly show a tremendous genomic variation among the collection of co-occurring vibrio populations. Also, it was evident that isolates of different hosts and places had high mutual similarity, indicating no biogeographical effect on the distribution of vibrio populations at the scale of metres and kilometres. Genome size variation was shown in V. splendidus in a study of the vibrioplankton of the North Atlantic Ocean (Thompson et al. 2003). The factors leading to the genomic variation observed in this study are unknown at present, but might involve both intracellular processes (such as rearrangements and point mutations) and horizontal gene transfer processes within the coral mucus. Another interesting aspect of vibrio diversification disclosed in this study, is the seemingly absence of species specificity of the vibrios in different hosts.
The rep-PCR analysis confirmed the grouping obtained by pyrH, recA and 16S rDNA gene sequences. However, it is evident that strain identification within the vibrio core group will require the use of a multigene approach. Overall, the rep-PCR analysis provided a finer discrimination between isolates than the pyrH gene sequences. A negative correlation founded between the nucleotide substitutions in the pyrH sequences and the rep-PCR suggests that pyrH gene sequences can be used to predict whole genome similarity among vibrios. In a collection of papers on the taxonomy of Lactic Acid Bacteria, Naser et al. (2005, 2007) have shown the reliability of using single locus for species identification. The loci used by these authors (i.e. atpA, pheS and rpoA) were carefully chosen for providing species differentiation. pheS alone provided the discrimination of closely related species. In this study, we show that the taxonomic resolution provided by pyrH (but not recA) appears to be enough for identifying species of vibrios and for disclosing putative new taxa. The simplified version of the MLSA used in this study correlates well with rep-PCR analysis, but it was clear that the use of recA and 16SrDNA decreased the taxonomic resolution of our analysis.
The authors acknowledge grants from FAPERJ, FAPESP, CNPq, IFS and FOSEMARNAT. L. A. Chimetto acknowledges a PhD scholarship provided by CNPq. We thank Álvaro Migotto (CEBIMAR-USP) for technical assistance and valuable comments.