Sulfur-oxidizing bacterial populations within cyanobacterial dominated coral disease lesions

Sulfur-oxidizing bacterial populations withincyanobacterial dominated coral disease lesions

David G. Bourne,1* Marc J. J. van der Zee,1

Emmanuelle S. Botté1 and Yui Sato1,2

1Australian Institute of Marine Science, Townsville, Qld,Australia.2ARC Centre of Excellence for Coral Reef Studies andSchool of Marine and Tropical Biology, James CookUniversity, Townsville 4811, Qld, Australia.

Summary

This study investigated the diversity and quantitativeshifts of sulfur-oxidizing bacteria (SOB) during theonset of black band disease (BBD) in corals usingquantitative PCR (qPCR) and cloning approaches tar-geting the soxB gene, involved in sulfur oxidation.Four Montipora sp. coral colonies identified withlesions previously termed cyanobacterial patches(CP) (comprising microbial communities differentfrom those of BBD lesions), was monitored in situas CP developed into BBD. The overall abundanceof SOB in both CP and BBD lesions were very lowand near the detection limit of the qPCR assay,although consistently indicated that SOB populationsdecreased as the lesions transitioned from CP toBBD. Phylogenetic assessment of retrieved soxBgenes showed that SOB in both CP and BBD lesionswere dominated by one sequence type, represent-ing > 70% of all soxB gene sequences and affiliatedwith members of the Rhodobacteraceae within thea-Proteobacteria. This study represents the firstassessment targeting SOB within BBD lesions andclearly shows that SOB are not highly diverse orabundant in this complex microbial mat. The lack ofoxidation of reduced sulfur compounds by SOB likelyaids the accumulation of high levels of sulfide at thebase of the BBD mat, a compound contributing to thepathogenicity of BBD lesions.

Introduction

Black band disease (BBD) in corals manifests as acomplex microbial mat dominated by phototrophic fila-mentous cyanobacteria and other diverse heterotrophic

bacterial groups (Richardson, 1997; Cooney et al.,2002b; Frias-Lopez et al., 2004; Viehman et al., 2006).This darkly pigmented microbial consortium migratesover live coral colonies, causing necrosis of the under-lying tissue due to the anoxic and sulfide-rich environ-ment in the lower parts of the mat (Carlton andRichardson, 1995; Richardson et al., 1997; Glas et al.,2012). In addition to the dominant cyanobacterialbiomass, large numbers of sulfate-reducing bacterial(SRB) populations from the genus Desulfovibrio havebeen detected (Frias-Lopez et al., 2002; 2004; Cooneyet al., 2002b; Barneah et al., 2007) and quantified in themicrobial mat (Bourne et al., 2011). Given the highsulfide concentration in the lower parts of the mat, it canbe hypothesized that sulfur-oxidizing bacteria (SOB) areresponsible for the recycling of inorganic sulfur com-pounds (sulfide, sulfite and thiosulfate) to sulfate, pro-viding the necessary compounds for SRB in the matsuch as Desulfovibrio species (Stal, 1995). Although thegeneral microbial diversity of the BBD has been studiedin detail (as reviewed in Miller and Richardson, 2011),there is little evidence directly demonstrating that SOBactually occur in the microbial mat of BBD. Based onmicroscopic evidence, previous studies suggested thatthe sulfur-oxidizing bacterium Beggiatoa is present inBBD lesions (Rutzler and Santavy, 1983; Richardson,1996; Richardson et al., 2009) and acts as stabilizers ofsulfide dynamics (Richardson, 2004). However, morerecent molecular-based studies targeting bacterial 16SrRNA genes have not retrieved sequences affiliated withBeggiatoa spp. from the BBD microbial mat (Frias-Lopezet al., 2002; 2004; Cooney et al., 2002a; Barneah et al.,2007). A study by Sekar and colleagues (2008) reporteda Beggiatoa spp.-related 16S rRNA gene sequence,although this is not supported by direct sequencecomparisons (< 85% sequence identity to Beggiatoasequences). The present study therefore directly inves-tigated the diversity and abundance of SOB populationsin BBD lesions found at an established disease outbreaksite on the fringing reefs of Pelorus Island in the centralGreat Barrier Reef (GBR). Ecological studies at this sitehave identified the role of light and temperature in thedynamics of BBD prevalence (Sato et al., 2009) andcharacterized an earlier lesion termed ‘cyanobacterialpatches (CP)’ which precedes BBD in some cases (Satoet al., 2010). During the transition from CP to BBD, a

Received 13 December, 2012; accepted 20 March, 2013. *For cor-respondence. E-mail [email protected]; Tel. (+61) 747534139;Fax (+61) 747725852.

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Environmental Microbiology Reports (2013) doi:10.1111/1758-2229.12055

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shift in the dominant cyanobacterial species occurs inaddition to changes in heterotrophic bacterial communi-ties (Sato et al., 2010), SRB populations (Bourne et al.,2011), and oxygen and sulfide dynamics within microbialmats (Glas et al., 2012). Virulence of lesions (linearprogression rate) increases during the development ofBBD from CP, and thus comparisons between CP andBBD facilitate identification of factors important for BBDpathogenicity.

To clarify the putative occurrence of SOB on diseasedcoral colonies from Pelorus Island, we specifically inves-tigated the diversity of SOB within the CP and BBDlesions and provided a quantitative assessment of thesepopulations during the progressive development of BBDfrom CP. We developed a quantitative real-time PCRapproach targeting the soxB gene, which encodes thesoxB component of a multienzyme complex involved inthe oxidation of thiosulfate to sulfate and responsiblefor complete oxidation of sulfide (Friedrich et al., 2001;Kappler and Dahl, 2001; Azai et al., 2009). The soxB geneis an excellent marker for targeting SOB populations sinceit contains highly conserved regions and is present in allknown SOB organisms (Friedrich et al., 2001; Meyeret al., 2007). Due to variability in bacterial populationdiversity and sampled biomass within individual corallesions, the SOB population was determined as a per-centage of the total bacterial population by including aquantitative real-time PCR assay targeting the bacterial16S rRNA gene (Nadkarni et al., 2002). Here we demon-strate the existence, in low abundance, of novel commu-nities of SOB populations within the microbial lesionsduring the onset of BBD.

Results and discussion

Four individually tagged Montipora hispida coral coloniesbearing cyanobacterial patches (CP) were monitored at3 m depth at fringing reefs of Pelorus Island (centralinshore GBR; 18°33′S, 146°30′E) at approximately2-week intervals as CP developed into characteristic BBDsigns (see Sato et al., 2010 for detailed description ofchanges in macroscopic signs from CP to BBD). For eachstage (CP and BBD), biomass from the microbial mat andunderlying skeleton (approximately 5 mm in diameter,2 mm in depth) was collected from the progression front ofthe lesion using a sterilized stainless steel chisel andprocessed for total DNA extraction as outlined previously(Sato et al., 2010). The diversity of soxB genes from theindividual stages was determined through PCR amplifica-tion using primers soxB-F + (5′-ATCGGNCARGCNTTYCCNTA-3′) and soxB-R (5′-CATGTCNCCNCCRTGYTG-3′)(Krishnani et al., 2010). Amplified products (~ 750 bp insize) were cloned (TOPO TA Kit, Invitrogen, CA, USA)and sequenced. A total of 271 soxB DNA sequences

derived from four CP and four BBD samples wereretrieved from the eight clone libraries. Protein sequenceswere generated and operational protein units (OPUs)determined using the MOTHUR program (Schloss et al.,2009) at the 90% sequence identity level. A total of 20OPUs were identified, 11 shared between CP and BBDlesions, seven unique to CP and three unique to BBDsamples (Fig. 1 and Fig. S1). Many OPUs contained onlya single or small number of sequences from the CP and/orBBD lesions. Rarefaction analysis revealed that diversityof SOB within the lesions was relatively well representedfor both CP and BBD lesion types using the soxB specificprimers employed, although SOB in CP samples wereslightly more diverse (Fig. S2).

Phylogenetic analysis (see Supporting information fordetailed methods) of OPU sequences derived from CPand BBD libraries showed that the majority of identifiedsequences had less than 90% amino acid sequenceidentity (range from 67% to 91%) to previously identi-fied sulfate thioesterase/sulfate thiohydrolase proteinsequences present in the NCBI-nr protein database(Fig. 1). OPU-1 was dominant in all CP and BBD clonelibraries, containing 134 of the 170 sequences derivedfrom CP libraries and 68 of the 104 sequences from BBDlibraries (Fig. 1). There was no change in the diversity ofdominant SoxB sequences retrieved between the CP andBBD lesion stages. This is in contrast with previous inves-tigations which demonstrated a shift in dominant Cyano-bacteria, heterotrophic bacteria and SRB populations asthe lesions transitioned from CP into BBD (Sato et al.,2010; Bourne et al., 2011). A rank abundance analysis ofall identified OPUs revealed the dominance of this OPU1relative to the other 19 identified OPUs (Fig. S1). OPU-1exhibited ~ 89% sequence identity (over 250 amino acids)with sulfate thioesterase/sulfate thiohydrolase proteinsderived from members of the Rhodobacteraceae, afamily within the Rhodobacterales and belonging to thea-Proteobacteria class. A recent study by Lenk andcolleagues (2012) identified that members of the Roseo-bacter clade bacteria (RCB), previously identified asabundant members of the bacterioplankton belongingto the Roseobacter lineage (Buchan et al., 2005;Wagner-Döbler and Biebl, 2006), are also abundant incoastal sediments. Other OPUs (in addition to OPU-1,e.g. OPUs 3, 10, 14, 15, 16 and 17) were similarly relatedwith SoxB sequences of RBC recovered from the study ofLenk and colleagues (2012). These RCB are commonlyassociated with marine invertebrates (Wagner-Döbler andBiebl, 2006) and have also been observed in CP andBBD lesions (Sato et al., 2010). RCB encode a novelcombination of sulfur-oxidizing genes, potentially havingimportant functional roles in cycling sulfur compounds inboth oxic and anoxic sediments (Lenk et al., 2012). There-fore the dominance of SoxB sequences affiliated to RCB

2 D. G. Bourne, M. J. J. van der Zee, E. S. Botté and Y. Sato

© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology Reports

SoxB_OPU-16 (KC494296)

uncultured bacterium (AFA 55003)


Sulfitobacter sp. NAS-14 (ZP 00963529)



AFA55017 uncultured bacterium (AFA55017)




Roseovarius sp. 217 (ZP 01037116)

Pelagibaca bermudensis HTCC2601 (ZP 01445923)

Rhodovulum adriaticum (ABR67362)


uncultured bacterium (CBH30921)


Bradyrhizobium sp. ORS 278 (YP 001205215)

Methylobacterium extorquens DM4 (YP 003066439)


Magnetospirillum.gryphiswaldense MSR-1 (CAM76242)





uncultured bacterium HTCC2080 (ZP 01627095)



Thiobacillus aquaesulis (ABR67367)

Ralstonia eutropha JMP134 (YP 297452)


Beggiatoa sp. B2 (AEL13418)

Thiocystis violacea (ABR67343)

Thiorhodococcus minor (ABR67376)



uncultured bacterium (AFA55002)

Congregibacter litoralis KT71 (ZP 01102556)

gammaproteobacterium IMCC3088 (ZP 08271487)




uncultured bacterium (AFA55008)

Sulfurimonas denitrificans DSM 1251 (ABB43545)

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521 00

0.15<X<10

X>50

X<5Number of sequences in OPU

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9 5

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Fig. 1. Neighbour-Joining phylogenetic tree for the soxB gene subunit of the Sox multienzyme complex involved in oxidizing thiosulfate tosulfate and conserved within SOB. Phylogenetic relationships between sequences obtained from CP and BBD lesion samples and referencesequences of SOB obtained from GenBank. Sequences were grouped into OPUs based on a 90% sequence identity cut-off. Representativesequences of OPU groups identified in this study are presented in bold text and numbers in the adjacent box are indicative of the numberof sequences within each OPU group. These sequences were also deposited in the GenBank database with accession numbers (KC494281to KC494300) also represented in brackets. Total numbers of retrieved sequences were 170 for CP and 104 for BBD. The scale bar is anestimate of sequence divergence (10%).

Sulfur-oxidizing bacterial populations of black band diseased corals 3


suggests that these organisms may also be involved insulfur cycling in oxic and anoxic microenvironments of CPand BBD microbial mats.

A number of OPUs found in both CP and BBD lesionsaffiliated with sulfate thioesterase/sulfate thiohydrolaseproteins from bacteria within the b-Proteobacteria andg-Proteobacteria classes and all showed only between67% and 91% sequence identity to these previouslyidentified sequences. The low sequence homologies iden-tified in this study highlight the diverse and potentiallyunique metabolic nature of the bacteria present within thestructured microbial mats of CP and BBD coral diseaselesions. Major phylogenetic branching of many SoxBOPUs and other sulfate thioesterase/sulfate thiohydrolaseproteins sequences are supported by high bootstrapvalues and consistent with taxonomic classification(Fig. 1). A few exceptions were observed however,including the incongruent placement of an unculturedg-proteobacterium partial protein sequence (strainHTCC2080 ZP_01627095) within the b-proteobacterialcluster, likely due to multiple lateral soxB gene transfersthroughout evolution (Meyer et al., 2007).

Interestingly, only one SoxB OPU sequence (OPU11representing two sequences) was affiliated to Beggiatoaspp. sulfate thioesterase/sulfate thiohydrolase proteinsequences, with direct comparison revealing ~ 80%sequence identity (over 256 aa). This indicates thatBeggiatoa spp. are not abundant in BBD lesions at ourstudy site and may not be a common component ofthe BBD microbial community, contrasting with the previ-ous report of their presence based on microscopic iden-tification (Rutzler and Santavy, 1983; Richardson, 1996).

Preisler and colleagues (2007) studied the contribution ofsulfide-oxidizing Beggiatoa spp. for removal of sulfidefrom coastal sediments. These organisms were responsi-ble for only a small fraction of the total sulfide removal,and sulfide was mostly removed by chemical processes.In addition, the authors reported sulfide to be a repellentfor Beggiatoa (Preisler et al., 2007), and therefore thehigh total sulfide in the BBD lesion may also explain whyfew Beggiatoa spp.-affiliated soxB sequences weredetected from BBD lesions.

To quantitatively assess SOB populations within micro-bial consortia of CP and BBD, we employed a quantita-tive real-time PCR (qPCR) assay targeting the dominantsoxB gene coding OPU-1. Primers soxB-OTU1-F125 (5′-GGCTTTGACGTTGACAAGA-3′) and soxB-OTU1-R275(5′-CGCGGGTCACAAATTTAC-3′) were designed usingPrimer3 online software (available at http://primer3.wi.mit.edu/) and following recommendations by Applied Bio-systems (CA, USA). The TaqMan® chemistry (AppliedBiosystems) and an additional internal probe [soxB-OTU1-H163 (HEX)-5′-AGGCATCGACGTGATCTTGTCG-3′-(TAMARA)] were used for increased specificity anddiscrimination in quantification of the target soxB geneswithin samples (see Supporting information for detailedoptimized qPCR reaction conditions). The raw copynumber of soxB genes detected in three independentreplicate CP lesions was ranging from 100 to 800 copiesper ng DNA, although it was near the detection limit ofthe assay in BBD samples (between 6 and 70 copies perng DNA). The appropriate negative controls were simul-taneously run, ensuring that DNA contamination wasabsent and that our results were robust even near thedetection limit of qPCR. The abundance of soxB geneswas normalized against the abundance of bacterial 16SrRNA-coding DNA to avoid misleading interpretationof relative abundance of soxB copy number per totalDNA, which can be influenced by the large amount ofco-extracted eukaryotic DNA (e.g. degrading coral tissueand zooxanthellae). To this end, an additional real-timeTaqMan® PCR assay was performed for quantification ofthe bacterial abundance by targeting the 16S rRNA geneas outlined in Bourne and colleagues (2011). Conse-quently, the soxB gene copy number was expressedas a proportion of dominant SOB (OPU-1 type) withinthe total bacterial population of the lesions. The averagepercentage of the dominant SOB was ~ 0.002% of thetotal bacterial population in CP lesions and an order ofmagnitude lower in BBD samples (< 0.0002%) (Fig. 2).The results demonstrated that (i) SOB populationsaccounted for low proportion of Bacteria in both CP andBBD and (ii) these SOB populations decreased in BBDlesions relative to CP. Although the decrease of SOBpopulations within lesions was not statistically signi-ficant due to sample variation, qPCR results indicated

CP BBD

Per

cent

age

popu

lati

on S

OB

0.0000

0.0005

0.0010

0.0015

0.0020

Fig. 2. Quantitative real-time PCR assessment of the number ofthe dominant soxB genes within CP and BBD coral diseaselesions. The number of soxB genes (OPU-1 type) was calculatedas gene copies per ng extracted DNA and normalized relative tothe total bacterial population determined by quantitative real-timePCR targeting 16S rRNA genes. Results are shown as averagepercentage (n = 3) of dominant SOB within the total bacterialcommunity.



http://primer3.wi.mit.edu/

http://primer3.wi.mit.edu/

consistent trends across all replicate sample sets (n = 3colonies; Fig. S3).

A previous study on this CP-BBD model system identi-fied that SRB populations increase in abundance as thelesion transitions from CP to BBD (Bourne et al., 2011).An increase in SRB and decrease in SOB (as detected inthis current study) would both contribute to the accumu-lation of sulfide in BBD lesions, confirmed by previousmicrosensor studies which detected extremely high con-centrations of sulfide in BBD lesions (~ 5000 mM) and littleor no sulfide in the CP microbial mat (Glas et al., 2012).Preliminary data derived from metagenomic studies of CPand BBD lesions also demonstrate a relative decrease ingenes involved in sulfur oxidation (results not shown).Low abundance of SOB populations in BBD lesions isalso supported by the many studies that have docu-mented a highly diverse bacterial community althoughretrieved few sequences affiliated with prominent SOB(Frias-Lopez et al., 2002; 2004; Cooney et al., 2002a;Barneah et al., 2007; Sato et al., 2010).

Other thiosulfate-oxidizing enzymatic systems/enzymes, for example an incomplete Sox complex plusthe reversible Dsr and a thiosulfate dehydrogenase (seeHensen et al., 2006), may also occur in BBD lesions tofacilitate the cycling of the high sulfide levels. Sulfur-oxidizing organisms responsible for these enzymaticprocesses may therefore be better adapted to the physi-ochemical conditions of the microbial lesions and contrib-ute to the overall low abundance of SOB populations.Attempts were made to further identify SOB within thelesions using the specific amplification and cloning-basedsequencing of the sat gene coding for a sulfate adenylyl-transferase, converting Adenosine-5-phosphosulfate intosulfate (see Supplementary material and methods). Thisenzyme is the only other protein-encoding gene known toconvert a precursor into sulfate in the sulfide-oxidizingpathway, along with soxB (Frigaard and Dahl, 2009;Gregersen et al., 2011); however, it is found in both SOBand SRB populations. Analysis of > 200 sat genesequences derived from both CP and BBD samplesrevealed that the vast majority of sequences (> 95%) wereaffiliated with SRB bacteria and not SOB (data notshown). Protein sequences translated from sat genesequences showed sequence identity (~ 90%) with Des-ulfovibrio spp. These results are consistent with the obser-vation that SRB affiliated with Desulfovibrio spp. dominatethe SRB community of both CP and BBD lesions and thatSOB populations (the dominant OPU type) have a farlower relative abundance within the lesions (< 0.0002%)compared with SRB (over 7% of the total bacterial popu-lation in some samples; see Bourne et al., 2011).

The present study provides the first direct investigationof SOB populations within cyanobacterial dominated coraldisease lesions. Within both CP and BBD lesions, SOB

have a very low abundance, suggesting that they onlyplay a minor role in oxidizing reduced sulfur compounds.Sulfide derived from bacterial sulfate reduction and des-ulfuration of degrading coral tissue accumulates at thebase of the BBD microbial mat, reaching levels up to~ 5000 mM (Glas et al., 2012). The low level of oxidation ofthe reduced sulfide compounds mediated by SOB is likelyto accentuate this accumulation. Although of low abun-dance, SOB were detected in the disease lesions andphylogenetic comparisons based on the SoxB proteinsequences identified the dominant SOB as members ofthe Rhodobacteraceae within the a-Proteobacteria,similar to SoxB sequences retrieved from the Roseo-bacter lineage and which have a potential important rolein cycling sulfur in marine sediments (Lenk et al., 2012).Overall, the present study reports for the first time both aqualitative and a quantitative assessment of SOB popu-lations in BBD-associated coral lesions, thereby providinga further step towards a better understanding of thisdisease.

Acknowledgements

The authors thank staff of Orpheus Island Research Station(JCU) for logistic support and field volunteers for their assist-ance during sampling. This study was supported by the Aus-tralian Institute of Marine Science and a research grant fromMitsubishi Corporation, and logistically supported by Earth-watch Institute Australia, AIMS@JCU, and the QueenslandGovernment funded Centre for Marine Microbiology andGenetics at AIMS.

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Supporting information

Additional Supporting Information may be found in the onlineversion of this article at the publisher’s web-site:

Supplementary materials and methods.Fig. S1. Rank abundance charts of OPUs retrieved from CPand BBD lesion samples. OPU-1 dominated retrievedsequences and therefore for clarity, the rank abundance ofOPUs 2–20 are inserted in an additional rank abundancedisplay.



Fig. S2. Rarefaction curves based on OPUs retrieved fromCP and BBD lesion samples and defined at 90% proteinsequence similarity.Fig. S3. Quantitative real-time PCR assessment of thenumber of the dominant soxB genes within individual CP and

BBD coral disease lesions. The number of soxB genes(OPU-1 type) was calculated as gene copies per ng extractedDNA and normalized relative to the total bacterial populationdetermined by quantitative real-time PCR targeting 16SrRNA genes.



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Sulfur-oxidizing bacterial populations within cyanobacterial dominated coral disease lesions