Culture and Identification of Desulfovibrio Spp. from Corals Infected by Black Band Disease on Dominican and Florida Keys Reefs

DISEASES OF AQUATIC ORGANISMS Vol. 69: 119–127, 2006 Published March 23 Dis Aquat Org

Culture and identification of Desulfovibrio spp. from corals infected by black band disease on Dominican and Florida Keys reefs

S. Viehman1, 3,*, D. K. Mills1, G. W. Meichel2, L. L. Richardson1

1Department of Biological Sciences and 2Southeast Environmental Research Center, Florida International University, University Park, Miami, Florida 33199, USA

3Present address: NOAA, National Ocean Service, Center for Coastal Fisheries and Habitat Research, 101 Pivers Island Road, Beaufort, North Carolina 28516, USA

ABSTRACT: Black band disease (BBD) of corals is characterized as a pathogenic microbial consortium composed of a wide variety of microorganisms. Together, many of these microorganisms con- tribute to an active sulfur cycle that produces anoxia and high levels of sulfide adjacent to the coral surface, conditions that are lethal to coral tissue. Sulfate-reducing bacteria, as sulfide producers, are an important component of the sulfur cycle and the black band community. Previous molecular survey studies have shown multiple Desulfovibrio species present in BBD but with limited consistency between bacterial species and infections. In this study we compared 16S rRNA gene sequences of sulfate-reducing bacteria selectively cultured from 6 BBD bands on 4 coral species, Diploria clivosa, D. strigosa, D. labyrinthiformes, and Siderastrea siderea, in the Florida Keys and Dominica. The 16S rRNA gene sequences were obtained through direct sequencing of PCR products or by cloning. A BLAST search revealed that 8 out of 10 cultures sequenced were highly homologous to Desulfovibrio sp. strain TBP-1, a strain originally isolated from marine sediment. Although the remaining 2 sequences were less homologous to Desulfovibrio sp. strain TBP-1, they did not match any other sulfate-reducing (or other) species in GenBank.

KEY WORDS: Coral disease · Black band disease · Sulfate-reducing bacteria · Desulfovibrio · Florida Keys · Dominica

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INTRODUCTION consortium (Richardson et al. 1997, Bythell et al. 2002, Richardson 2004). The microbial consortium is domi- Black band disease (BBD) of scleractinian corals is nated by cyanobacteria (Rützler & Santavy 1983, considered to be an important factor in the decline and Bythell et al. 2002, Cooney et al. 2002, Frias-Lopez et al. community restructuring of coral reefs (Rosenberg & 2003, Richardson 2004), sulfide-oxidizing bacteria pre- Loya 2004). This disease directly causes coral tissue sumed to be Beggiatoa spp. (Ducklow & Mitchell 1979, lysis as it progresses over an infected coral colony, lead- Viehman & Richardson 2002), marine fungi (Ramos- ing to either partial or complete death of the colony. Not Flores 1983), sulfate-reducing bacteria (SRB) including only is coral tissue destroyed by the disease, but it has Desulfovibrio (Chet & Mitchell 1975, Garrett & Duck- been shown that scleractinian coral recruitment onto low 1975, Cooney et al. 2002, Frias-Lopez et al. 2002), the newly available substrate of exposed coral skeleton and many other heterotrophic microbes (Cooney et al. produced by BBD is negligible (Edmunds 1991, Bruck- 2002, Frias-Lopez et al. 2002). Few members of the con- ner & Bruckner 1997, Kuta & Richardson 1997), further sortium have been cultured and no single or primary reducing coral cover on reefs (Kuta & Richardson 1996). pathogen has been identified. Characterized by a distinct black or dark red band, The complexity of the BBD microbial community has BBD is composed of a complex and distinct microbial recently been explored using molecular techniques,

*Email: [email protected] © Inter-Research 2006 · www.int-res.com 120 Dis Aquat Org 69: 119–127, 2006

which revealed a high degree of microbial diversity strains were <97.5% similar in sequence homology to and variability among infections (Cooney et al. 2002, the closest related GenBank deposit, and only partial Frias-Lopez et al. 2002, 2003). However, the molecular gene sequences were used. Therefore, these results techniques used in these studies, while providing a are subject to some sequence bias and are probably broad survey of the BBD microbial community, may not indicative of a strict identification at the species not account for all bacteria present, particularly those level (Stackebrandt & Goebel 1994). Bacterial strains present at low densities. with >97.5% homology to Desulfovibrio salexigens BBD has an active sulfur cycle similar to that found in and Desulfovibrio sp. strain TBP-1 were found in one microbial mats in extreme environments such as hot- survey of partial bacterial sequences cloned from 3 dif- spring outflows and hypersaline ponds (Carlton & ferent BBD infections on different coral species in Richardson 1995). Both BBD and such microbial mats Curaçao (Frias-Lopez et al. 2002). No single Desul- contain anoxic and sulfide-rich microenvironments at fovibrio species was common to the different infec- depth in the mat. In the case of BBD, this zone is adja- tions. cent to the coral tissue, a condition which has been SRB were also found on denuded coral skeleton shown to cause coral tissue death (Richardson et al. behind BBD infections (Frias-Lopez et al. 2002). One 1997). In BBD, the source of this sulfide is biological, clone, sequenced from the bacterial community on the result of dissimilatory sulfate reduction (Richard- denuded coral skeleton, had 99% homology to Des- son 2004). SRB are ecologically significant in marine or ulfovibrio sp. strain TBP-1. Although multiple De- hypersaline microbial mat environments where, as the sulfovibrio species were found per sample from both dominant bacteria in the anaerobic zone, they may BBD and denuded coral skeleton behind the band, no oxidize up to 50% of total organic carbon in support single Desulfovibrio species was found on more than 1 of sulfate respiration (Howarth & Hobbie 1982, Jør- infection (Frias-Lopez et al. 2002). Additional clones gensen 1982). In seawater, the concentration of sulfate were reported as <97.5% homologous to D. zosterae is abundant at approximately 28 mM (Hansen 1993, and D. alaskensis, respectively. Clones identified Odom 1993), providing a sufficient sulfate supply for as most closely matching other genera of SRB were SRB to outcompete methanogens in an anaerobic also sequenced from denuded coral skeleton behind microenvironment, and thus act as terminal oxidizers the band, but again at <97.5% homology, thus not in the anaerobic degradation of organic matter to CO2 sufficient for a positive identification. These included (Jørgensen & Fenchel 1974). In a marine anoxic envi- Desulfofrigus oceanense strain Asv26, Desulfobotu- ronment, the influx of organic matter can be a control- lus sp. strain BG14, Desulfocella halophila, Desul- ling factor for the rate of sulfate reduction (Jørgensen forhopalus singaporensis, Desulfofrigus fragile, and 1982). Desulfobacterium catecholicum (Frias-Lopez et al. Desulfovibrio spp. are commonly found in marine, 2002). estuarine and freshwater environments that are both Perhaps the most obvious explanation for the varia- anaerobic and reducing (Widdel & Pfennig 1984). tion in Desulfovibrio species among BBD infections is Although SRB carry out a potentially integral role in that SRB fill an ecophysiological niche rather than a the etiology of BBD by producing toxic sulfide, charac- species-specific niche in BBD. Given the bias inherent terization of SRB associated with BBD has been limited in the techniques used, more Desulfovibrio species to date. SRB in BBD were first postulated to be Desul- may, in fact, have been present in the disease band fovibrio spp. in the 1970s (Garrett & Ducklow 1975). than were cloned or sequenced; thus only a subsample This was supported with fluorescence in situ of species present were detected in each sample. hybridization analyses using SRB probes and confocal While theoretically providing a broad survey of the laser microscopy of the BBD microbial community composition and variability of the BBD community, the (Schnell et al. 1996). Maximal growth of enrichment polymerase chain reaction (PCR) based approach cultures of SRB from BBD on hydrogen and lactate, employed by these studies has an inherent bias for when compared with other electron donors, supported under-representation of bacteria that may be present their identification as Desulfovibrio spp. (Schnell et al. at low densities. The question thus remains as to 1996). which, if any, microbial components of the disease are Two studies using culture-independent cloning of conserved between individual BBD infections and the 16S rRNA genes from BBD-associated microbial among various geographic areas. communities have shown a high diversity of bacteria The objective of this study was to culture and specif- within the disease consortium (Cooney et al. 2002, ically select for Desulfovibrio spp. from different BBD Frias-Lopez et al. 2002). These studies identified a infections on reefs in different regions of the variety of Desulfovibrio and other sulfate-reducing Caribbean. The 16S rRNA gene sequences were used genera present in BBD infections. Most of the cloned to compare the samples from this study to those found Viehman et al.: Desulfovibrio present in BBD of corals 121

in previous BBD studies as well as Table 1. Sampling location and host coral species for black band disease (BBD) to environmental SRB not associated sulfate-reducing bacteria culturing and sequencing with BBD. Sampling location Coral species Culture identification

MATERIALS AND METHODS Picture Reef, Biscayne Diploria clivosa BBD-2 National Park, Florida BBD-3.1 Field sampling. Active BBD was D. strigosa BBD-6 sampled in 2 locations in the Flo- Dominica Siderastrea siderea 1 BBD-10 rida Keys — Picture Reef in Biscayne BBD-11a National Park (BNP) and Algae Reef in S. siderea 2 BBD-13.1 the Florida Keys National Marine Sanc- BBD-15 tuary (FKNMS) — plus 1 reef in S. siderea 3 BBD-16 BBD-19 Dominica: Tarou Point (Fig. 1). Six infected corals of 4 coral species, Diplo- Algae Reef, Florida Keys Diploria labyrinthiformis BBD-22 National Marine ria clivosa, D. labyrinthiformes, D. Sanctuary, Florida strigosa, and Siderastrea siderea, were aBBD-11.2 (not included here) was from the same culture as BBD-11 sampled (Table 1). BBD samples were

collected in situ on the reef by a SCUBA diver in July and September 2000. Sampling was conducted using sterile 10 ml syringes to directly collect BBD. Upon return to the boat, samples in sealed syringes were maintained at ambient seawater temperature in cool- ers with low light, and then transported or shipped to the laboratory for immediate culture. Enrichment and isolation of sulfate-reducers. The contents of the sampling syringes were inoculated directly into anoxic vials of Sulfate API Broth (Difco Laboratories) made with natural seawater. Prior to autoclaving and inoculation, air was removed from the

sealed vials in alternating cycles of vacuum and N2 injected through a syringe apparatus. Media were always reduced prior to inoculation with either tita-

nium NTA or 200 mM H2S (final concentration) until a resazaurin indicator in the media indicated reducing (–Eh) conditions. Sulfate API media contains, per liter

of distilled H2O, bacto yeast extract (1.0 g), ascorbic acid (0.1 g), sodium lactate (5.3 g), magnesium sulfate (0.2 g), dipotassium phosphate (0.01 g), ferrous ammo- nium sulfate (0.1g), and sodium chloride (10.0g). Inoc- ulated vials were visually monitored for an intense black FeS precipitate, indicative of bacterial sulfate reduction. Standard microbiological techniques were used for culture of SRB from BBD. Subsampling of original in- oculations and plating were carried out within a Coy anaerobic chamber (Coy Laboratory Products) contain-

ing approximately 90% N2:10% H2 and trace amounts of CO2. Subsamples were inoculated into new vials of Sulfate API Broth. For isolation of individual colonies, agar plates were made with Sulfate API Broth, modified iron sulfate (Postgate 1984), or Post- Fig. 1. Sampling sites (A) in the Florida Keys and (B) on the island of Dominica. BNP: Biscayne National Park; FKNMS: gate’s B media (Postgate 1979), each using filtered Florida Keys National Marine Sanctuary (Whatman no. 1) or autoclaved seawater, Instant 122 Dis Aquat Org 69: 119–127, 2006

Ocean, or distilled water with addition of NaCl. Single Sequence analysis. The gene sequences obtained in colonies producing a strong FeS precipitate were this study from both cloning and direct-PCR were picked using a sterile glass pipette and transferred to compared with those in the GenBank database using Sulfate API Broth. Broth cultures that produced an FeS the basic local alignment search tool (BLAST) precipitate were re-inoculated onto modified iron sul- (www.ncbi.nlm.nih.gov). Only cloned sequences fate agar plates and again subsampled into sealed obtained in this study were used for phylogenic com- anaerobic vials of Sulfate API Broth prior to removal parisons. Twenty-eight sequences of Desulfovibrio from the anaerobic chamber. spp. and 8 sequences of delta proteobacterium were DNA extraction and sequencing. Total DNA was ex- extracted from GenBank and used in phylogenetic tracted from each SRB culture using a Soil DNA Isolation comparisons. Seven sequences of SRB were also Kit (MoBio Laboratories) and was PCR amplified on an included for use as outgroups. Many of these MJ Research PTC 200 thermal cylinder. The PCR reac- sequences were chosen because they were homolo- tion consisted of 1× buffer; 2.5 mM MgSO4; 0.25 µM gous to sequences from this study or to sequences dNTPs; 0.5 µM forward primer; 0.5 µM reverse primer; obtained in previous BBD molecular studies (Cooney 5U µl–1 Tfl or Taq polymerase (Promega); 0.1% bovine et al. 2002, Frias-Lopez et al. 2002). Others were cho- serum albumin (BSA, fraction V) and DEPC H2O to 20 µl. sen as a random sample of complete or nearly com- To each reaction tube, 1 µl DNA was added. Cycling plete Desulfovibrio spp. 16S rRNA gene sequences parameters were: 94°C for 2 min; 30 cycles of: 94°C for present in the GenBank database. In addition, 8 spe- 2 min, 55°C for 15 s, and 72°C for 30 s; followed by a final cies of unidentified bacterial 16S rRNA genes from extension at 72°C for 10 min. Universal bacterial previous molecular studies of BBD and from bacteria polymerase chain reaction (PCR) primers used were found on denuded coral adjacent to the band were [5’→3’] forward primer: FD1 (AGAGTTTGATCCTG- extracted from GenBank based on the published GCTCAG) and reverse primer: RD1 (AAGGAGGT- results of Cooney et al. (2002) and Frias-Lopez et al. GATCCAGCC) (Weisburg et al. 1991), obtained from (2002). All 16S rRNA gene sequences that were the Gibco Life Technologies. Negative controls contained no most homologous to Desulfovibrio found in BBD infec- DNA. Positive controls included cultures from this study tions in this study and in other BBD studies (Cooney et that had previously been amplified with the given al. 2002, Frias-Lopez et al. 2002) were compared to primer set, and a known Desulfovibrio strain, Desulfo- Desulfovibrio sequences in GenBank. vibrio desulfuricans subspecies desulfuricans DSM 1924 Desulfovibrio spp. sequences were aligned on (DSMZ – Deutsche Sammlung von Mikroorganismen ClustalX (Thompson et al. 1997) with the cloned se- und Zellkulturen). PCR products were visualized on a quences obtained from this study. Alignments were 1% agarose gel in TBE buffer stained with ethidium bro- visually checked on both ClustalX and MacClade (Mad- mide. PCR fragments were purified for sequencing using dison & Maddison 2000). Analyses and tree construction Qiagen Qiaquick PCR Purification Kit (Qiagen). Three were done using PAUP 4.0 (Swofford 1999). Heuristic cultures that were partially sequenced from direct PCR search parameters were as follows: starting trees were were also cloned to avoid any bias in phylogenetic analy- obtained via neighbor-joining, sequences were added sis from culture impurity. In each case the 16S rRNA as-is, keeping best trees only, and the tree-bisection-re- gene PCR fragment from FD1 to RD1 was cloned using a combination (TBR) branch swapping algorithm and TOPO TA cloning kit (Invitrogen) following the manu- steepest descent were used. Bootstrap support was ob- facturer’s recommended protocols. The plasmid insert tained for 1000 replicates. Outgroups were assigned to was sequenced with M13F and M13R primers (Invitro- be species other than Desulfo vibrio. These included gen). Other primers used to sequence the PCR product Desulfofrigus fragile Desulfobotulus sp. BG14, Desulfu- included: [5’→3’] FD1, 519R (G(G/T)ATTACCGCGGC ricans oceanense strain Asv26, Desulfocella halophila, (A/T)GCTG) (Lane et al. 1985), 926F (AAACTYAA Desulfobacterium catecholicum, and Desulforhopalus AG(A/T)AATTGACGG) (Britschgi & Fallon 1994), and singaporensis. A neighbor-joining algorithm with a RD1 (Weisburg et al. 1991). Optimal primers were de- Jukes-Cantor distance measure was used. signed using the OLIGO Primer Analysis Software (Mol- Reference 16S rDNA sequences extracted from the ecular Biology Insights) and obtained from Gibco Life GenBank database used in phylogenetic analysis follow: Technologies. These were [5’→3’] 514F (CACTG (1) Desulfovibrio spp.: Desulfovibrio. sp. D1 A318380, GGCGTA AAGC), 1040R (CTCGTTGCGGGACTT), Desulfovibrio sp. D4 AF192155, Desulfovibrio sp. FD1 and 1056F (AAGTCCCGCAACGAG). Cycle sequenc- AJ251631, Desulfovibrio sp. strain KH2 X93147, Desul- ing was done on an MJ Research PTC 200 using the Big fovibrio sp. Met 82 AY062930, Desulfovibrio sp. PCP1 Dye terminator Cycle Sequencing kit version L AF194417, Desulfovibrio sp. TBP-1 AF090830, Desul- (Perkin-Elmer) and run on an ABI Prism™ 377 Auto- fovibrio sp. ‘Bendigo A’ AF131234, Desulfovibrio sp. mated DNA Sequencer (Perkin-Elmer). strain STL 1 X99501, D. acrylicus U32578, D. aespoe- Viehman et al.: Desulfovibrio present in BBD of corals 123

ensis X95230, D. africanus X99236, D. alaskensis TBP-1, and 95% homologous to D. acrylicus (Table 2). Y11984, D. alcoholovorans AF053751, D. aminophilus BBD-11.2, D. acrylicus, and Desulfovibrio sp. strain AF067964, D. desulfuricans subsp. desulfuricans strain TBP-1 grouped separately, but with a low degree of Essex 6 AF192153, D. desulfuricans subsp. desulfuricans support to each other (Fig. 2). The degree of separation strain MB AF192154, D. fairfieldensis U42221, D. gabo- between the cluster containing BBD-11.2, D. acrylicus, nensis U31080, D. giganteus AF418170, D. halophilus and Desulfovibrio sp. strain TBP-1 and that containing U48243, D. indonensis Y09504, D. magneticus D43944, BBD-3.1, 13.1, CD12A12, and CD12B13 remains an D. mexicoense AF227984, D. oryzae AF273083, D. pro- unresolved polytomy. This is indicative of the lack of fundus AF418172, D. salexigens M34401, D. vietnamen- resolution within this genus when using the 16S rRNA sis X93994, D. zosterae Y18049. (2) Other bacterial gene as a phylogenetic marker. species: CD12A12 AF441965, CD13B10 AF441876, delta Sequencing directly from PCR products yielded 8 proteobacterium RS15 AJ289743, delta proteobacterium partial 16S rRNA gene sequences from SRB cultures of RS64 AJ289756, SRB LZK1 AF216645, uncultured bac- 6 different BBD infections from 4 different coral spe- terium 128-3-12 AF473924, uncultured delta proteobac- cies. Six of these sequences, representing 5 out of terium 128-11 AF473908, uncultured delta proteo- 6 infections sampled, revealed >97% homology in a bacterium 128-12 AF473909, uncultured delta pro- BLAST search to 2 uncultured delta proteobacterium teobacterium 34-35 AF473968, Desulfobotulus sp. clones, CD13B10 and CD12A12, both cloned from BBD BG14 U85470, Desolfocella halophila AF022936, De- on Montastraea cavernosa in Curaçao by Frias-Lopez sulfobacterium catecholicum AJ237602, Desulfofrigus et al. (2002). Only 1 BBD sample, BBD-15 cultured from fragile AF099065, Desulfofrigus oceanense strain ASv26 BBD on Siderastrea siderea in Dominica, did not yield AF099064, Desulforhopalus singaporensis AF118453 a SRB with a high degree of homology (<97.5%) to Desulfovibrio spp. in GenBank. No relationship was supported between infected coral species and BBD- RESULTS SRB species cultured. The partial sequences obtained in this study from direct PCR were not used in phyloge- Successful enrichment cultures of SRB from BBD netic analysis to avoid compounding bias from trying turned black after approximately 2 to 3 d, indicating to extrapolate relatedness of short sequences to longer the precipitation of FeS. The best culture results were or more complete sequences. obtained using modified iron sulfate media in natural seawater. Although every BBD sample inoculated into enrichment media eventually indicated sulfate- DISCUSSION reduction, the limiting factor for this study was the appearance of single colonies for selection. Although a The use of culture techniques to assess microorgan- variety of BBD bacteria grew on the media, only single isms in natural samples vastly underestimates micro- black colonies were selected on solid media for trans- bial community diversity (Furhman & Campbell 1998). feral to new media. Ten cultures were used in this Possibly as few as ≤1% of bacteria present in nature can study. be cultured (Hicks et al. 1992). In this study, selective The 16S rRNA gene sequences were obtained from 3 culturing techniques were used to specifically enrich cloned isolates, and 8 sequences were obtained for BBD SRB with similar growth requirements (i.e. directly from the PCR product from cultures. Of the 3 members of the genus Desulfovibrio) from different cloned 16S rRNA gene sequences, 2 nearly complete BBD infections. Although this approach may have arti- sequences were obtained. When aligned, both of these ficially selected for Desulfovibrio spp. found at back- (BBD-3.1 from Biscayne National Park and BBD-11.2 ground levels in BBD infections, perhaps at low levels from Dominica, cultured from Diploria clivosa and not necessarily detectable by molecular survey tech- Siderastrea siderea, respectively; see Table 1) were niques, we believe that this approach addresses the hy- 99% homologous to Desulfovibrio sp. TBP-1, and 98% pothesis that individual Desulfovibrio spp. specifically homologous to D. acrylicus and Desulfovibrio sp. PCP- fill an ecological niche within BBD in different infec- 1 (Table 2). BBD-3.1 from this study was very closely tions. Additional species of SRB may also be important related to 2 partial bacterial sequences cloned from in the BBD microbial consortium, but were not assessed BBD in a previous study (Frias-Lopez et al. 2002) and in this study. We found the presence of bacterial species deposited as CD12A12 and CD13B10 (Fig. 2). BBD- with a high degree of homology to the same Desul- 13.1 showed a lower degree of relatedness to the same fovibrio species, cultured from both Florida Keys sam- group. The BLAST result for the 3rd cloned 633 bp ples and from Dominica samples as well as sequenced sequence (BBD-13.1 cultured from BBD on S. siderea from a sample in Curaçao (Frias-Lopez et al. 2002). in Dominica) was 96% homologous to Desulfovibrio sp. Thus our results suggest that this species may be com- 124 Dis Aquat Org 69: 119–127, 2006

Table 2. 16S rRNA gene sequence homologies from BBD sulfate-reducing bacteria. Both full and partial sequences were used

Sample Primers No. GenBank % homology Source of GenBank sequence identification of bases accession no. with highest homology

BBD-2 FD1 487 AY907525 97 Uncultured delta proteobacterium clone CD13B10b RD1 358 AY907526 97 Uncultured delta proteobacterium clone CD12A12b 96–97 Desulfovibrio acrylicus BBD-3.1a Various 1476 AY750147 99 Desulfovibrio sp. strain TBP-1 98 D. acrylicus BBD-6 FD1 486 AY907527 98–99 Uncultured delta proteobacterium clone CD13B10b RD1 432 DQ020004 97–99 Uncultured delta proteobacterium clone CD12A12b 97–99 Desulfovibrio sp. strain TBP-1 97–99 D. acrylicus BBD-10 FD1 211 AY907528 99 Uncultured delta proteobacterium clone CD13B10b RD1 407 AY907529 99 Uncultured delta proteobacterium clone CD12A12b 96–99 Desulfovibrio sp. strain TBP-1 95–98 D. acrylicus BBD-11 FD1 560 AY907530 96–99 Desulfovibrio sp. strain TBP-1 519R 421 95–98 D. acrylicus BBD-11.2a Various 1536 AY750148 99 Desulfovibrio sp. TBP-1 98 D. acrylicus BBD-13.1a M13 633 AY750149 96 Desulfovibrio sp. TBP-1 FD1 95 D. acrylicus BBD-15 519R 367 AY907531 94 Delta proteobacterium RS64 94 D. acrylicus 94 Desulfovibrio sp. strain TBP-1 BBD-16 FD1 471 AY907532 98–99 Uncultured delta proteobacterium clone CD13B10b RD1 533 AY907533 98–99 Uncultured delta proteobacterium clone CD12A12b 926F 293 AY907534 97–99 Desulfovibrio sp. strain TBP-1 97–99 D. acrylicus BBD-19 RD1 664 AY907535 98 Uncultured delta proteobacterium clone CD13B10b 98 Uncultured delta proteobacterium clone CD12A12b 97 Desulfovibrio sp. strain TBP-1 97 D. acrylicus BBD-22 FD1 241 AY907536 98 Uncultured delta proteobacterium clone CD13B10b 98 Uncultured delta proteobacterium clone CD12A12b 98 Desulfovibrio sp. strain TBP-1 98 D. acrylicus aSequence was cloned. All other sequences were obtained from PCR products bThis sequence was also identified in other BBD samples reported in Frias-Lopez et al. (2002) mon to the BBD microbial community and may play the ent areas of the wider Caribbean. All but 2 of the cul- role of a required BBD consortium member. tures had 16S rRNA gene sequences (partial to almost The Desulfovibrio cultures obtained in this study were complete) with >97.5% homologies to Desulfovibrio sp. presumably able to outcompete other SRB present be- TBP-1 and D. acrylicus (Table 2). Six of these sequences cause of the provision of much higher levels of lactate as were also highly homologous to the uncultured delta opposed to yeast extract. Use of lactate as a preferred proteobacterium clones CD13B10 and CD12A12, both carbon source is characteristic of the genus Desulfovib- obtained from BBD (Frias-Lopez et al. 2002). Although rio. Although the yeast extract present in the media is an 2 of our 10 sequences, both from the same Siderastrea additional or alternative carbon source, it was present in siderea infection in Dominica, were most homologous a much lower amount and thus would not confer a selec- to Desulfovibrio sp. TBP-1 and D. acrylicus, they had tive advantage for growth of additional BBD sulfate re- <97.5% homology (Stackebrandt & Goebel 1994). ducing genera that may be present. Presumably, a wide Only partial sequences were obtained from direct variety of carbon compounds are produced during the ly- PCR sequencing from 8 of our 10 cultures, and in 1 of sis of coral tissue that may potentially be used by repre- the 3 clones analyzed in this study. Therefore, direct sentatives of the different genera of sulfate-reducers. comparisons of our results to those of the earlier stud- In this study we obtained 10 cultures of SRB from 6 ies (Cooney et al. 2002, Frias-Lopez et al. 2002) are different BBD infections, from 3 different reefs in differ- complicated by the fact that the location of the Viehman et al.: Desulfovibrio present in BBD of corals 125

Fig. 2. Phylogenetic tree of Desulfovibrio spp. sequenced in this study, Desulfovibrio spp. from the environment (GenBank), and Desulfovibrio and other sulfate-reducing bacteria (SRB) cloned from black band disease (BBD) infections in other studies, from 1000 bootstrap replicates with Jukes-Cantor distance measure. Only bootstrap support values above 50% are presented. SRB sequences of clones from this study are in boxes. SRB sequences of clones from Frias-Lopez et al. (2002) are underlined sequences of the 16S rRNA gene may not overlap with high degree of homology to Desulfovibrio sp. TBP-1 the exact sequence locations in each study. For exam- and D. acrylicus we cannot conclude that each partial ple, the SRB BBD-15 (Table 2) was sequenced using sequence represents the same species because the primer 519R (thus sequencing more toward the 5’ end sequences were located in different regions of the 16S of the gene), and therefore was not comparable to the rRNA genes. sequences of the 3’ end from the uncultured delta pro- An alternative aspect of using partial gene sequence teobacteria (CD12A12 and CD13B10) cloned from BBD data to study microbial communities is the problem of by Frias-Lopez et al. (2002). This lack of overlap is the ubiquitous species that may be routinely identified, but reason why partial sequences obtained from direct falsely interpreted as important constituents in the PCR were not included in phylogenetic analysis. niche being studied. For example, both Desulfo- Therefore, even though all 3 of these sequences had a vibrio sp. TBP-1 and D. acrylicus that were originally 126 Dis Aquat Org 69: 119–127, 2006

deposited in GenBank came from marine or estuarine regard to rRNA structure, even though such variation sediment samples (van der Maarel et al. 1996, Boyle et considerably influences evolution (Yang 1996). al. 1999) and, therefore, may be ubiquitous in marine In this study, only limited phylogenetic clustering sediments. Frias-Lopez et al. (2002) found Desulfovib- was found between non-BBD related Desulfovibrio rio sp. TBP-1 in 1 out of 3 samples of the sediment on species and those found in (or highly homologous to the denuded coral surface behind the disease band. An those found in) BBD. In addition, no relationships were additional problem can arise when the partial se- shown between Desulfovibrio species from BBD and quences surveyed are highly conserved within the the species of coral infected. Two Desulfovibrio spp. genus and/or species. This can lead to misinterpreta- cultured in this study, BBD-3.1 and BBD-13.1 (Fig. 2), tion of the true identity of the species when the genetic were closely related to 2 clones from a BBD infection marker of choice (i.e. 16S rRNA) is not completely on a Montastraea cavernosa in Curaçao (Frias-Lopez sequenced. Therefore, results from partial sequences et al. 2002). However, this group clustered separately must be cautiously interpreted when assigning niche (although via an unresolved polytomy) from the cluster and/or community status to such data. including D. acrylicus, Desulfovibrio sp. D1, Desul- Desulfovibrio sp. TBP-1 and D. acrylicus share fovibrio sp. strain TBP-1, BBD-11.2 from this study, and 98.9% sequence homology to each other, and both the non-BBD delta proteobacterium RS15, despite high were implicated as important, and perhaps common, degrees of sequence homology. members of the BBD microbial consortium. However, In summary, we found evidence that BBD may con- these 2 species have different physiological character- tain specific SRB species common to BBD infections istics. D. acrylicus, unlike Desulfovibrio sp. strain both on different coral hosts and in different regions of TBP-1, can reduce acrylate but cannot grow on lactate the wider Caribbean. While our findings support the and acrylate with >4% NaCl in growth media. hypothesis that SRB may be filling an ecological niche Desulfovibrio sp. TBP-1, unlike D. acrylicus, can also in BBD, and that there is no coral species specificity, grow on fumarate and sulfate (Boyle et al. 1999). This our results can also be interpreted as evidence that illustrates another limitation of the exclusive use of 16S there is in fact microbial species specificity. We believe rRNA gene sequences for identification and analysis of that additional physiological studies aimed at culturing microbial communities in that when more than one specific functional groups, genera, or even species, species is implicated as present, knowledge of the supplemented by molecular studies targeting func- physiological properities of known species should be tional gene expression, must be carried out to support considered in interpreting the molecular data. Within BBD microbial community assessment using PCR. this study, clones and PCR products of samples were Another promising approach is use of real time PCR to analyzed in terms of homology to only the 16S rRNA quantify the number of microbial species present. Only gene. Sequence homology to functional or physiologi- in this way can we determine which microbial species cally related genes would provide more information to are functionally important in the natural environment. more accurately identify species present. The lack of resolution at so many branches in the phylogenetic tree further indicates the lack of resolution of the Acknowledgements. We thank J. Borger, D. Diresta, R. Curry, and M. Walker for sample assistance. We also thank Biscayne 16S rRNA gene for this genus. However, the 16S rRNA National Park (BNP) and the Florida Keys National Marine gene was targeted to allow comparison of our results to Sanctuary (FKNMS) for research vessel support. Sample col- other studies of BBD SRB. lection in the FKNMS was carried out under National Marine Other factors to consider in these types of studies, in Sanctuary Permit FKNMS-2000-056 and in BNP was included particular when different research groups find signifi- under National Park Service Permit BISC 00–032. We thank E. Weil, G. Smith, D. Gil-Agudelo, G. Piniak and 3 anonymous cant differences in species homologies and low levels reviewers for helpful comments on the manuscript. This of bootstrap support, could be based on the inherent research was funded by an EPA STAR graduate fellowship biases of the phylogenetic methods used. The Jukes- (U-91562001 to S.V.). Funding from NIH (NIH/NIGMS Cantor distance analysis has the underlying assump- SO6GM8205 to L.L.R. and D.K.M.) and NSF (NSF ADVANCE tion that all bases are equally likely to vary at the same 0340695 to D.K.M.) assisted in presenting and producing this document. 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Submitted: September 15, 2004; Accepted: November 7, 2005 Proofs received from author(s): December 12, 2005